Pathogens occuring in the winter pea - maize

PATHOGENS OCCURRING IN THE WINTER PEA – MAIZE – WINTER

WHEAT ROTATION, THEIR HOST SPECIFICITY AND THE

POTENTIAL OF COMPOST IN SUPPRESSING FOOT AND ROOT

DISEASE OF PEAS

DISSERTATION

ZUR ERLANGUNG DES AKADEMISCHEN GRADES EINES DOKTORS DER AGRARWISSENSCHAFTEN (DR. AGR.)

EINGEREICHT AM FACHBEREICH ÖKOLOGISCHE AGRARWISSENSCHAFTEN DER UNIVERSITÄT KASSEL

VON

JELENA BAĆANOVIĆ

January 2015

Supervisor:

1 Prof. Dr. Maria R. Finckh

2 Prof. Dr. Gunter Backes

Defense date: 15.04.2015

To a loving memory of my sister

Milena Baćanović

(10.01.1985 – 07.04. 2009)

You will always be in my heart and my thoughts

TABLE OF CONTENTS

IV

Table of Contents

Table of Contents ............................................................................................................... IV

List of Abbreviations......................................................................................................... VII

List of Figures ................................................................................................................. VIII

List of Tables .................................................................................................................. XIII

1. GENERAL INTRODUCTION ............................................................................................ 1

1.1 Effects of modern agriculture and climate change on soil health and quality ........... 1

1.2 Possibilities to enhance soil health and quality and system resilience ..................... 3

1.3 Legumes as important components in sustainable agricultural systems .................. 4

1.4 Objectives and thesis outline ................................................................................... 6

1.5 References .............................................................................................................. 7

2. INCIDENCE OF FUSARIUM SPP. IN THE CROP ROTATION WINTER PEA – MAIZE – WINTER WHEAT AND THE POTENTIAL OF YARD WASTE COMPOST TO SUPPRESS FOOT AND ROOT ROT OF PEAS UNDER FIELD CONDITIONS .......................................10

Abstract ............................................................................................................................10

2.1 Introduction ............................................................................................................11

2.2 Material and Methods .............................................................................................13

2.2.1 Experimental site and setup ............................................................................13

Inoculum preparation and inoculation ............................................................................17

2.2.2 Assessments ........................................................................................................19

Soil N-dynamics ............................................................................................................19

Plant development and yields ........................................................................................19

Disease assessments, pathogen isolation and identification .........................................19

2.3 Data processing and analysis ................................................................................22

2.4 Results ..................................................................................................................23

2.4.1 Weather data ........................................................................................................23

2.4.2 Crop performance .................................................................................................25

2.4.3 Foot and root rot of peas and wheat .....................................................................28

2.4.4 Occurrence of Fusarium spp. in the tree crops in the rotation ...............................30

2.5 Discussion ..................................................................................................................34

2.6 References .................................................................................................................37

3. AGGRESSIVENESS OF FOUR PATHOGENS CAUSING FOOT AND ROOT ROT OF PEA (PISUM SATIVUM L.) ON A SPRING AND A WINTER PEA CULTIVAR UNDER VARIABLE TEMPERATURE CONDITIONS ........................................................................40

Abstract ............................................................................................................................40

TABLE OF CONTENTS

V

3.1 Introduction .................................................................................................................41

3.2 Materials and Methods ................................................................................................45

3.2.1 Pathogenicity of Fusarium avenaceum, Fusarium solani, Phoma medicaginis and Didymella pinodes, in sterile sand and in non-sterilized field soil ...................................45

3.2.2 Effect of the temperature on the aggressiveness of F. avenaceum and P. medicaginis ...................................................................................................................47

3.2.3 Pathogenicity of F. avenaceum .........................................................................47

3.2.4 Measurement of plant biomass and assessment of disease .................................48

3.2.5 Data processing and analysis ...............................................................................48

3.3 Results ........................................................................................................................49

3.3.1 Comparison of aggressiveness of F. avenaceum, F. solani, P. medicaginis and D. pinodes on two pea varieties in sand and field soil ........................................................49

3.3.2 Effect of temperature on disease severity caused by F. avenaceum and P. medicaginis ...................................................................................................................56

3.3.3 Pathogenicity of Fusarium avenaceum .................................................................57

3.4 Discussion ..................................................................................................................60

3.5 References .................................................................................................................63

4. EFFECTS OF COMPOST APPLICATION ON FOOT AND ROOT ROT OF SPRING AND WINTER PEA VARIETIES CAUSED BY FUSARIUM AVENACEUM, FUSARIUM

SOLANI, PHOMA MEDICAGINIS AND DIDYMELLA PINODES .........................................66

Abstract ............................................................................................................................66

4.1 Introduction ............................................................................................................67

4.2 Material and Methods .............................................................................................69

4.2.1 Potential of yard waste compost to suppress foot and root rot of peas caused by F. avenaceum, F. solani, P. medicaginis and D. pinodes ...................................................69

4.2.2 Effect of compost application rate on disease suppression ...................................70

4.2.3 Effect of reduced temperature on compost induced disease suppression .............70

4.2.4 Measurement of plant biomass and assessment of disease .................................71

4.2.5 Data processing and analysis ...............................................................................71

4.3 Results ...................................................................................................................72

4.3.1 Potential of yard waste compost to suppress foot and root rot of peas caused by F. avenaceum, F. solani, P. medicaginis and D. pinodes ...................................................72

4.3.2 Effect of compost application rate on disease suppression ...................................75

4.3.3 Temperature effect on compost induced disease suppression .............................77

4.4 Discussion ..............................................................................................................79

4.5 References .................................................................................................................82

5. GENERAL DISCUSSION ..............................................................................................84

5.1 References .................................................................................................................89

TABLE OF CONTENTS

VI

APPENDIX ...........................................................................................................................90

Chapter 2 ..........................................................................................................................91

Chapter 3 ........................................................................................................................ 100

Chapter 4 ........................................................................................................................ 110

Summary ........................................................................................................................... 116

Acknowledgments ............................................................................................................ 120

Erklärung .......................................................................................................................... 121

LIST OF ABBREVIATIONS

VII

List of Abbreviations SOM Soil organic matter

YWC Yard waste compost

IPCC Intergovernmental Panel on Climate Change

EEG Renewable Energy Sources Act

FHB Fusarium head blight

TGW Thousand grain weight

OA Organic amendments

Nmin Mineral nitrogen

ANOVA Analysis of Variance

DI Disease index

HSD Honest significant differences

ÖNORM Österreichisches Normungsinstitut

VDLUFA Verband deutscher landwirtschaftlicher Untersuchungs- und

Forschungsanstalten

SD Standard deviation

Pm Phoma medicaginis

Dp Didymella pinodes

Fs Fusarium solani

Fo Fusarium oxysporum

Fa Fusarium avenaceum

BOFRU Projekt: „Steigerung der Wertschöpfung ökologisch angebauter

Marktfrüchte durch Optimierung des Managements der

Bodenfruchtbarkeit“

Df Degrees of freedom

PDA Potato dextrose agar

SNA Synthetic Nutrient Agar

PPA Pentachloronitrobenzene Peptone Agar

DM Dry matter

KLIFF KLimaFolgenForschung in Niedersachsen

LIST OF FIGURES

VIII

List of Figures

Figure 2.1. Section of the crop rotation that was studied in the field experiment. .................14

Figure 2.2. Main plot divided in four experimental plots presenting four different treatments

O=Control, I=Inoculated with P. medicaginis, C=Compost and I+C= Inoculated + Compost. 15

Figure 2.3. Inoculum of Phoma medicaginis grown on oat seeds.........................................17

Figure 2.4. Monthly mean temperatures (°C) and precipitation (mm) in the period between

2009 and 2013, compared with thirty year average (1970-2000). .........................................24

Figure 2.5. Percentage of the emerged pea plants that survived the winters 2010, 2011 and

2012. Treatments O = Control; I = Inoculated with P. medicaginis; C= Compost; I+C=

Inoculated with P. medicaginis + Compost. The error bars represent the ± 1 SD.................25

Figure 2.6. Dry matter yield of peas (A) and maize (B), and grain yield of wheat (14%

moisture) (C). For peas O= control, I= inoculated with P. medicaginis, C= compost, I+C=

inoculated with P. medicaginis + compost. For maize and wheat C= with compost, O=

without compost. Different small letters within one crop indicate significant differences in yield

among years at P < 0.05 (Tukey’s HSD test). The error bars represent the ± 1 SD. .............26

Figure 2.7. Effect of compost application on pea fresh matter yield in May 2010 and 2011..

.............................................................................................................................................27

Figure 2.8. Mineral nitrogen (Nmin) content in soil at 0-60 cm depth. The error bars

represent the ± 1 SD. n.a. stand for not available. ................................................................28

Figure 2.9. Foot Disease Index (DI) on peas in March and May 2010-2012. For the year

2012 disease was assessed only in May. Different small letters within one year indicate

significant differences in DI at P < 0.05 (Tukey’s HSD test). Different capital letters indicate

significant differences in DI in May among years at P < 0.05 (Tukey’s HSD test). The error

bars represent the ± 1 SD. ...................................................................................................29

Figure 2.10. Incidence of P. medicaginis (Pm), D. pinodes (Dp), F. solani (Fs), F. oxysporum

(Fo) and F. avenaceum (Fa) isolated from pea in May of 2010, 2011, 2012. Different small

letters within one pathogen indicate significant differences in incidence at P < 0.05 (Tukey’s

HSD test). .............................................................................................................................29

Figure 2.11. Foot rot disease of wheat caused by Fusarium spp., Pseudocercosporella

herpotrichoides and Rhizoctonia cerealis in treatments without (O) and with compost (C).

The error bars represent the ± 1 SD. ....................................................................................30

Figure 2.12. Incidence of Fusarium spp. isolated from three crops in the rotation. WP =

winter peas, M = maize and WW = winter wheat; time of samplings, month/year, is given in

the brackets. .........................................................................................................................32

Figure 3.1. Disease index (DI) of Santana and EFB33 plants grown in sand and soil and

inoculated with five isolates of F. avenaceum (A), F. solani (B), D. pinodes (C) and P.

LIST OF FIGURES

IX

medicaginis (D). Different small letters within variety and substrate are indicating significant

differences in DI at P < 0.05 (Tukey’s HSD test). The horizontal line in the boxplot shows the

median, the bottom and tops of the box the 25th and 75th percentiles and the vertical lines

the minimum and maximum values; outliers as single points. Mean values of DI are marked

with triangles. .......................................................................................................................50

Figure 3.2. Fresh weights of Santana and EFB33 plants inoculated with five isolates of F.

avenaceum and grown in sterile sand or non-sterilized field soil. Different letters within

variety and substrate are indicating significant differences in DI at P < 0.05 (Tukey’s HSD

test). The error bars represent the ± 1 SD. ...........................................................................51

Figure 3.3. Fresh weights of Santana and EFB33 plants inoculated with five isolates of P.

medicaginis and grown in sterile sand or non-sterilized field soil. Different letters within

variety and substrate are indicating significant differences in DI at P < 0.05 (Tukey’s HSD

test). The error bars represent the ± 1 SD. ...........................................................................51

Figure 3.4. Typical symptoms of F. solani infection on the stem base of Santana (A) and

EFB33 (B) plants. .................................................................................................................52

Figure 3.5. Wilting symptoms of Santana caused by F. avenaceum with the formation of

bright orange sporodochia (marked with the arrow). .............................................................53

Figure 3.6. Disease index (DI) of Santana and EFB33 plants grown in sand and soil and

inoculated with F. avenaceum (Fa), F. solani (Fs), P. medicaginis (Pm) and D. pinodes (Dp).

O is the non-inoculated control. The horizontal line in the boxplot shows the median, the

bottom and tops of the box the 25th and 75th percentiles and the vertical lines the minimum

and maximum values; outliers as single points. Mean values of DI are marked with triangles.

Different letters within variety and substrate are indicating significant differences in DI at P <

0.05 (Tukey’s HSD test). ......................................................................................................54

Figure 3.7. Fresh weights of Santana and EFB33 inoculated with F. avenaceum (Fa), F.

solani (Fs), P. medicaginis (Pm) and D. pinodes (Dp). Different letters within variety and

substrate are indicating significant differences in DI at P < 0.05 (Tukey’s HSD test). The error

bars represent the ± 1 SD. ...................................................................................................54

Figure 3.8. Disease index of Santana and EFB33 inoculated with F. avenaceum (Fa) and P.

medicaginis (Pm) and grown under different temperature regimes. O is the non-inoculated

control. The horizontal line in the boxplot shows the median, the bottom and tops of the box

the 25th and 75th percentiles and the vertical lines the minimum and maximum values;

outliers as single points. Mean values of DI are marked with triangles. Different letters within

variety and temperature are indicating significant differences in DI at P < 0.05 (Tukey’s HSD

test). .....................................................................................................................................56

Figure 3.9. Mean values of fresh weights of Santana and EFB33 plants grown at three

temperature regimes and inoculated with F. avenaceum (Fa) and P. medicaginis (Pm). O is

LIST OF FIGURES

X

the non-inoculated control. Different capital letters within one variety indicate significant

differences in fresh weights among temperature regimes, whereas small letters indicate

significant differences among treatments within variety and temperature regime at P < 0.05

(Tukey’s HSD test). The error bars represent the ± 1 SD. ....................................................57

Figure 4.1. Effect of compost application (no-without, N-not sterilized and S-sterilized

compost) on fresh weight per plant of Santana in sand and soil inoculated with F. avenaceum

(Fa), F. solani (Fs), P. medicaginis (Pm) and D. pinodes (Dp). Different letters within one

substrate and one pathogen indicate significant differences in fresh weights among compost

treatments at P < 0.05 (Tukey’s HSD test). The error bars represent the ± 1 SD. .................74

Figure 4.2. Disease index of Santana and EFB33 inoculated with F. avenaceum (Fa), F.

solani (Fs), P. medicaginis (Pm) and D. pinodes (Dp) and amended with compost (no-

without, low- 3.5% v/v and high- 20% v/v). O is the non-inoculated control. The horizontal line

in the boxplot shows the median, the bottom and tops of the box the 25th and 75th

percentiles and the vertical lines the minimum and maximum values; outliers as single points.

Mean values of DI are marked with triangles. Different letters within variety and pathogen are

indicating significant differences in DI at P < 0.05 (Tukey’s HSD test). .................................75

Figure 4.3. Effect of compost application rate (no-without, low- 3.5% v/v and high- 20% v/v)

on the fresh weight of (A) Santana and (B) EFB33 plants inoculated with 105 spores g-1 of

substrate of F. avenaceum (Fa), F. solani (Fs), P. medicaginis (Pm) and D. pinodes (Dp).

Different letters within pathogen are indicating significant differences in fresh weights at P <

0.05 (Tukey’s HSD test). The error bars represent the ± 1 SD. .............................................76

Figure 4.4. Effect of compost application (no-without, compost- 20% v/v) on the fresh

weights of Santana (A) and EFB33 (B) plants inoculated with F. avenaceum (Fa) and P.

medicaginis (Pm) and grown under different temperature regimes (low - 16/12°C and high -

19/16°C). Asterisk indicates significant differences at P < 0.05 among treatments with and

without compost within one temperature regime and pathogen. The error bars represent the

± 1 SD. .................................................................................................................................78

Figure A 2.1. Effect of treatments on pea emergence one month after sowing presented as

percentage of sowing densities. The error bars represent the ± 1 SD. ..................................92

Figure A. 2.2. Emergence (Autumn) and winter survival (Spring) of peas as a percentage of

sown plants in 2011/12. The error bars represent the ± 1 SD. ..............................................92

Figure A. 2.3. Fresh weight per plant of maize in 2010, 2011 and 2012. Different letters

indicate significant differences at P < 0.05 (Tukey’s HSD test). The error bars represent the ±

1 SD. ....................................................................................................................................94

Figure A. 2.4. Thousand grain weights (g) (A) and number of the heads per m- 2 (B) of wheat

in treatments with (+ compost) and without compost (- compost) in three experimental years

LIST OF FIGURES

XI

(2011 - 2013). Different letters indicate significant differences at P < 0.05 (Tukey’s HSD test).

The error bars represent the ± 1 SD. ....................................................................................94

Figure A. 2.5. Incidence (%) of pathogens isolated from pea in different treatments in March

2010 and 2011. O = Control; I = Inoculated with P. medicaginis; C= Compost; I+C=

Inoculated with P. medicaginis + Compost. ..........................................................................96

Figure A. 2.6. Incidence (%) of pathogens isolated from pea in different treatments in May of

three experimental years. O = Control; I = Inoculated with P. medicaginis; C= Compost; I+C=

Inoculated with P. medicaginis + Compost. ..........................................................................96

Figure A. 2.7. Pathogen incidence on pea in March and May of 2010 and 2011. Pm = P.

medicaginis, Dp = D. pinodes, Fs = F. solani and Fo = F. oxysporum. .................................98

Figure A. 2.8. External (A) and Internal (B) disease scores of pea in May of all three

experimental years. Score 1 stands for healthy plant; score 9 for dead plant. O = Control; I =

Inoculated with P. medicaginis; C= Compost; I+C= Inoculated with P. medicaginis +

Compost. ..............................................................................................................................97

Figure A. 2.9. External (A) and Internal (B) disease scores of pea in March and May 2010.

Score 1 stands for healthy plant; score 9 for dead. O = Control; I = Inoculated with P.

medicaginis; C= Compost; I+C= Inoculated with P. medicaginis + Compost. .......................98

Figure A. 2.10. External (A) and Internal (B) disease scores of pea in March and May 2011.

Score 1 stands for healthy plant; score 9 for dead plant. O = Control; I = Inoculated with P.

medicaginis; C= Compost; I+C= Inoculated with P. medicaginis + Compost. .......................99

Figure A. 2.11. Incidence of different Fusarium spp. isolated from maize (A) and wheat (B) in

treatments with (+ compost) and without compost (- compost) from 2009-2013. For wheat

n=30 in 2011 and n=20 in 2012 and 2013. For maize n=60 for all three years. ................... 100

Figure A. 3.1. External lesion scores for all of the isolates of all tested pathogens on

Santana .............................................................................................................................. 103

Figure A. 3.2. External lesion scores for all of the isolates of all tested pathogens on EFB33

........................................................................................................................................... 104

Figure A. 3.3. Fresh weight of Santana and EFB33 plants inoculated with five isolates of F.

solani and grown in sterile sand or non-sterilized field soil. The error bars represent the ± 1

SD. ..................................................................................................................................... 105

Figure A. 3.4. Fresh weight of Santana and EFB33 plants inoculated with five isolates of D.

pinodes and grown in sterile sand or non-sterilized field soil. The error bars represent the ± 1

SD. ..................................................................................................................................... 105

Figure A. 3.5. Correlation between fresh weight and disease severity of Santana grown in

sterile sand (A) and non-sterilized field soil (B) and inoculated with different pathogens. Only

significant regressions are indicated with regression lines. ................................................. 108

LIST OF FIGURES

XII

Figure A. 3.6. Correlation between fresh weight and disease severity of EFB33 grown in

sterile sand (A) and non-sterilized field soil (B) and inoculated with different pathogens. Only

significant regressions are indicated with regression lines. ................................................. 109

Figure A. 4.1. Disease index of EFB33 in sand and soil inoculated with F. avenaceum (Fa),

F. solani (Fs), P. medicaginis (Pm) and D. pinodes (Dp) and amended with compost (no-

without, N-not sterilized and S-sterilized compost). The horizontal line in the boxplot shows

the median, the bottom and tops of the box the 25th and 75th percentiles and the vertical

lines the minimum and maximum values; outliers as single points. Mean values of DI are

marked with triangles.......................................................................................................... 111

Figure A. 4.2. Effect of compost application (no-without, N-not sterilized and S-sterilized

compost) on fresh weight per plant of EFB33 in sterile sand and non-sterilized field soil


Different letters within one substrate and one pathogen indicate significant differences in

fresh weights among compost treatments at P < 0.05 (Tukey’s HSD test). The error bars

represent the ± 1 SD. ......................................................................................................... 111


compost) on dry weight per plant of Santana in sterile sand and non-sterilized field soil




represent the ± 1 SD. ......................................................................................................... 112


compost) on dry weight per plant of EFB33 in sterile sand and non-sterilized field soil




represent the ± 1 SD. ......................................................................................................... 112

Figure A. 4.5. Effect of compost application rate (no-without, low- 3.5% v/v and high- 20%

v/v) on the dry weight per plant of Santana and EFB33 plants inoculated with F. avenaceum

(Fa), F. solani (Fs), P. medicaginis (Pm) and D. pinodes (Dp). Different letters within one

variety and one pathogen indicate significant differences in fresh weights among compost

treatments at P < 0.05 (Tukey’s HSD test). The error bars represent the ± 1 SD. ............... 114

Figure A. 4.6. Effect of compost application (no-without, compost- 20% v/v) on the dry

weights of Santana (A) and EFB33 (B) plants inoculated with F. avenaceum (Fa) and P.

medicaginis (Pm) and grown under different temperature regimes (low – 12/16°C, high –

16/19°C). Asterisk indicates significant differences in fresh weights between two compost

treatments within one pathogen. ......................................................................................... 115

LIST OF TABLES

XIII

List of Tables

Table 2.1. Chemical characteristic of composts used in the field from 2009-2013. ...............16

Table 2.2. Treatments applied in the field experiment. .........................................................17

Table 2.3. Timing of field operations. ...................................................................................18

Table 2.4. Scoring scheme for assessment of root and foot rot of peas (Pflughöft, 2008). ...21

Table 2.5. Number and average temperatures of the frost and icing days in February. ........23

Table 2.6. Incidence of different Fusarium spp. in the maize and wheat in 2010 - 2013.

Different letters within one species indicate significant differences in incidence among three

experimental years at P < 0.05 (Tukey’s HSD test). .............................................................33

Table 3.1. Pathogens and isolates used in experiments. ......................................................46

Table 3.2. Temperature regimes used in the experiment .....................................................47

Table 3.3. Plant species, varieties and sowing density used in the experiment. ...................47

Table 3.4. Disease Index (DI), coefficient of determination (R2) and significance for the

correlation between Disease Index and fresh weights of EFB33 and Santana plants grown in

sterile sand and not sterilized field soil. ................................................................................55

Table 3.5. Emergence, percentage of emerged plants that wilted, fresh weight per pot and

plant height of nine plant species inoculated with two F. avenaceum isolates, (Fa1 – isolate

from wheat, Fa4 – isolate from pea). ....................................................................................59

Table 4.1. Chemical properties of substrates and composts used in the experiment. ...........70

Table 4.2. Disease Index (DI) of Santana in sterile sand and non-sterilized field soil in non-

inoculated control and treatments inoculated with F. avenaceum, F. solani, P. medicaginis

and D. pinodes and amended with compost (no-without, N-not sterilized and S-sterilized

compost). Different letters within one pathogen and one substrate indicate significant

differences in DI among compost treatments at P < 0.05 (Tukey’s HSD test). ......................73

Table 4.3. Disease index of Santana and EFB33 inoculated with F. avenaceum and P.

medicaginis, amended with compost (no-without, compost - 20% v/v) and grown under two

temperature regimes (low - 16/12°C and high - 19/16°C). Different letters within one variety,

temperature and pathogen are indicating significant differences in DI among compost

treatments at P < 0.05 (Tukey’s HSD test). Percent change presents difference in DI in low

temperature treatment compared to high. .............................................................................77

Table A. 2.1. Mineral nitrogen (Nmin) content in the soil at 0-60 cm depth. O=Control,

I=Inoculated, C=Compost, I+C=Inoculated+Compost. ..........................................................93

Table A. 2.2. Fresh and dry weights of pea and weeds in 2010 and 2011. O = Control, I =

Inoculated, C = Compost, I+C = Inoculated + Compost ........................................................93

Table A. 2.3. F-values for dry matter yields of peas and maize, and grain yield of wheat in

field experiment from 2009-2013. .........................................................................................94

LIST OF TABLES

XIV

Table A. 2.4. F-values for the disease index (DI) for root and foot rot of peas in 2010 and

2011. ....................................................................................................................................95

Table A. 3.1. F-values for DI for isolate comparison of four pathogens in three-way ANOVA.

........................................................................................................................................... 101

Table A. 3.2. F-values for DI of Santana and EFB33 inoculated with five isolates of four

pathogens in sand and soil. ................................................................................................ 101

Table A. 3.3. F-values for fresh weights of Santana and EFB33 grown in sand and soil and

inoculated with five isolates of four pathogens. ................................................................... 101

Table A. 3.4. F-values for dry weights of Santana and EFB33 grown in sand and soil and

inoculated with five isolates of four pathogens. ................................................................... 102

Table A. 3.5. F-values for the DI of pea inoculated with four different pathogens in three-way

ANOVA............................................................................................................................... 106

Table A. 3.6. F-values for DI for pathogen comparison for Santana and EFB33 in sterile sand

and non-sterilized field soil. ................................................................................................ 106

Table A. 3.7. ANOVA table and F values for fresh and dry weights per plant of Santana and

EFB33 inoculated with four different pathogens and grown in sterile sand and non-sterilized

field soil. ............................................................................................................................. 106

Table A. 3.8. F-values for DI for Santana and EFB33 inoculated with P. medicaginis and F.

avenaceum and grown under three different temperature regimes. .................................... 107

Table A. 3.9. F-values for fresh and dry weights for Santana and EFB33 inoculated with P.

medicaginis and F. avenaceum and grown under three different temperature regimes. ..... 107

Table A. 4.1. F-values for fresh and dry weights and DI for Santana and EFB33 inoculated

with F. avenaceum, F. solani, P. medicaginis and D. pinodes and amended with 20% v/v of

non-sterilized and γ sterilized YWC and grown in sterile sand and non-sterilized field soil. 110

Table A. 4.2. F-values for DI, fresh and dry weights of Santana and EFB33 inoculated with

four pathogens and amended with two rates of compost (low – 3.5% v/v and high – 20% v/v)

and grown in sterile sand in three-way ANOVA. ................................................................. 113

Table A. 4.3. F-values for DI, fresh and dry weights of Santana and EFB33 inoculated with

four pathogens and amended with two rates of compost (low – 3.5% v/v and high – 20% v/v)

and grown in sterile sand. ................................................................................................... 113

Table A. 4.4. F-values for DI, fresh and dry weights of Santana and EFB33 inoculated with F.

avenaceum and P. medicaginis, amended with 20% v/v YWC and grown under two

temperature regimes (low - 16/12°C and high – 19/16°C) in sterile sand in three-way ANOVA

………………………………………………………………………………………………………114

GENERAL INTRODUCTION

1

1. GENERAL INTRODUCTION

1.1 Effects of modern agriculture and climate change on soil health and quality

Agricultural systems are considered to be sustainable if they can sustain themselves over a

long period of time, meaning they are economically viable, environmentally safe and socially

fair (Lichtfouse et al., 2009). At the center of each sustainable system is the soil, a resource

that is finite, unequally distributed, nonrenewable and prone to degradation (Lal, 2009).

Over the past half century, agriculture has become highly dependent on synthetically

produced agrochemicals resulting in changes in agricultural practices with respect to soil

management that have led to a general decline in soil quality (Bailey and Lazarovits, 2003).

Thus, in many modern industrialized agricultural systems traditional practices of crop rotation

and the use of animal manures or plant residues have been abandoned in favor of short

rotations, monocultures, intensive tillage, synthetic fertilizers and pesticides. In the long term,

this approach adversely affects soil fertility, quality and health (Sturz and Christie, 2003) as

the balance between beneficial and detrimental members of the resident microbial population

in the soil shifts in favor of the latter (Bailey and Lazarovits, 2003; Hoitink and Boehm, 1999;

Katan, 1997), leading to a decline in system productivity and an increase in plant diseases

(Doran and Zeiss, 2000). Nearly 40 % of the world’s agricultural land is showing human-

induced degradation as a result of soil erosion, pollution, over-grazing, salinization,

desertification, etc. (Doran, 2002). Because of these adverse effects of industrialized

agriculture on the environment, soil, water, and air quality there is a need to change the

existing short term approach in modern agriculture into long-term oriented, holistic and

sustainable systems geared towards improved soil quality and health.

Soil quality has been defined as “the capacity of a specific kind of soil to function, within

natural or managed ecosystem boundaries, to sustain plant and animal productivity, maintain

or enhance water and air quality, and support human health and habitation” (Schjønning et

al., 2004). Similarly, soil health has been defined as “the continued capacity of soil to function

as a vital living system, within ecosystem and land-use boundaries, to sustain biological

productivity, maintain the quality of air and water environments, and promote plant, animal,

and human health” (Doran et al., 1996). These definitions emphasize the multiple functions

soil resources have to fulfil in the modern world – i) production of food to meet the growing

needs of the world’s population, ii) energy production through growth of energy plants, iii)

sequestration of carbon, iv) improvement of water quality and use efficiency, v) waste

disposal medium, vi) habitat for diverse organisms, etc. (Lal, 2008). Soil organic matter

(SOM) is a key for maintaining soil quality and essential for the sustainability of agricultural

systems (Dick and Gregorich, 2004; Lal, 2009). Many soil properties such as microbial


2

activity, soil structure, cation exchange capacity, water to air ratio, etc., are directly or

indirectly affected by SOM content (Dick and Gregorich, 2004).

In addition to the need for high and stable production to feed a growing world population,

current agriculture also has to cope with global climatic changes and extreme weather events

of considerable magnitude (Crutzen, 2002; Walther et al., 2002; Parmesan and Yohe, 2003;

Root et al., 2003) that require mitigation and adaptation strategies. This needs to be based

on a solid understanding of soil processes and how these are affected by climate and

agricultural practices. The impacts of a changing climate on the system productivity are a

combination of direct effects on the growing environment, changes in the geography and

prevalence of pests and diseases, as well as changes in fertility and biological function of

soils (Jarvis et al., 2010). Thus, any environmental change that alters soil nutrient availability

changes the trophic structure in the soil and can affect crop productivity (Pritchard, 2011).

Moreover, these changes lead to changes in geographic distribution of host plants and their

pathogens and to changes in host-pathogen interactions (Coakley et al., 1999).

Climate change and climate variability are important drivers in the epidemiology of plant

diseases (Juroszek and Tiedemann, 2011). Working group I reported in the Third

Assessment Report of The Intergovernmental Panel on Climate Change (IPCC) that average

diurnal temperature had increased by 0.6°C in the 20th century. Sea levels increased

between 0.1 and 0.2 m. Overall annual changes in precipitation have been 1 % per decade.

However, frequency and intensity of heavy rainfall events as well as occurrence of extreme

weather conditions and cloud cover increased (IPCC, 2001). Climate change scenarios for

Germany exhibit a definite warming trend. By the year 2080 the annual average temperature

is predicted to increase by 1.6 – 3.8°C, compared to 1990. Although only small changes in

average annual precipitation are predicted, significant changes in the annual distribution of

precipitation are expected. Winter precipitation will increase between about 7 and 30%, while

summer precipitation is expected to decrease by about 5 to 33% by 2080 (Schröter et al.,

2005).

Factors that are most likely to influence disease development, spread and severity are

increased atmospheric CO2, extreme weather conditions (heavy rains, sudden drops of

temperature, drought, cyclones) and warmer winter temperatures (Cannon, 1998;

Chakraborty et al., 2000; Pimentel et al., 2001). Changed environmental conditions cause

alteration in morphology and physiology of plants such as stoma morphology, increased net

photosynthesis, carbohydrate accumulation in plant tissues, increased fiber content and

cuticular waxes, etc. that can modify host resistance (Chakraborty et al., 2000; Juroszek and

Tiedemann, 2011).


3

Despite the considerable body of literature available on the potential effects of the global

environmental changes on the above ground components of the ecosystem, by 2004, less

than 3 % of these articles were considering the belowground organisms or processes

(Pritchard, 2011; Wardle et al., 2004). Soil inhabiting organisms play an important role in the

dynamics of SOM decomposition and nutrient cycling. Thus, there is a need to understand

how changes in climate will affect the soil community. Predicted climatic changes will affect

soil microorganisms directly and indirectly through effects on plant growth and physiology.

Soil borne pathogens are already playing an important role, limiting agricultural production

worldwide (Pritchard, 2011). Increased winter temperatures will most likely reduce the

latency period and allow a higher number of generations of pathogens per season that can

result in more rapid buildup of pathogen populations in early spring (Garrett et al., 2006;

Pariaud et al., 2009). Effects of climate change on soil microbial processes are also likely to

affect pathogens. In moist and warm soils microbial diversity and biomass are higher and

that can lead to faster microbial turnover of organic material.

(Wardle et al., 2004) state that composition and functioning of soil organisms are closely

related to aboveground plant composition. Introduction of novel plant species into existing

ecosystems may alter the timing and type of inputs by rhizodeposition, introduce new

antimicrobial compounds, alter soil structure, and change the existing nutrient cycles (Wolfe

and Klironomos, 2005). Moreover, it can lead to introduction of new pests and pathogens

(Chakraborty et al., 2000). Newly introduced crops can act as alternative hosts to already

present pathogens and increase disease pressure for the succeeding crops. For example,

incidence of Fusarium graminearum a pathogen of Fusarium Head Blight (FHB) of wheat in

Western Europe has increased as a result of inclusion of maize in crop rotations (West et al.,

2012).

1.2 Possibilities to enhance soil health and quality and system resilience

Soil resilience and resistance are two fundamental components of soil quality (Seybold et al.,

1999). Soil resistance is “the capacity of a soil to continue to function without change

throughout a disturbance” (Herrick and Wander, 1997). Resilience is defined as “the capacity

of a system to absorb disturbance and re-organize while undergoing change so as to still

retain essentially the same function, structure, identity and feedbacks” (Walker et al., 2004).

A resilient soil can replace and rebuild functions that may have been compromised by

management and environmental changes. Both properties depend on the soil type, diversity

at all levels, land use, level of the disturbance, and climate (Seybold et al., 1999). Resilient

and resistant soils are bases on which resilient and sustainable agricultural systems should

be built on.


4

In recent decades, awareness of the dependence of sustainable agriculture on the

management of soil quality resulted in changes in soil tillage practices, introduction of cover

crops, living or dead mulches, application of organic amendments such as compost, green

manures or manures in the production system (Abawi and Widmer, 2000). These practices

that maintain or increase SOM content are improving soil functions and contributing to

sustainability of the system (Lal, 2008) as a soil’s biological, chemical, and physical

properties are functions of SOM (Abawi and Widmer, 2000). Soil is also the environment that

hosts soil borne pathogens; diseases caused by these are the result of a disturbed balance

between beneficial and harmful parts of the soil microbial community (Alabouvette et al.,

2004). Incorporation of different organic amendments not only increases SOM, provides

plant nutrients, and improves physical properties of soil, but it can also introduce beneficial

organisms which contribute to soil health and disease suppression (Boutler et al., 2000; Dick

and Gregorich, 2004; Hoitink et al., 1997; Hoitink and Fahy, 1986).

Composting of different biodegradable materials is an efficient and environmentally safe way

of dealing with organic wastes (Boutler et al., 2000) and, at the same time, the final product

is stable compost rich in humus, nutrients, and beneficial microorganisms. Compost induced

disease suppression can be attributed mainly to the biological properties of composts

(Hadar, 2011; Hoitink and Boehm, 1999). Characteristics of pathogen inhibiting microbial

populations in composts depend on the type of material used and conditions under which

composting was conducted (Boutler et al., 2000; Recycled Organics Unit, 2006).

Depending if the disease suppression is the result of the compost’s overall microbial biomass

or a specific group of microorganisms, two categories of disease suppression can be

distinguished – general and specific. General disease suppression is the result of total

microbial activity in a medium after compost addition which increases nutrient depletion

resulting in microstasis of pathogens (Aviles et al., 2011). In specific suppression, one or

more organisms are responsible for disease suppression. A range of different mechanisms

responsible for this type of suppression, such as competition for nutrients, infection sites,

direct antagonism, antibiotic production, parasitism, etc., have been described. Which of

these prevails depends on the type of pathogen and antagonistic organisms present in the

system (Hoitink et al., 1997; Hoitink and Fahy, 1986; Recycled Organics Unit, 2006).

1.3 Legumes as important components in sustainable agricultural systems

Enhanced diversity contributes to system resilience. Promoting the biodiversity in

agroecosystems, below ground as well as aboveground, provides ecological services that

are necessary for long-term sustainability of the system. However, this is only possible if the

diversification strategy takes into account the site specific conditions and is adjusted to local

conditions. Plants that not only have a role as a crop but also provide an ecological service to


5

the system are of great importance for the sustainability of agricultural systems. Because of

their ability for biological nitrogen fixation, leguminous plants can generally contribute to soil

fertility thus delivering an important ecological service. Due to the restrictions on the use of

mineral nitrogen fertilizers organic farming is highly dependent on legumes to build soil

fertility (Corre-Hellou and Crozat, 2005) and requires crop rotations with high frequencies of

leguminous crops. The amount of nitrogen fixed by different leguminous species is estimated

to be in the range between 20 and 350 kg N ha-1 (Karpenstein-Machan and Stuelpnagel,

2000; Peoples et al., 1995). In addition to nitrogen, legumes can also mobilize and

remineralize other nutrients (Eriksen and Thorup-Kristensen, 2002), thereby preventing their

leaching and making them available for following crops (Mueller and Thorup-Kristensen,

2001). Furthermore, used as a cover crop they can prevent soil erosion (Langdale et al.,

1991), increase water infiltration (Meisinger et al., 1991), improve the soil structure (Roldán

et al., 2003), and contribute to weed suppression (Hartwig and Ammon, 2002; Teasdale et

al., 2007).

Peas (Pisum sativum L.) together with faba beans (Vicia faba) are the most common grain

legumes grown in Germany (Pflughöft, 2008). Besides their ecological services provided in

crop rotations, peas and faba beans are important sources of proteins for humans and

animals. Despite their importance, production of peas is declining in Germany and many

other European countries (Sass, 2009; Urbatzka et al., 2011) and the same trend is present

in organic agriculture (Boehm, 2009). Low and instable yields together with low prices are

discouraging organic farmers from growing peas (Charles et al., 2007). The yield instability is

due to a range of abiotic and biotic factors (Fuchs et al., 2014). Abiotic factors limiting pea

productivity are unfavorable soil structure (Allmaras et al., 2003, 1988; Tu, 1994), lack of

nutrients (Fuchs et al., 2014), and toxic compounds in the soil (Narawal, 2000). Moreover,

the commonly used spring pea varieties are highly susceptible to soil-borne diseases

(Urbatzka et al., 2011a), compete poorly with weeds (Corre-Hellou et al., 2011), and they are

prone to lodging (Graß, 2003). Thus, legumes, peas in particular, are in principle able to

provide multiple services to the German agroecosystem, however, there are severe

constraints to pea production in Germany and in general. There is an urgent need to find

options to minimize the numerous factors limiting pea production and to optimize the

beneficial aspects of their inclusion into the crop rotation.

Growing peas in mixtures with cereals (Corre-Hellou et al., 2011) can be useful to a certain

extent, mainly for the purposes of better weed control and also improved N use efficiency

(Bedoussac and Justes, 2010; Saeed, 2013). Secondly, substitution of commonly grown

spring peas with winter pea varieties can be a good alternative as winter peas efficiently

compete with weeds and have a higher yield potential and stability (Stoddard et al., 2006;

Urbatzka et al., 2011a). As a catch crop they may prevent leaching of nitrogen during the


6

winter (Graß, 2003). Urbatzka et al., (2011b) showed that winter peas fix higher amounts of

N2 than spring varieties. However, little is known about their susceptibility to common soil

borne pathogens that currently present a major constraint to spring pea production

(Pflughöft, 2008).

1.4 Objectives and thesis outline

The main focus of the work presented in this thesis is soil health management with a specific

focus on soilborne diseases of peas. The central part of the research is an organic rotation

encompassing winter peas as green manure crop followed by maize and wheat. In the field

and in the laboratory the potential of improving system health with compost application was

explored. The results of the field experiments are presented in Chapter 2.

The key for the success of the field rotation described above is the ability of peas to fix

enough nitrogen. This depends crucially on pea health and until now little is known about the

importance and specificity of pathogens affecting winter peas in the German climate. Also,

there are open questions about the role of peas as alternative host for mycotoxin producing

Fusarium spp. pathogens of maize and wheat. Therefore, the most important pathogens of

the root and foot rot complex on pea identified in the field experiment were tested for their

aggressiveness on spring and winter peas under controlled conditions (Chapter 3). The

aggressiveness of Phoma medicaginis var pinodella, Didymella pinodes, Fusarium solani f.

sp. pisi and Fusarium avenaceum was assessed in sterile sand and in non-sterilized field

soil. Also, the pathogenicity of F. avenacuem on nine different plant species (including two

pea varieties) commonly grown in Germany was determined. P. medicaginis and F.

avenaceum were chosen because of their high virulence and wide host range for further

tests of temperature effects on the level of aggressiveness.

Chapter 4 deals with the potential of Yard Waste Compost (YWC) to suppress root and foot

rot of peas caused by P. medicaginis var pinodella, D. pinodes, F. solani f. sp. pisi and F.

avenaceum. In addition, effects of compost amounts and temperature were studied.

In the final Chapter 5, the results of Chapters 2 to 4 are summarized and discussed.

The research presented in this dissertation was conducted as the part of the “Climate change and production of healthy crops - processes and adaptation strategies by the

year 2030” in the frame of the research project “KLIFF - Climate Impact Research in

Lower Saxony".


7

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Allmaras, R.R., Fritz, V.A., Pfleger, F.L., Copeland, S.M., 2003. Impaired internal drainage and Aphanomyces euteiches root rot of pea caused by soil compaction in a fine-textured soil. Soil Tillage Res. 70, 41–52.

Allmaras, R.R., Kraft, J.M., Miller, D.E., 1988. Effects of soil compaction and incorporated crop residue on root health. Annu. Rev. Phytopathol. 26, 219–243.

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Hadar, Y., 2011. Suppressive compost: when plant pathology met microbial ecology. Phytoparasitica 39, 311–314.

Herrick, J.E., Wander, M.M., 1997. Relationships between soil organic carbon and soil quality in cropped and rangeland soils: the importance of distribution, composition, and soil biological activity. Soil Process. Carbon Cycle 405–425.

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Hoitink, H.A.J., Stone, A.G., Han, D.Y., 1997. Suppression of plant disease by composts. HortScience 32, 184–189.

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Jarvis, A., Ramirez, J., Anderson, B., Leibing, C., Aggarwal, P., 2010. Scenarios of climate change within the context of agriculture, in: Reynolds, M.P. (Ed.), Climate Change and Crop Production. CABI.

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Karpenstein-Machan, M., Stuelpnagel, R., 2000. Biomass yield and nitrogen fixation of legumes monocropped and intercropped with rye and rotation effects on a subsequent maize crop. Plant Soil 218, 215–232.

Katan, J., 1997. Soil disinfestation: environmental problems and solutions, in: Rosen, D., Tel-Or, E., Hadar, Y., Chen, Y. (Eds.), Modern Agriculture and the Environment, Developments in Plant and Soil Sciences. Springer Netherlands, pp. 41–45.

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Thomas, A.W., Tyler, D.D., Williams, J.R., 1991. Cover crop effects on soil erosion by wind and water, in: Cover Crops for Clean Water. Jackson, Tennessee, pp. 15–40.

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Meisinger, J.J., Hargrove, W.L., Mikkelsen, R.L., Williams, J.R., Benson, V.W., 1991. Effects of cover crops on groundwater quality. Cover Crops Clean Water Soil Water Conserv. Soc. Ankeny Iowa 266, 793–799.

Mueller, T., Thorup-Kristensen, K., 2001. N-Fixation of Selected Green Manure Plants in an Organic Crop Rotation. Biol. Agric. Hortic. 18, 345–363.

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Pritchard, S.G., 2011. Soil organisms and global climate change. Plant Pathol. 60, 82–99. Recycled Organics Unit, 2006. Compost use for pest and disease suppression in NSW. The

University of New South Wales, Australia. Roldán, A., Caravaca, F., Hernández, M.T., Garcia, C., Sánchez-Brito, C., Velásquez, M.,

Tiscareño, M., 2003. No-tillage, crop residue additions, and legume cover cropping effects on soil quality characteristics under maize in Patzcuaro watershed (Mexico). Soil Tillage Res. 72, 65–73.

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Saeed, M.F., 2013. Seed health in organic peas and faba beans and management approaches to improve pea production in organic rotations. University of Kassel, Witzenhausen.

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FUSARIUM IN THE CROP ROTATION AND YWC DISEASE SUPPRESSION IN THE FIELD

10

2. INCIDENCE OF FUSARIUM SPP. IN THE CROP ROTATION WINTER PEA – MAIZE – WINTER WHEAT AND THE POTENTIAL OF YARD WASTE COMPOST TO SUPPRESS FOOT AND ROOT ROT OF PEAS UNDER FIELD CONDITIONS

“If it is green, there is some Fusarium that can grow on it, in it, or with it”

Leslie and Summerel, 2006

Abstract

Field experiments were carried out from 2009 until 2013 to assess crop performance and pathogen occurrence in the rotation winter pea-maize-winter wheat and if the application of composts can improve system performance. The winter peas were left untreated or inoculated with Phoma medicaginis, in the presence or absence of yard waste compost at rate of 5 t dry matter ha-1. A second application of compost was made to the winter wheat. Fusarium ssp. were isolated and identified from the roots of all three crops. In addition, the Ascochyta complex pathogens on peas were identified. Overall crop performance of peas, maize and wheat and N dynamics were strongly affected by seasonal climatic conditions but not by experimental treatments. Dry summers impeded N-uptake by maize resulting in relatively high soil N contents in fall and consequent risk of leaching. Application of composts overall stabilized crop performance but it did not lead to yield increases nor did it affect pathogen composition and occurrence. Pathogen occurrence was highly variable across the experimental field and among years. Foot rot of peas was moderate in 2010 and 2011 and severe in 2012 when the crop failed. Five different pathogens were isolated and identified from symptomatic plants with Phoma medicaginis dominating. It appears that higher winter temperatures combined with lower rainfall favored P. medicaginis on peas over other pathogens in 2011 and 2012 in comparison to 2010. In 2012, surviving pea plants were severely affected by Fusarium avenaceum. More than 15 different Fusarium species were isolated from maize and wheat. Overall frequencies of Fusarium spp. on maize and winter wheat were highest in 2011. Dominating species on maize in all three years were F.

graminearum and F. culmorum. In addition, F. proliferatum, Microdochium nivale, F.

crookwellense, F. sambucinum, F. equiseti were frequently isolated species. The most frequently isolated species from winter wheat were F. oxysporum, F. culmorum, F.

avenaceum and M. nivale. The Fusarium species dominating on pea plants were F.

oxysporum, F. avenaceum and F. solani, and they were isolated most frequent in 2012. The Fusarium species occurring in the three subsequent crops did not correlate well. Suggesting factors other than the crops themselves influencing species dominance in a given season and crop.

Keywords: Fusarium spp., Ascochyta, Phoma medicaginis, winter peas, maize, wheat, yard waste compost


11

2.1 Introduction

As a result of the Renewable Energy Sources Act (EEG) of the German Government in

2009, biogas production gained high economic competitiveness. By 2012, this led to an

increase in silage maize production of nearly 60% compared with the year 2000 (Meyer and

Priefer, 2012). Maize production in organic agriculture has also increased in the last decade

although to a lesser extent. Organically produced maize is partly used for biogas production

but mainly as a protein and energy rich animal feed. High frequency of maize in the crop

rotation can lead to various environmental problems such as nitrogen leaching, soil erosion

and need for intensive and expensive weed control (Graß, 2003) and to an increase of

Fusarium head blight in wheat (West et al., 2012).

The “Double cropping” system (Zweikulturnutzungssystem) is a production system consisting

of a winter pea catch and cover crop before maize that was developed in order to optimize

benefits of growing a winter legume in the crop rotation and at the same time minimize the

potential negative aspects of growing maize (Graß, 2003). Winter peas are sown in

September or October, grown overwinter until the end of May and then either mulched or

incorporated into the soil as green manure or harvested for use as animal feed or biogas.

Winter soil cover provided by peas prevents soil erosion, reduces early weed infestation in

the spring and preserves winter moisture. After pea harvest, maize can be sown directly or

after shallow tillage. Maize utilizes the available N left after peas preventing it from leaching.

Due to the reduced tillage intensity production costs are also reduced. Maize is harvested at

the end of September or beginning of October and used either for silage or in biogas

production. Under temperate conditions, winter wheat is often the only crop that can be sown

after maize. With respect to soil and plant health the pea-maize-wheat rotation is a system

that might increase the risk of infection with Fusarium fungi and thus mycotoxin

contamination (Dill-Macky and Jones, 2000).

Fusarium head blight of wheat (FHB) and Fusarium ear rot of maize are economically

important diseases causing yield losses and losses in quality due to contamination of the

grains with mycotoxins (Vigier et al., 1997). Stalk and crown rot of maize and wheat are also

caused by Fusarium spp. and although of less economic importance, they are wide spread

wherever these crops are grown (Ares et al., 2004; Gatch and Munkvold, 2002; Osunlaja,

1990). In the case of severe infection and symptoms, foot rot of wheat can lead to lodging or

emergence of sterile ears leading to yield reduction (T. Pettitt et al., 1996). Infection of roots

and stem bases with Fusarium spp., also enhances later survival of the fungi in the soil and

plant residues and increases soil borne inoculum (Cotten and Munkvold, 1998; Leplat et al.,

2013; Suárez-Estrella et al., 2004).


12

As well ear as foot diseases are caused by a complex of different Fusarium spp. Fusarium

graminearum, F. culmorum, F. verticillioides, F.crookwellense, F. subglutinans, F.

avenaceum and F. equiseti are the most commonly isolated species from stalk rot of maize

in Europe (Scauflaire et al., 2011). Fusarium crown rot of wheat is commonly caused by F.

pseudograminearum, F. graminearum, F. culmorum, F. avenaceum and Microdochium nivale

(Backhouse, 2014; Khudhair et al., 2014; Liu et al., 2010; Melloy et al., 2014; Moya-Elizondo

et al., 2011; Poole et al., 2013). F. avenaceum and F. culmorum together with F. solani f. sp

pisi, F. oxysporum f.sp pisi, are part of the pathogen complex causing foot and root rot of

peas, limiting pea production worldwide (Ali et al., 1993; Bretag and Ramsey, 2001; Kraft et

al., 1998; Oyarzun, 1993).

Prevalence and dominance of different species in the above mentioned pathogen complexes

are affected by environmental conditions, tillage and cultural practices (Backhouse, 2014;

Gaurilckiene and Cesnuleviciene, 2013; Khudhair et al., 2014; Munkvold, 2003; Munkvold et

al., 1997; Persson et al., 1997). Most Fusarium spp. are soil and seed borne fungi, with little

host specification, usually with saprophytic and parasitic phases in their life cycle. Many are

able to produce survival structures like chlamydospores that enable long survival in the soil,

making control and management of root and stem rot diseases of pea, maize and wheat

difficult. In addition, lack of an effective management option for the control of the numerous

wide host range Fusarium spp. affecting pea, maize and wheat undermine the effectiveness

of the rotation. As the diseases are caused by a dynamic complex rather than one pathogen,

finding resistant cultivars is a challenge. Removal or incorporation of maize residues may

decrease the risk of Fusarium ear infection on subsequent wheat (Kong, 2014). However, the

retention of crop residues on or near the soil surface positively affects soil quality by

increasing the soil microbial biomass, increasing soil organic matter (SOM) content,

improving nutrient cycling and soil structure (Kong, 2014; Liu et al., 2010). Moreover, residue

incorporation requires several tillage operations increasing production costs.

An alternative way to suppress soil borne diseases is the enhancement of microbial activity

e.g. through the use of organic amendments (OA) (Bailey and Lazarovits, 2003; Bonanomi et

al., 2007; Bulluck III and Ristaino, 2002; Osunlaja, 1990). In a review of 250 articles

published between 1940 and 2006, in 45% of the cases OA suppressed disease, in 35% of

the cases there were no significant effects, while in 20% disease incidence was enhanced

(Bonanomi et al., 2007). Among the OA tested, composts were the most effective and

increase in disease incidence and/or severity was rarely observed.

In pot trials successful disease suppression usually follows application of 30 to 50% v/v of

compost to the potting mixes (Noble and Coventry, 2005; Termorshuizen et al., 2006). In

contrast to greenhouse trials, the amounts of compost that can be applied in the field are


13

considerably lower, e.g. in organic farming in Germany field application of off-farm compost

is limited to 5 t DM ha-1 and year. (Litterick et al., 2004) reviewed the literature about the role

of different uncomposted and composted organic materials in the reduction of pests and

diseases of different horticultural and agricultural crops. They suggested that besides

compost composition, application rates in the field are usually inadequate to achieve the

desired level of suppression. However, even at 5 t DM ha-1 of high quality yard waste

compost in the field can successfully reduce severity of Rhizoctonia solani on potatoes

(Bohne et al., 2013; Schulte-Geldermann et al., 2009). Field experiments with row

applications of compost and the highly susceptible pea variety Santana were variable in

outcome though (Bruns, 2013).

The field experiments described above were all based on one time application of compost to

the fields. In organic farming systems OA are typically applied on a regular basis, changing

overall microbial dynamics. We therefore set up field experiments with the rotation winter

pea-maize-wheat including a yearly application of compost. The two-year rotation was run

three times in the period between 2009 and 2013, with the main focus on identifying

Fusarium species present on the root and/or stem base of all three crops. In addition, the

potential of the yard waste compost (YWC) application to suppress foot and root rot of peas

and improve the system health and performance was investigated. The following questions

were addressed:

1) What are the effects of compost applications on pea and wheat foot and root

diseases in the rotation?

2) How does the spectrum of Fusarium spp. on the crops change in the course of the

rotation and are there effects of compost applications?


14

2.2 Material and Methods

2.2.1 Experimental site and setup

The field experiments were conducted on the experimental farm of the University of Kassel in

Neu Eichenberg, at 51°22’ N.L. and 9°54’ E.L, at an elevation of 247 m asl. The soil type is a

deep Haplic Luvisol from loess with 78 soil points (Wildhagen, 1998). Average annual

temperature is 7.9°C and average annual precipitation is 619 mm. The experimental farm is

managed organically since 1984.

The experiments took place within a crop rotation of six years consisting of two years grass-

clover followed by winter wheat a cover crop of winter pea – maize – winter wheat followed

by a spring cereal. Since 2005, each year all crops of the rotation are grown in large plots of

17.5 x 50 m resulting in a total of 24 plots arranged in four blocks of six plots. The sequence

starting with a winter pea cover crop until the end of the second wheat crop was studied

three times starting in 2009, 2010 and 2011 (Fig. 2.1). The winter pea variety EFB33, the

maize hybrid Fabregas and wheat variety Achat were used in all three years. In the second

experimental year due to lack of seeds, Fabregas was sown in the three middle rows of the

plots and hybrids Ronaldino and Atletico were used for the rest of plots. All the sampling and

analysis were done only on Fabregas.

Figure 2.1. Section of the crop rotation that was studied in the field experiment.

Within the large plots four treatments were arranged as plots of 7.5 x 20 m (Fig. 2.2). A total

of 5 t of dry matter ha-1 yard waste compost (YWC) was applied by hand two times in each

rotation. The first application was on the day before sowing peas and the second right before

wheat was sown (Fig 2.1). Nutrient contents and other properties of the YWC are listed in

Table 2.1.

C+ YWC 1 C+ YWC 2

C+ YWC 2 C+ YWC 3

C+ YWC 3 C+ YWC 4


15

Figure 2.2. Main plot divided in four experimental plots presenting four different treatments O=Control, I=Inoculated with P. medicaginis, C=Compost and I+C= Inoculated + Compost.

In addition to the compost application, half of the plots were inoculated with laboratory

prepared inoculum of Phoma medicaginis (Fig. 2.3). Peas were cut in May before maize was

sown. In 2010, plant material was removed; in 2011 all of the material was incorporated into

the top 5 cm of the soil during seed bed preparation for maize. In 2012, the peas failed due

to the severe frost. Maize was harvested in the second week of October, and the same plots

that had already received compost before peas received compost before wheat was sown

(Tab. 2.2). All field operations are listed in the Table 2.3.

I

I+C O

C


16

Table 2.1. Chemical characteristic of composts used in the field from 2009-2013.

Compost

Dry matter

content

(%)

Bulk

density

(g/L)

pH EC

(µS/cm)

K

(mg/kg)

P

(mg/kg)

Total N

(%)

Total

C C/N ratio Nmin

YWC 1 67 408 6.9 533 3397 477 1.18 26.93 22.87 0

YWC 2 95 375 6.6 758 5347 541 1.09 12.51 11.43 0

YWC 3 63 339 6.6 617 4598 637 1.31 35.45 27.08 0

YWC 4 85 389 7.5 498 3104 541 1.81 29.01 16.04 0


17

Table 2.2. Treatments applied in the field experiment.

Compost

application Inoculation Treatment name

Pea

- Compost - P. medicaginis Control (O)

+ P. medicaginis Inoculated (I)

+ Compost - P. medicaginis Compost (C)

+ P. medicaginis Inoculated + Compost (I+C)

Maize - Compost -* Control (O)

+ Compost - Compost (C)

Wheat - Compost - Control (O)

+ Compost - Compost (C)

*Compost was not directly applied before maize, but indicates plots that have received compost in the pea crop (see Fig. 2.1)

Inoculum preparation and inoculation

Inoculum of P. medicaginis, isolate Pm1, (Tab. 3.1, Chapter 3.2.1) was prepared on broken

oat grains. Approximately 40 kg of grains were soaked in water for 24 hours, autoclaved at

121°C for 15 minutes, and after another 24 hours autoclaved a second time. Agar plugs, 1

cm in diameter, of fungal culture from two fully covered Petri dishes were added and

incubated for two weeks at room temperature. During this time the material was regularly

stirred by hand to ensure homogenous fungal growth and prevent anaerobic conditions (Fig.

2.3). Inoculation in the field was done together with compost application by hand

broadcasting approximately 4 kg of inoculum per plot.

Figure 2.3. Inoculum of Phoma medicaginis grown on oat seeds.


18

Table 2.3. Timing of field operations.

Crop Rotation I

(2009/11)

Rotation II

(2010/12)

Rotation III

(2011/13)

Pea

Plough (15 cm)

Rotary disc

26.09.2009

29.09.2009

01.10.2010

04.10.2010

05.10.2011

05.10.2011

Compost application/ inoculation 28.09.2009 08.10.2010 17.10.2011

Sowing 29.09.2009 10.10. 2010 18.10.2011

Seed/m2 80 120 120

Emergence assessment 18.11.2009 26.11.2010 28.11.2011

Overwintering assessment 03.03.2010 05.04.2011 30.04.2012

Sampling for disease assessment

and pathogen ID

29.03.2010

18.05.2011

04.03.2011

18.05.2011

- 1

05.05.2012

Soil sampling for Nmin 19.03.2010

26.05.2010

05.04.2011

16.05.2011

26.04.2012

- 2

Harvest date 25.05.2010 18.05. 2011 No harvest 1

Maize

Plough (15 cm)

05.06.2010

18.05., 20.05.,

25.05.2011 09.05.2012

Sowing date 06.06.2010 25.05.2011 10.05.2012.

Sowing density in seed number/m2 11 11 16

Fertilization (hair pellets 14%N)

06.06.2010

56 kg N ha-1

in seed row

25.05.2011

56 kg N ha-1

in seed row

30.05.2012

42 kg N ha-1

broadcast

Weed control (hoe) 10.07.2010 29.06.2011 25.06.2012

Harvest date 15.10.2010 14.10.2011 03.10.2012.

Soil samples for Nmin - 3 15.10.2011 04.10.2012.

Wheat

Plough (15 cm)

Rotary disc

27.10.2010

28.10.2010

21.10.2011

23.10.2011

08.10.2012

11.10.2012

Compost application 28.10.2010 24.10.2011 11.10.2012

Sowing date 29.10.2010 25.10.2011 12.10.2012

Seed /m2 350 350 350

Sampling for pathogen ID 23.03.2011 26.03.2012 11.03.2013

Assessment foot disease 07.07.2011 16.07.2012 18.07.2013

Harvest date 29.07.2011 02.08.2012 15.08.2013

Soil samples for Nmin 02.08.2011 17.08.2012 22.08.2013

1 peas failed in 2011/12 due to severe frost

2 no early spring Nmin sampling was possible due to frozen soil

3 not measured


19

2.2.2 Assessments

Soil N-dynamics

Mineral nitrogen contents (Nmin) in soil depths of 0-30 cm and 30-60 cm were usually

measured in March in the peas, before and after maize and after wheat harvest (Tab. 2.3).

Ten soil cores were taken throughout each plot following a “W” sampling shape; bulk

samples were made and directly cooled down in the field. In the laboratory samples were

frozen at -18°C until analysis. Extraction and determination of Nmin was carried out according

to ÖNORM L-1091 (Österreichisches Normungsinstitut, 1999) and VDLUFA A6.1.4.1

(VDLUFA Methodenbuch, 1991). Extraction was done with 0.0125 M CaCl2 solution, and

measurement of NOx-N and NH4-N was done photometrically by absorption at 540 nm and

660 nm, respectively.

Plant development and yields

The number of pea and wheat plants was counted 2 to 4 weeks after sowing, depending on

the year. In addition, pea overwintering rate was recorded in early spring. The number of pea

plants per 0.1 m2 was counted in five randomly selected subplots per plot and average plant

density per m2 was calculated. For wheat, before harvest the number of head bearing tillers

was counted in five randomly chosen 1.1 m long rows and the mean density calculated.

Fresh mass of peas and weeds was measured right before mulching from three times 1 m2

per plot. After separation of weeds and peas they were dried at 105°C for 48 to 72 h for

determination of dry weights. Wheat was combine harvested and grain yield calculated

based on 14% moisture content and thousand grains weight determined. Maize was

harvested at BBCH 73-75 (Meier, 1997). Three 1 m row sections were cut by hand per plot,

number of plants counted and weighted. All harvested plants from one plot were afterwards

chopped into pieces of approximately 1-3 cm length and mixed well. Dry weights were

determined from subsamples of 300-500 g.

Disease assessments, pathogen isolation and identification

Pea plants were sampled two times for the assessment of root and foot rot severity and

pathogen identification. The first sampling was at BBCH stage 15/17, i.e. with five to seven

fully developed leaves (middle to end March) and the second at the beginning of flowering,

BBCH 59-61 (middle to end of May) (Tab. 2.3). Twenty plants together with roots were

selected following a “W” sampling route throughout each plot and taken to the laboratory for

the assessment and isolation of pathogens. Root and foot rot on peas was assessed using

the key of (Pflughöft, 2008) (Tab. 2.4). For pathogen isolations plants were surface

disinfected by placing them in 3% NaClO for 10 s. Afterwards they were thoroughly rinsed

under running distilled water and placed on filter paper under a laminar flow hood for 1 h to

dry. From the stem base and root of each plant three 1 cm pieces were cut, placed on

Coon’s agar media (Maltose 4 g, KNO3 2 g, MgSO4 1.20 g, KH2PO4 2.68 g, Agar 20 g , H2O


20

1L) and incubated for a week under UV light at 23 ± 3°C. After isolation, pathogenic fungi

belonging to Fusarium spp. and the Ascochyta complex, i.e. Ascochyta pisi, Phoma

medicaginis, Didymella pinodes, were identified to the species level. For that purpose the

one-week old mycelium from the original Coon’s plate was subcultured and incubated again

under the same conditions. Subculturing was done on half strength Potato Dextrose Agar

(PDA), for colony formation, and on Synthetic Nutrient Agar (SNA) medium (KH2PO4 1 g,

KNO3 1 g, MgSO4•7H2O 0.5 g, KCl 0.5 g, Glucose 0.2 g, Sucrose 0.2 g, Agar 20 g, H2O 1L)

for the sporulation of Fusarium spp., whereas for Ascochyta complex pathogens Coon’s agar

was used again. After 10 to 14 days pathogens were identified based on the colony

characteristic and microscopically based on the characteristics of spores (Leslie and

Summerell, 2006; Nasir et al., 1992).

At maize harvest, five to ten plants per plot were randomly selected and the lowest 20 cm of

the stem was cut, placed in paper bags and left to dry at room temperature. The sampling for

isolation and identification of Fusarium spp. on the stem basis and roots of wheat was done

in early spring in the seedling stage, BBCH 19-21. Ten to 15 plants per plot were taken to the

laboratory. At the late milk (BBCH 77) to early dough stage (BBCH 83) foot diseases on the

stem basis were assessed visually after removing all leaf materials. Symptoms caused by

the foot rot pathogens Fusarium spp., Pseudocercosporella herpotrichoides and Rhizoctonia

cerealis were assessed separately on the stem bases and severity scores from 0 to 3 were

given where 0 = no visible symptoms; 1 = less than 50% of the stem circumference affected;

2 = 50-100 % of stem circumference covered by a lesion; and 3 = stem is rotten or broken.

For Fusarium spp. isolation from wheat and maize, plants were washed with running tap

water for 1 h and placed under the laminar flow hood for 1 h to dry. Fusarium spp. selective

Pentachloronitrobenzene Peptone Agar (PPA) (Peptone 15 g, KH2PO4 1 g, MgSO4•7H2O 0.5

g, Pentachloronitrobenzene 750 mg, Agar 20 g, H2O 1 L) (Leslie and Summerell, 2006) was

used for the isolation. About 1 cm long plant pieces (three per plant) were placed on agar

media and incubated for 3 to 5 days under UV light at 23 ± 3°C, then subcultured on half

strength PDA and on SNA and again incubated for 10 to 14 days before identification as

described above.


21

Table 2.4. Scoring scheme for assessment of root and foot rot of peas (Pflughöft, 2008).

Score State of plant external tissues at root and stem

State of plant internal tissues at root and stem by cutting through the lesion

1 No visible symptoms No visible symptoms

2 Streaks at the transition zone or at epicotyl

or hypocotyl (not the discoloration of the seed and seed remaining attached)

Epidermis/rhizodermis is brownish to black

3 Brownish lesion covers <50% perimeter Brownish discoloration on cortical tissues

4 Brownish-black lesion covers >50%

perimeter Cortical tissues partially black, but center and

endodermis are still brownish or healthy 5 Black lesion covers 100% of stem Cortex tissue is completely black

6 Intensive color and black spread of lesion Cortex tissue begins to rot (bursting of

epicotyl or rhizodermis on the root)

7 Lesions spread up to the first lower leaf or

<3 cm on the tap root Cortex tissue is completely rotten

8 Lesions spread above the first lower leaf

and/or >3 cm on the tap root Shedding of the cortex tissue of endodermis

9 Dead plant Dead plant


22

2.3 Data processing and analysis As each pea plant was assessed with an external and internal score, a disease index was

calculated as the mean of external and internal lesion score and further analyses were done

on these values.

For foot rot of wheat a disease index (DI) based on a weighted mean was calculated as

follows:

�� = # �� + # �� ∗ + # �� ℎ� �� ∗n∗

where n= total number of assessed plants per sample

This results in DI values ranging between 0 and 100.

Data were analyzed with the statistical program R version 2.15.2. Analysis of Variance

(ANOVA) was conducted to analyze the effect of experimental factors on the plant growth

parameters, severity of disease and pathogen frequencies. Prior to ANOVA, data were

checked for normality with the Shapiro-Wilks-W-Test. Data for the pathogen incidence (%)

and DI were arcsine square root transformed to fulfil ANOVA requirements. Mean

separations were made by Tukey honest significant differences (HSD) test at P < 0.05.


23

2.4 Results

2.4.1 Weather data

Overall, temperatures measured from October 2009 until September 2013 followed the thirty

years trend (1970-2000) with a few extremes mainly in the winter months (Fig. 2.4). The

distribution of the precipitation differed strongly from the long-term average, with pronounced

dry periods at the end of winter and the beginning of spring in 2011-13. In contrast, summer

precipitation was generally higher with one or two extremely wet months. An exception was

2013, when the wet period occurred in May and was followed by a dry summer (Fig. 2.4).

A sudden drop of the temperature in the first two weeks of February 2012 after a relatively

warm December and January followed by a warm and dry March resulted in an almost

complete failure of the pea crop in the season 2011/12. The number of frost days (daily

minimum temperature below 0°C) in February 2012 was similar to 2010 and 2011; however,

the daily minimum temperatures were considerably lower on average. Also, the number of

icing days (daily maximum temperature below 0°C) was highest in 2012, and in this period

average maximal temperature was only -5.7°C (Tab. 2.5). The temperatures increased

sharply starting from 15th of February and this continued in March. The mean monthly

temperature of 7.5°C in March was 3°C higher than the long term average and at the same

time precipitation was 14.7 mm, only 26% of the thirty year average.

Table 2.5. Number and average temperatures of the frost and icing days in February.

Year Number of frost

days

Average

minimum

temperature (°C)

Number of icing

days

Average

maximum

temperature (°C)

2010 18 - 5.7 8 -3.4

2011 20 -3.8 7 -2.1

2012 19 -9.9 13 -5.7

2013 16 -2.3 9 -1.0


24

Figure 2.4. Monthly mean temperatures (°C) and precipitation (mm) in the period between 2009 and 2013, compared with thirty year average (1970-

2000).


25

2.4.2 Crop performance

There were no statistically significant differences in pea emergence as a result of treatments

or year. Overall plant emergence was in the range of 80 to 110% of sown seeds (see

Appendix Fig. A. 2.1.). The combination of extreme weather conditions in the winter

2011/2012 (Fig. 2.4) was devastating for pea. In contrast to 2010 and 2011, only around 30%

to 40% of the plants that emerged in autumn survived the winter (Fig. 2.5). It was not

possible to measure any yield parameter in this crop and in early May plots were ploughed

and prepared for sowing of maize. In contrast to peas, winter wheat was not strongly affected

by this cold period. In 2012, the mean number of heads per m2 one month before harvesting

was 234 compared to 275 and 265 in 2011 and 2013, respectively (see Appendix Fig. A.

2.4B).

Figure 2.5. Percentage of the emerged pea plants that survived the winters 2010, 2011 and 2012. Treatments O = Control; I = Inoculated with P. medicaginis; C= Compost; I+C= Inoculated with P. medicaginis + Compost. The error bars represent the ± 1 SD.

Fresh and dry matter yields followed the same pattern; therefore only dry matter data are

shown. The mean dry matter yield of peas was higher in 2010 compared to 2011, although

the sowing density was 80 seeds m-2 in 2009/10, and 120 seeds m-2 in 2010/11 (Fig. 2.6A).

The share of weeds in total biomass produced in 2010 was about 13% and 16% in 2011 (see

Appendix Tab. A. 2.2). In contrast, increasing the sowing densities of maize from 11 to 16

plants per m2 in 2012 resulted in increased dry matter yields (Fig. 2.6B) of 18.2 t ha-1 in 2012,

compared to 15.2 t ha-1 and 11.3 t ha-1 in 2010 and 2011, respectively.


26

Figure 2.6. Dry matter yield of peas (A) and maize (B), and grain yield of wheat (14% moisture) (C). For peas O= control, I= inoculated with P. medicaginis, C= compost, I+C= inoculated with P.

medicaginis + compost. For maize and wheat C= with compost, O= without compost. Different small letters within one crop indicate significant differences in yield among years at P < 0.05 (Tukey’s HSD test). The error bars represent the ± 1 SD.

a b

A)

B)

C)

b c a

b b a


27

The weight per plant in 2012 was significantly lower than in 2010 when plant density had

been lower. In contrast, during the dry season 2011 plant weights were also lower than in

2010 despite the lower density (see Appendix Fig. A. 2.3). The years had statistically

significant effects on the grain yield of wheat (Fig. 6C) and thousand grain weight (TGW)

(see Appendix Fig. A. 2.4A). The highest yield of wheat was achieved in 2013 with 5.8 t ha-1

(TGW = 48 g) followed by 4.6 t ha-1 (TGW = 54 g) in 2011 and 4.3 t ha-1 (TGW = 50 g) in

2012.

Compost application did not have any statistically significant effect on yields of any of the

three crops in the rotation. Also, neither inoculation nor compost did have any measurable

effects on disease on peas. However, in 2009/10 performance of the peas varied among

main plots from 750 g m-2 fresh matter to 1800 g m-2. In the main plots with the lowest mean

yield the control yielded 930 g m-2 while the inoculated peas yielded only 212 g m-2. Plots that

had received compost did markedly better (Fig 2.7.) The differences were less marked for

the high yielding main plots. In 2011 overall yields were lower and less variable among plots.

Figure 2.7. Effect of compost application on pea fresh matter yield in May 2010 and 2011. Treatments: Control ; Inoculated ; Compost ; Inoculated + Compost .

Overall, the lowest contents of soil mineral N were measured in the first rotation sequence at

all sampling times (Fig. 2.8). In 2011 and 2012, relatively high amounts of mineral N were left

in the soil after maize was harvested, average 55 and 40 kg N ha-1, respectively, and about

30 kg N ha-1 after wheat in both rotations.

2010

2011


28

Figure 2.8. Mineral nitrogen (Nmin) content in soil at 0-60 cm depth. The error bars represent the ± 1 SD. n.a. stand for not available. For exact dates of sampling see Table 2.3.

2.4.3 Foot and root rot of peas and wheat

In 2012, sampling for assessment of foot and root rot diseases of peas could be done only

once, due to the severe winter kill. The severity of the disease was highest in May 2012 and

the disease index (DI) was significantly higher than in 2010 and 2011 (Fig. 2.9). In the latter

two years DI were higher at the second sampling time in May than in March. Neither

inoculation nor compost applications affected the disease severity on peas (see Appendix

Tab. A.2.4). P. medicaginis was the most frequently isolated pathogen in all three years, as

well in March (see Appendix Fig. A. 2.5) followed by D. pinodes. Incidence of Fusarium spp.

was variable over years. Year had a statistically significant effect on the incidence of pea

pathogens in May, except for P. medicaginis (Fig. 2.10). Lowest incidence of D. pinodes, F.

oxysporum and F. solani was in 2011. F. avenaceum was not isolated in 2010 from peas. For

2010 and 2011, there was a significant interaction between year and sampling time for

incidence of P. medicaginis, D. pinodes, F. solani and F. oxysporum (see Appendix Fig. A.

2.7). In 2010, incidences of Ascochyta pathogens were higher in May compared to March,

whereas for Fusarium spp. it was opposite. In 2011, all pathogens were more frequently

isolated on the first sampling date. In 2011, incidence of F. avenaceum was 5% in May while

in 2012 in May it was 40%. Again, inoculation and compost application had no effect on the

spectrum and incidence of pathogens isolated from peas (data not shown).

n.a n.a

2011-2013

2009-2011

2010-2012


29

Figure 2.9. Foot Disease Index (DI) on peas in March and May 2010-2012. For the year 2012 disease was assessed only in May. Different small letters within one year indicate significant differences in DI at P < 0.05 (Tukey’s HSD test). Different capital letters indicate significant differences in DI in May among years at P < 0.05 (Tukey’s HSD test). The error bars represent the ± 1 SD.

Figure 2.10. Incidence of P. medicaginis (Pm), D. pinodes (Dp), F. solani (Fs), F. oxysporum (Fo) and F. avenaceum (Fa) isolated from pea in May of 2010, 2011, 2012. Different small letters within one pathogen indicate significant differences in incidence at P < 0.05 (Tukey’s HSD test).

The severity of foot diseases on wheat caused by Fusarium spp, Pseudocercosporella

herpotrichoides and Rhizoctonia cerealis was overall low (Fig. 2.11). The lowest disease

severity in all three years was caused by Rhizoctonia cerealis. In 2011, Fusarium spp.

dominated the complex, whereas in 2012 and 2013 they were on the same level as P.

ns

a b b

a a b c a b

a a b

b

a a

b

B B A


30

herpotrichoides. Compost application did not significantly affect the DI. Disease severity

caused by P. herpotrichoides was significantly lower in 2011 compared with 2012 and 2013.

There was no year effect on the two other diseases.

Figure 2.11. Foot rot disease of wheat caused by Fusarium spp., Pseudocercosporella

herpotrichoides and Rhizoctonia cerealis in treatments without (O) and with compost (C). The error bars represent the ± 1 SD.

2.4.4 Occurrence of Fusarium spp. in the tree crops in the rotation

More than 10,000 Fusarium isolates were obtained from peas, wheat and maize over the

years. A total of 20 different Fusarium spp. were isolated and identified from maize and

wheat, whereas only three species were commonly found on pea. The incidence of the

different species varied greatly from year to year (Fig. 2.12). Compost application did not

have any statistically significant effects on Fusarium spp. incidence.

In 2010, F. oxysporum was isolated from 38% of the analyzed pea plants, whereas F. solani

from 18%. Overall, the lowest incidence of Fusarium spp isolated from peas was in the

second rotation in 2011 with the incidence of F. oxysporum, F. solani and F. avenaceum

<10%. In the third rotation in 2012, F. avenaceum was isolated the most frequently (40% of

the sampled plants) compared with the other two species (Fig. 2.12).

In all three maize years, all maize samples were infected with at least one of the Fusarium

species causing stalk rot of maize. The predominant species isolated were F. graminearum


31

and F. culmorum (Fig. 2.12). Dominance among them varied over years. In the first rotation

in 2010, F. graminearum was isolated from nearly 50% of the samples and F. culmorum from

30%. In contrast, F. culmorum was isolated more frequently than F. graminearum, and in

2012 incidence of both pathogens was similar at about 40%. In 2012, with the exception of

those two species, overall incidence of Fusarium species isolated from maize was lowest

(Fig. 2.12). Microdochium nivale, F. proliferatum, F. oxysporum, F. sambucinum, F.

verticilioides and F. equiseti were commonly found on maize but at lower frequencies.

F. culmorum, F. oxysporum, F. avenaceum and F. equiseti were the most frequently isolated

species from wheat seedlings (Fig. 2.12). However, dominance and incidence of different

Fusarium spp. varied greatly among years. In the first rotation in 2011, incidence of all

isolated species was below 20% with dominance of F. proliferatum and F. oxysporum. In the

two subsequent years, incidence of F. aveanceum, F. culmorum, F. equiseti and M. nivale

increased reaching the maximum incidence in the last rotation sequence in 2013. There was

no obvious relationship between Fusarium communities found on maize and wheat neither

within year nor within rotation (Tab. 2.6) For example, incidence of F. graminearum on maize

was high and did not vary statistically significantly among years. On the following wheat it

was much lower and differences among years were statistically significant. F. crookwellense

occurred always in maize but only in 2013 on wheat. F. avenaceum was more prominent on

maize (15%) than on wheat (2.5%) in 2010/11, while in 2012/13 it was low on maize (5%)

and high on wheat (33%) (Tab. 2.6).


32

Figure 2.12. Incidence of Fusarium spp. isolated from three crops in the rotation. WP = winter peas, M = maize and WW = winter wheat; time of samplings, month/year, is given in the brackets.


33

Table 2.6. Incidence of different Fusarium spp. on maize and wheat in 2010 - 2013. Different letters within one species indicate significant differences in incidence among three experimental years at P < 0.05 (Tukey’s HSD test). Same colored cells show the frequencies in the crop sequence.

Fusarium spp. Crop 2010 2011 2012 2013

F. graminearum maize 49.2 50.0 33.8

wheat

5.4 ab 0.6 b 9.4 a

F. culmorum maize 40.5 ab 62.5 a 36.3 b

wheat

9.2 b 28.1 a 43.8 a

F. crookwellense maize 27.9 35.0 13.8

wheat

0 b 0 b 16.9 a

F. oxysporum maize 36.7 a 26.3 ab 13.8 b

wheat

18.4 b 39.4 a 16.9 b

F. avenaceum maize 14.6 a 1.3 b 5.0 b

wheat

2.5 b 20.6 a 32.5 a

F. verticilioides maize 18.3 a 5.0 b 0 b

wheat

1.7 ab 5.0 a 0 b

F. solani maize 15.8 a 8.8 a 1.3 b

wheat

5.4 b 5.6 b 11.3 a


34

2.5 Discussion Overall crop performance of peas, maize and wheat was strongly affected by seasonal

climatic conditions but not by experimental treatments. As a result of drought in the summer

2011 yield of the maize was relatively low. Plants were not able to utilize available nitrogen

and high amounts were left in the soil after maize harvest. In the rotation with wheat following

maize, high nitrogen contents in the soil in the autumn pose an increased risk of leaching

losses as the demands of wheat for N in the early growth stages are relatively low. Excessive

N in late fall in wheat production may lead to N leaching to ground water, as wheat does not

take up much N before winter (Brown and Petrie, 2006).

Application of yard waste compost overall did not reduce the severity of foot rot of peas or

wheat in our study. Bruns et al. (2013) showed that concentrated application of compost in

the seed row together with pea seeds can increase efficiency of compost in suppressing root

rot on the susceptible spring variety Santana. Application of compost had a positive effect on

the pea biomass especially in the low performing plots in the trial in 2010. The winter and

early spring 2009/10 were unusually wet compared to the long-term average and also

considerably wetter than in the following years. The low productivity of peas in one of main

plots (Fig 2.7.) was most likely due to water logging that was apparent in this part of the field

but not in the others. It seems that under these conditions the inoculated peas did especially

poorly and that compost application helped to improve the situation.

Foot rot of peas was moderate in 2010 and 2011 and severe in 2012 when the crop failed.

Five different pathogens were isolated and identified from symptomatic plants with Phoma

medicaginis dominating. In most of the cases foot rot of peas is caused by mixed infections

with Ascochyta complex pathogens and several Fusarium spp. (Ali et al., 1993; Gaurilckiene

and Cesnuleviciene, 2013; Kraft et al., 1998; Oyarzun, 1993; Persson et al., 1997) and

dominance and importance of single pathogens in the complex vary greatly depending on

location, climate, and agricultural practice (Gaurilckiene and Cesnuleviciene, 2013; Jensen et

al., 2004; Persson et al., 1997). Severe foot rot in 2012 occurred on the few remaining plants

after the frost in early February. Especially the incidence of F. avenacuem increased

significantly from <10% in the previous years to 40% (Fig. 2.12). (Bateman et al., 1997)

found that poor survival of lupines in the UK was due to inadequate cold tolerance followed

by fungal infestation. F. avenaceum is a wide host range pathogen that can survive

saprotrophically in the soil. In a healthy and active soil, it is usually not posing a problem, as

it is suppressed by fast growing soil saprophytes (Fletcher et al., 1991). However, plants

under environmental stress are more susceptible to this opportunistic pathogen (Leslie et al.,

1990). F. avenaceum is together with F. tricinctum, F. poae, F. culmorum and F.

graminearum part of the complex causing Fusarium Head Blight (FHB) of small grain cereals


35

in Europe. Moreover, it has been shown that in recent years in Northern Europe F.

avenaceum is the dominating species of the complex (Uhlig et al., 2007). In our study, F.

avenaceum was isolated from all three crops in the crop rotation. Beside causing wilting and

rot symptoms in infected plants, F. avenaceum produces mycotoxins moniliformin, enniatin,

beauvericin and zearalenone (Bottalico, 1998; Langseth, 1998; Logrieco et al., 2002) that are

harmful for animals and humans. Mycotoxin production of fungi that cause diseases on roots

and stem bases of plants is usually not of a great concern, because they stay in the field as

plant residues after harvest. However, in the case of F. avenaceum the lack of host

specialization and saprophytic survival on plant residues increases the risk of building up

inoculum that can lead to subsequent infection and mycotoxin contamination of cereals.

More than fifteen Fusarium species were isolated from maize and wheat in our study and

most of them are mycotoxin producers. The spectrum of species found did not differ among

years; however, the incidence of different species of the complex did. In all three seasons F.

graminearum and F. culmorum dominated the pathogen complex on maize. They were found

on 28 to 62% of the sampled plants, depending on the year. These results are in agreement

with findings of (Scauflaire et al., 2011) who reported these species dominating in the

pathogen complex causing stalk rot of maize in Northern Europe. F. graminearum and F.

culmorum were also found on wheat seedlings but at much lower frequencies and the

dominance was neither connected to the Fusarium spp. in the preceding maize crop nor to

the communities found on maize in the same year (Fig. 2.12, Tab. 2.6). F. culmorum was

always isolated at higher frequencies than F. graminearum from wheat seedlings. In all three

rotations, the frequency of F. graminearum strongly decreased from maize to wheat. This

species was more often associated with FHB in Australia and North America, whereas stem-

base disease of wheat is mainly related with F. culmorum. F. culmorum has been dominating

the Fusarium complex on wheat in the cooler and humid conditions of Northern Europe and

America (Backhouse et al., 2001; Poole et al., 2013). These results are confirmed by our

data.

Maize residues are considered to be an important source of inoculum for the infection of

wheat ears with FHB pathogens (Osborne and Stein, 2007). The low incidence (< 10%) of F.

graminearum on wheat seedlings in our study should not be interpreted as an indicator of low

risk for FHB later in the season or as an indicator of low inoculum potential of maize

residues. It is known that growth and sporulation of F. graminearum as well as its teleomorph

Gibberella zeae is favored by warm and humid conditions (Melloy et al., 2014; Pettitt et al.,

2003; T. R. Pettitt et al., 1996). The optimum temperature for mycelial growth and sporulation

is 25°C, and growth of the fungus stops below 5°C (Leplat et al., 2013). Therefore, the low

level of infestation of wheat seedlings with F. graminearum in early spring is likely the result


36

of reduced activity of the fungi due to low temperatures rather than low inoculum potential.

The role of maize residues and the level of risk it poses for later infection of wheat ears are

highly dependent on a combination of agronomic practices and climatic conditions at the time

of anthesis (Kong, 2014; Munkvold, 2003).

The other species found on maize, F. sambucinum, F. crookwellense, F. equisety, F.

proliferatum and F. verticilioides have been reported as part of the pathogen complex

causing maize stalk rot worldwide (Cotten and Munkvold, 1998; Leslie et al., 1990; Leslie

and Summerell, 2006; Osborne and Stein, 2007; Scauflaire et al., 2011). The spectrum of

Fusarium species on wheat seedlings was also diverse and variable over years. Besides the

above mentioned F. avenaceum and F. culmorum, F. oxysporum, F. equiseti and M. nivale

were part of the pathogen complex on wheat seedlings. In our study, F. equiseti was found at

relatively high frequencies in the second and third rotation sequence on both maize and

wheat. It is a cosmopolitan soil-borne species that was for a long time considered a

saprophyte or weak pathogen of different plants (Gerlach and Nirenberg, 1982). However, F.

equiseti has been confirmed as a pathogen causing stalk rot of maize (Leslie and Summerell,

2006), root rot and FHB of wheat (Bottalico, 1998; Logrieco et al., 2002), blights and pre

emergence damping-off of cucurbits (Palmero et al., 2011) and tomato (Palmero et al.,

2011). Although it is usually associated with warmer areas, in the last decade it has

commonly been part of the Fusarium complex in Northern Europe and America (Leslie and

Summerell, 2006).

It is common that many different Fusarium species occur simultaneously in plants. Overall

diversity as well as relative prevalence of species in specific regions of the world is affected

by cultural practices, plant cultivars and weather patterns (Gatch and Munkvold, 2002;

Munkvold, 2003; Osunlaja, 1990; Scauflaire et al., 2011). The changing climate with more

frequent extreme weather events brings new complexity into an already complicated system.

General increase or decrease in temperatures and precipitation will affect certain Fusarium

species adversely and favor others. However, this should not be observed outside of the

existing agricultural systems that will also have to change as a result of climate change.

Changes in tillage practices, sowing times, crop and cultivar choice among others, make the

prediction of disease risk and pathogens associated with climate change difficult. Current

expansion of maize production in Germany is likely to increase the risk of damage caused by

Fusarium spp. and their mycotoxins in crop rotation as many of the species have a wide host

range (Meissle et al., 2009). Our results emphasize the importance of further research on the

effect of environmental and cultural practices on the Fusarium complex. This may include the

optimization of compost applications or other organic amendments in order to maximize

suppressiveness to soil-borne diseases.


37

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FOOT AND ROOT ROT OF PEAS

40

3. AGGRESSIVENESS OF FOUR PATHOGENS CAUSING FOOT AND ROOT ROT OF PEA (PISUM SATIVUM L.) ON A SPRING AND A WINTER PEA CULTIVAR UNDER VARIABLE TEMPERATURE CONDITIONS

Abstract

A series of greenhouse trials was carried out to evaluate the susceptibility of the spring pea variety Santana and the winter variety EFB33 to Fusarium avenaceum, F. solani f. sp pisi, D.

pinodes and P. medicaginis under controlled conditions in sterile sand and field soil. Moreover, the effect of temperature on the aggressiveness of F. avenaceum and P.

medicaginis and pathogenicity of F. avenaceum on the agricultural crops that are commonly grown in rotation with peas in Germany was studied. Three weeks after sowing and inoculation disease symptoms were assessed and plant growth parameters measured. Regardless of pathogen and isolate used, as well as pea variety, plants grown in non-sterilized field soil were healthier than the plants grown in sterile sand. Santana was more susceptible than EFB33. All of the tested pathogens resulted in disease development. F.

avenaceum was the most aggressive pathogen causing severe wilting symptoms on Santana in sand (disease incidence (DI) 7.4). A strong negative correlation was observed between external root tissue damage and fresh weight for both varieties in both substrates. P.

medicaginis was the second most aggressive pathogen on Santana in sand (DI 5.7) and reduced fresh weight by 20% compared to control. Although the DI caused by D. pinodes

was somewhat lower than that of P. medicaginis, it caused significantly higher reductions of fresh weight in both substrates on Santana and in soil on EFB33. F. solani was the least aggressive of the four pathogens tested. Although there were no statistically significant effects of temperature on disease severity, there was a tendency that with decreasing temperature disease severity also decreased. In all treatments both varieties produced the highest biomass at the highest temperature. Out of eight plant species tested maize and wheat were least affected by inoculation with F. avenaceum. Stunted growth and reduction in biomass was observed for black oats, peas (Santana and EFB33), subterranean clover and Indian mustard, while white clover and white mustard exhibited severe wilting. This study implies that the tested pathogens differ in their aggressiveness and that the wide host range pathogen F. avenaceum can be highly aggressive causing severe disease and reduction of biomass on leguminous and no-leguminous hosts, especially Brassicaceae. Winter pea EFB33 is more tolerant to the pathogens tested and could be considered as alternative to susceptible summer varieties commonly grown in Germany and potentially contribute in overcoming recent decline in pea production in organic farming in Germany. Keywords: F. avenaceum, P. medicaginis, D. pinodes, F. solani, peas, temperature, foot rot


41

3.1 Introduction

Root and foot rot limits pea production worldwide (Ali et al., 1993; Bretag and Ramsey, 2001)

and is likely one of the reasons for the decline of pea production in Germany (Pflughöft et al.,

2012). It limits water and nutrient uptake, nitrogen fixation and causes stagnation of plant

growth (Oyarzun, 1993), preventing peas from reaching their full genetic potential (Kraft et

al., 1988). Yield losses of 10 - 30% are commonly reported for legume crops affected with

root rot, but yield loss potential under favorable conditions is even higher (Foroud et al.,

2014). A complex of more than 20 different species of soil-borne fungal pathogens can

cause foot and root rot of peas (Blume and Harman, 1979; Bodker et al., 1993; Kraft et al.,

1988; Olofsson, 1967; Persson et al., 1997). Many of the pathogens involved also attack a

range of legumes including chickpea, lentil, faba bean and lupine.

The most important species causing foot and root rot on peas are Didymella pinodes (syn

Mycospharella pinodes), Phoma medicaginis, Fusarium spp. (F. solani f. sp pisi, F.

oxysporum f.sp pisi, F. avenaceum, F. culmorum), Pythium spp., Thielaviopsis basicola,

Aphanomyces euteiches, and Rhizoctonia solani (Feng et al., 2010; Kraft et al., 1988; Nasir

et al., 1992; Oyarzun, 1993; Persson et al., 1997; Pflughöft et al., 2012).

Didymella pinodes (Berk. & A. Bloxam) Vestergr. (anamorph Ascochyta pinodes L. K. Jones)

together with Phoma medicaginis var. pinodela (Jones) and Ascochyta pisi Lib. are grouped

together in the “Ascochyta comlex” due to similar symptoms caused on pea (Wallen, 1974).

However, Ascochyta pisi rarely causes root rot. It is seed borne, does not produce

chlamydospores and causes mainly leaf and pod spot. The other two pathogens are seed

and soil borne and cause lesions on leaves, stems, and pods as well as foot rot (Ali et al.,

1993; Tivoli and Banniza, 2007).

Wallen (1974) stresses the importance of D. pinodes as a root rot pathogen on peas, as

infection of young seedlings can result in damping-off and increase lodging of older plants.

The main source of inoculum is infected stubble, in which the fungus can persist for several

cropping seasons (Gossen et al., 2010; Zhang et al., 2003). In seed, D. pinodes is able to

survive up to four years (Corbière et al., 1994) and in severe cases it may infect the embryo.

However, seed to seedling transmission is overall low (Bretag et al., 1995; Gossen et al.,

2010). Moussart et al. (1998) showed that low temperatures increase the frequency of this

type of transmission in peas. Even though seed infection is apparently of minor importance

for the epidemic development, it is often responsible for the introduction of the pathogen to

new areas (Gossen et al., 2010). In the case of blight, i.e. foliar and stem infection, air-borne

ascospores are considered to be the most important source of inoculum (Gossen et al.,

2010; Roger and Tivoli, 1996). Cool and wet conditions favor blight epidemics caused by D.

pinodes because they enhance ascospore dispersal and promote infection. Severe


42

epidemics can lead to yield losses of up to 50% (Bretag et al., 1995; Setti et al., 2009;

Wallen, 1974), reduce seed quality and limit the area in which field peas can be grown (Ali et

al., 1978; Le May et al., 2009; Roger et al., 1999).

Phoma medicaginis var. pinodella (teleomorph Didymella sp.) is also seedborne (Tivoli and

Banniza, 2007) but the main sources of inoculum in the field are pycnidiospores and

chlamydospores on crop debris in soil. There they can survive up to 3 years (Carr and Smith

1988). The main means of their distribution is rain splash. Ascospores produced by

Didymella sp. can be spread by wind over longer distances, and can lead to development of

severe epidemics (Bowen et al., 1997). P. medicagnis can cause severe foot rot symptoms

that can reduce plant growth and subsequent yield loss can reach 25% (Wallen, 1974). Ali et

al. (1994) report that P. medicaginis var pinodella isolates from soil are weakly virulent until

passage through the host plant and that is the reason why it is not usually epiphytotic in

nature. It has a wide host range within the Fabaceae family, causing the blights and root rot

on different Medicago, Vicia, Pisum, Melilotus, Glycine, Lupinus and Trifolium spp (Farr and

Rosmann, 2015).

F. solani f. sp pisi together with F. oxysporum f.sp pisi have been described as the most

frequent and most aggressive root rot causing fungi on peas worldwide in the last three

decades (Kraft et al., 1988; Oyarzun, 1993; Persson et al., 1997; Pflughöft et al., 2012). They

can reduce pea yields by 10% to 50% (Kraft et al., 1988; Rush and Kraft, 1986). Other

Fusarium spp. such as F. avenaceum and F. culmorum, are also frequently isolated from

roots (Persson et al., 1997).

F. oxysporum Schlecht. f. sp. pisi (Lindford) Snyder & Hansen occurs worldwide but the

severity of the damage depends on the race of the pathogen (Pflughöft, 2008). F. oxysporum

is a vascular wilt pathogen that usually infects plants through the side roots (Kraft et al.,

1988; Kraft and Pfleger, 2001). No sexual state has been found so far (Skovgaard et al.,

2002). This pathogen survives in the soil in the form of chlamydospores (Leslie and

Summerell, 2006).

F. solani (Mart.) Sacc. f. sp. pisi (Jones) Snyder & Hansen (teleomorph: Nectria

haematococca) is a very variable species that can be both saprophytic and parasitic (Ondrej

et al., 2008). It infects plants early with seed germination but symptoms usually appear later.

Biotic and abiotic stress conditions affect the incubation time of pathogen (Rush and Kraft,

1986). Initial symptoms on the roots consist of reddish-brown streaks starting from the

cotyledonary attachment area, which later spread and form black lesions on the roots and

stem base (Kraft et al., 1988; Kraft and Pfleger, 2001; Ondrej et al., 2008). Severely infected

plants are chlorotic and have stunted growth (Tu, 1987). In the plants infected with F. solani,


43

vascular tissue often develops intensive red color and this can be used as an indicator of its

presence. The pathogen survives in soil in the form of chlamydospores.

F. avenaceum (Fries) Sacc (sexual stage Gibberella avenacea) is predominantly a soilborne

fungus, that may be also seedborne on some legumes (Leslie and Summerell, 2006). It is

common in temperate regions throughout the world. F. avenaceum has a wide host range,

does not typically segregate into host-specific pathotypes (Schneider, 1958; Hwang et al.,

1994), and causes disease on a range of agricultural crops. F. avenaceum is morphologically

variable (Schneider, 1958) and genetically diverse (Miedaner et al., 2008). It has been

reported as a pathogen causing economically important diseases of canola (Berkenkamp

and Vaartnou, 1972; Hwang et al., 2000), lentil (Fletcher et al., 1991; Hwang et al., 2000),

subterranean clover (Greenhalgh and Siobhan,1984), red clover (Yli-Mattila et al. 2010), pea

(Feng et al., 2010; Fletcher et al., 1991b; Hwang et al., 2000; Persson et al., 1997), soybean

(Zhang et al., 2013), alfalfa (Cormack, 1951; Hwang et al., 2000), wheat, barley, oat (Cook,

1980; Hwang et al., 2000; Smiley and Patterson, 1996), etc.

F. avenaceum rarely produces chlamydospores and survives saprophyticaly in soils or on

plant residues (Forbes and Dickinson, 1977; Leslie and Summerell, 2006). It produces

secondary metabolites such as beauvericin, fusarin C, moniliformin, and enniatins A, B, and

B1 (Leslie and Summerell, 2006), which are toxic to animals and humans. Pre-emergence

damping off, post-emergence root rots, yellowing, stunting and wilting are symptoms of

infection with F. avenaceum (Bateman et al., 2007).

Usually, several pathogens are isolated from symptomatic pea roots. The role of single

pathogens in disease development and severity is not clear. Prevalence, dominance and

importance of the single pathogens vary greatly depending on location, climate, and

agricultural practice (Gaurilckiene and Cesnuleviciene, 2013; Jensen et al., 2004; Persson et

al., 1997) and some shifts in importance have occurred over time. Thus, overall disease

severity and yield loss due to foot and root rot are the combined effects of the pathogens and

it is practically impossible to relate symptoms on roots and reduction of yield under field

conditions to single pathogens (Tu, 1987). For that reason a series of greenhouse trials was

conducted in order to:

1) Examine the pathogenic potential of the four foot and root rot pathogens, F.

avenaceum, F. solani, P. medicaginis and D. pinodes on a spring and winter pea

variety in two substrates;

2) Examine the effect of temperature on disease severity caused by F. avenaceum and

P. medicaginis;


44

3) Investigate the pathogenicity of F. avenaceum on the agricultural crops that are

commonly grown in rotation with peas in Germany.


45

3.2 Materials and Methods All experiments were replicated five times in a completely randomized design.

3.2.1 Pathogenicity of Fusarium avenaceum, Fusarium solani, Phoma medicaginis and

Didymella pinodes, in sterile sand and in non-sterilized field soil

The winter pea variety EFB33 and spring variety pea Santana (KWS LOCHOW, Gmbh) were

used in the experiments. EFB33 is botanically Pisum sativum L. ssp. sativum convar.

speciosum (Dierb.) Alef. (formerly Pisum arvense L.). It is a regularly leafed, small seeded

variety with thousand seed weight of about 130 g and violet flowers. Santana is a spring pea

widely grown in Germany. Botanically it belongs to Pisum sativum L. ssp. sativum convar.

sativum. This cultivar is semi-leafless, white flowering with white seed coat and a thousand

seed weight of 250 to 300 g.

Prior to use pea seeds were surface disinfected with 70% ethanol for five minutes, rinsed

with running distilled water and air dried under a laminar flow cabinet. All defective and

damaged seeds were discarded. Plants were grown in 6 cm diameter and 8 cm deep round

plastic pots filled with 150 mL (~200 g) of sterile yellow sand or non-sterilized field soil as

growing media. Sand was autoclaved for 20 minutes at 121°C and stored in a closed

container until the beginning of the trial. It had a water content of 7%, water holding capacity

of 27.5 g per 100g dry sand and maximum water absorption of 17.5 mL per 100 g. The field

soil for the trial was collected from the experimental field of the University of Kassel at Neu-

Eichenberg. Peas were grown in this field a year before the soil was used for the greenhouse

experiments. The soil type is a deep Haplic Luvisol from loess with 78 soil points (Wildhagen,

1998). Four seeds were sown in each pot at 2 cm depth. Plants were grown for 21 days with

a day/night temperature regime of 19/16°C. Pots with sand as a substrate were fertilized with

complex N:P:K fertilizer Wuxal Super (8:8:6 + microelements). A total of 100 mg of N L-1

substrate was divided in two equal portions and added 10 and 15 days after sowing. No

fertilizer was used in the field soil.

Five different isolates of each pathogen were used in the experiment (Tab. 3.1). Isolates of F.

solani were collected from pea plants on different organic farms in Germany, in the period

between 2010 and 2012. Two isolates of D. pinodes and two of P. medicaginis were kindly

provided by Dr. B. Tivoli from the National Institute of Agronomic Research (INRA) France.

One isolate of D. pinodes and one of P. medicaginis were kindly provided by the Plant

Pathology Department of the Georg-August-University in Göttingen. F. avenaceum was

represented in the experiment by two isolates from winter, one isolate from subterranean

clover seeds and two from pea plants collected between 2011 and 2012. D. pinodes and P.

medicaginis isolates were multiplied in Petri dishes containing COON’s agar (Maltose 4 g,

KNO3 2 g, MgSO4 1.20 g, KH2PO4 2.68 g, Agar 20 g , H2O 1L) while F. solani and F.


46

avenaceum cultures were grown on half strength PDA. Petri dishes were incubated at 20˚C

and subjected to 12 hour cycles of UV light and dark for 15 to 20 days.

Inoculation was done with spore suspensions at the time of sowing. Spores were prepared

by flooding the surface of cultures with distilled water and gently scraping them. The spore

concentrations were determined with a Fuchs Rosenthal haemocytometer and adjusted to

105 spores g-1 of growing substrate. After inoculation, pots were watered and placed in the

greenhouse. Irrigation was done regularly according to need.

Table 3.1. Pathogens and isolates used in experiments.

Pathogen Isolate name Plant species Origin

Didymella pinodes

Dp4 Dp 91.31.12 Pea INRA, France

Dp5 Dp Gö Pea Göttingen

Dp6 Dp KLIFF Pea Neu Eichenberg

Dp3 Dp 96Ca2 Pea INRA, France

Dp7 Dp B2 Pea Neu Eichenberg

Fusarium avenaceum

Fa1 Fa W/VIII.11 Wheat Neu Eichenberg

Fa6 Fa W/III.13.2 Wheat Neu Eichenberg

Fa4 Fa NEB.12 Pea Neu Eichenberg

Fa7 Fa DFH Pea Domene Frankenhausen

Fa8 Fa SC Subclover seeds Italy

Phoma medicaginis

Pm1 Pm GÖ Pea Göttingen

Pm2 Pm KLIFF 12 Pea Neu Eichenberg

Pm3 Pm 94.56.3 Pea INRA, France

Pm4 B1.10 Pea Neu Eichenberg

Pm5 Pm 94.14.11 Pea INRA, France

Fusarium solani

Fs2 Fs GER 10III10 Pea East Germany

Fs3 Fs LIN V.12 Pea BOFRU

Fs4 Fs MÖL V.12 Pea BOFRU

Fs1 Fs KLIFF IV.12 Pea Neu Eichenberg

Fs5 Fs T19 3III.10 Pea North of Germany


47

3.2.2 Effect of temperature on the aggressiveness of F. avenaceum and P. medicaginis

Effects of reduced growing temperatures (Tab. 3.2) on the aggressiveness of P. medicaginis

and F. avenaceum were studied in the period between March and May 2012. P. medicaginis

isolate Pm1 and F. avenaceum isolate Fa1 (Tab. 3.1) were chosen for this test. The same

pea varieties and sterile sand were used as in the previous trial. Preparation of seeds,

sowing, inoculation, fertilization, watering and duration of experiment was as described

above (see 3.2.1.). Square pots of 12 x 12 x 11 cm were filled with 1 L (~1.3 kg) of yellow

sterile sand and eight pea seeds were sown per pot.

Table 3.2. Temperature regimes used in the experiment

Treatment Day temperature (°C) Night temperature (°C)

Low 13 10

Medium 16 12

High 19 16

3.2.3 Pathogenicity of F. avenaceum

The pathogenicity of two F. avenaceum isolates, Fa1 from wheat and Fa4 from pea (Tab.

3.1) was tested on eight plant species in addition to peas (Tab. 3.3). Plants were inoculated

at the time of sowing with 104 spores g-1 substrate and grown at a day/night temperature

regime of 19/16°C. Square pots of 12 x 12 x 11 cm with 1 L (~1.3 kg) of yellow sterile sand

were used. Preparation of seeds, sowing, inoculation, fertilization, watering and duration of

experiment was as described above (see 3.2.1).

Table 3.3. Plant species, varieties and sowing density used in the experiment.

Plant species Variety No. plants per pot Maize (Zea mays) Fabregas 5

Winter wheat (Triticum aestivum) Achat 5

Oat (Avena sativa) Dominik 5

Black oat (Avena strigosa) Pratex 5

Pea (Pisum sativum L. convar. sativum) Santana 8

Pea (Pisum sativum L. convar. speciosum) EFB33 8

Subterranean clover (Trifolium subterraneum) Dalkeith 12

White clover (Trifolium repens) Liflex 12

Indian mustard (Brassica juncea) Terrafit 12

White mustard (Sinapis alba) Samba 12


48

3.2.4 Measurement of plant biomass and assessment of disease

Plants were harvested 21 days after sowing in all experiments. They were carefully pulled

out of the substrate. Roots were cut off, washed with running tap water, and stored at 4°C

until use. Above ground plant parts of each pot were weighted and dried at 105°C until

constant weight. Root disease severity was assessed according to the key of Pflughöft

(2008) (Tab. 2.4, Chapter 2). Assessments were always performed within 48 hours following

harvest. Symptoms on the external plant tissue were evaluated followed by the assessment

of damage of the internal tissue. In the test for pathogenicity of F. avenaceum on nine plant

species, in addition to above ground biomass, roots were also weighted. As the symptoms

on roots were uniform within different plant species, no detailed assessment of the symptoms

was performed.

3.2.5 Data processing and analysis

A disease index (DI) for each plant was calculated as the average of the external and internal

lesion score. As all plants in sand controls emerged, missing plants in the treatments were

considered dead and score 9 was given for both external and internal tissue. The mean DI of

the eight plants in each pot was calculated to present one replication, and further analyses

were done on these values. Analyses of variance (ANOVA) were applied in order to analyze

the effect of the pathogens, substrate and temperature on the DI, plant weights and survival

rates. Prior to ANOVA, data were checked for normality with Shapiro-Wilks-W-Test. Mean

separations were made by Tukey honest significant differences (HSD) test at the P < 0.05.

All statistical analyses were done in the statistical program R version 2.15.2. Regressions of

fresh weights and DI were analyzed with Excel 2007.


49

3.3 Results

3.3.1 Comparison of aggressiveness of F. avenaceum, F. solani, P. medicaginis and D.

pinodes on two pea varieties in sand and field soil

3.3.1.1 Isolate comparison

Regardless of pathogen and isolate used, as well as pea variety, plants grown in non-

sterilized field soil were healthier than the plants grown in sterile sand. Santana was more

susceptible than EFB33. DI varied rarely among isolates within pathogens depending on the

test system (Fig. 3.1). Statistically significant isolate effects occurred only for F. avenaceum

isolates on Santana in sand (Fig. 3.1A) and for D. pinodes isolates on Santana grown in soil,

and on EFB33 in both substrates (Fig. 3.1C).

All of the tested F. avenaceum isolates significantly reduced the weight of Santana plants

grown in sand (Fig. 3.2). In contrast, EFB33 was not affected. There was even a tendency

that weights of inoculated treatments were higher than control. However, this difference was

not statistically significant (Fig. 3.2).

Three out of the five P. medicaginis caused significant reductions of fresh and dry weights

(data not shown) of Santana plants in sand (Fig. 3.3). There were no significant differences

in fresh weights among different D. pinodes or F. solani isolates neither on Santana nor on

EFB33 (see Appendix Fig. A. 3.3 and A. 3.4).


50

Figure 3.1. Disease index (DI) of Santana and EFB33 plants grown in sand and soil and inoculated with five isolates of F. avenaceum (A), F. solani (B), D. pinodes (C) and P. medicaginis (D). Different small letters within variety and substrate are indicating significant differences in DI at P < 0.05 (Tukey’s HSD test). The horizontal line in the boxplot shows the median, the bottom and tops of the box the 25th and 75th percentiles and the vertical lines the minimum and maximum values; outliers as single points. Mean values of DI are marked with triangles.

ab

a ab b b

ab b a b ab ab b ab a ab

b b ab a ab

A B

C D


51

Figure 3.2. Fresh weights of Santana and EFB33 plants inoculated with five isolates of F.

avenaceum and grown in sterile sand or non-sterilized field soil. Different letters within variety and substrate are indicating significant differences in DI at P < 0.05 (Tukey’s HSD test). The error bars represent the ± 1 SD.

Figure 3.3. Fresh weights of Santana and EFB33 plants inoculated with five isolates of P.

medicaginis and grown in sterile sand or non-sterilized field soil. Different letters within variety and substrate are indicating significant differences in DI at P < 0.05 (Tukey’s HSD test). The error bars represent the ± 1 SD.

Santana EFB33

Santana EFB33

a

bc c

ab bc abc

a

bc b

b

c

b


52

3.3.1.2 Pathogen comparison

Unlike the other pathogens that caused compact black lesions that spread completely around

the plant stem accompanied by rot of epidermis/rhizodermis, symptoms of F. solani infection

appeared as a mass of single black streaks very close to each other but it was still possible

to see green healthy tissue in between (Fig. 3.4). During the disease assessment red color of

vascular tissue was observed in plants inoculated with both Fusarium spp.

Figure 3.4. Typical symptoms of F. solani infection on the stem base of Santana (A) and EFB33 (B) plants.

Interactions between pathogen and variety, as well as pathogen and substrate were highly

significant, P < 0.001 (see Appendix Tab. A. 3.5). F. avenaceum caused severe disease

symptoms on Santana plants grown in sand (Fig. 3.5) with mean DI of 7.38 (Fig. 3.6) and an

average reduction of fresh weight of 65.3% compared to the control (Fig. 3.7). Besides the

intensive black lesions on the epicotyl that spread up to the soil level, plants exhibited strong

wilting symptoms. First wilting symptoms usually appeared after 14 days and soon after the

plants died. Intensive orange sporodochia were formed on the dead plant tissue (Fig. 3.5).

A B


53

Figure 3.5. Wilting symptoms of Santana caused by F. avenaceum with the formation of bright orange sporodochia (marked with the arrow).

The second most aggressive pathogen on Santana in sand was P. medicaginis with a mean

DI of 5.7 (Fig. 3.6) and reduction of plant fresh weight by 20%. Although D. pinodes caused

less severe symptoms with DI 4.8, reduction of fresh weight caused by infection with this

pathogen was on average 34% (Fig. 3.7). F. solani was least aggressive and caused

moderate disease symptoms with DI 4.0 and an average fresh weight reduction of 8% (Fig.

3.7). Dry weights followed the same pattern (data not shown).


54

Figure 3.6. Disease index (DI) of Santana and EFB33 plants grown in sand and soil and inoculated with F. avenaceum (Fa), F. solani (Fs), P. medicaginis (Pm) and D. pinodes (Dp). O is the non-inoculated control. The horizontal line in the boxplot shows the median, the bottom and tops of the box the 25th and 75th percentiles and the vertical lines the minimum and maximum values; outliers as single points. Mean values of DI are marked with triangles. Different letters within variety and substrate are indicating significant differences in DI at P < 0.05 (Tukey’s HSD test).

Figure 3.7. Fresh weights of Santana and EFB33 inoculated with F. avenaceum (Fa), F. solani (Fs), P. medicaginis (Pm) and D. pinodes (Dp). Different letters within variety and substrate are indicating significant differences in DI at P < 0.05 (Tukey’s HSD test). The error bars represent the ± 1 SD.

c

ab b

a

c

a

ab b b c

b ab ab ab a

e d a

Santana EFB33

a

d

ab

c b

a ab

bc

a

c

ab

a

b ab a a a

b


55

There was no significant difference in disease severity caused by tested pathogens when

Santana was grown in soil (Fig. 3.6). Only D. pinodes reduced biomass yield significantly

(Fig. 3.7).

On EFB33, all four pathogens caused only weak disease symptoms (Fig. 3.6). In sand, P.

medicaginis was the most aggressive (mean DI 3.3), followed by F. avenaceum (3.0), D.

pinodes (2.5) and F. solani (2.5), respectively. As for Santana, D. pinodes was the most

aggressive pathogen on EFB33 plants grown in soil, although disease severity was low (DI

2.2). However, fresh weight was significantly reduced by 26% compared to control plants.

Disease index for the three remaining pathogens was below 2 and differences in fresh weight

in comparison to the non-inoculated control were low (Fig. 3.7).

There was a strong negative correlation (P < 0.001) between disease severity and fresh

weight of pea plants inoculated with F. avenaceum, for both pea varieties in both substrates

(Tab. 3.4). Reduction of fresh weight per unit of DI caused by D. pinodes was significant for

both pea varieties in sand but in soil only for Santana (Tab. 3.4). There was no significant

correlation between disease severity and fresh biomass when pea was inoculated with P.

medicaginis and for F. solani it was only significant for Santana in soil.

Table 3.4. Disease Index (DI), coefficient of determination (R2) and significance for the correlation between Disease Index and fresh weights of EFB33 and Santana plants grown in sterile sand and not sterilized field soil.

Sand Soil

DI R2 slope DI R2 slope

Santana

F. avenaceum 7.4 0.73*** -0.31 3.9 0.70*** -0.38

F. solani 4.0 0.13 -0.21 3.7 0.54*** -0.50

D. pinodes 4.8 0.42*** -0.33 4.2 0.28** -0.20

P. medicaginis 5.6 0.15 -0.16 3.0 0.07 -0.28

EFB33

F. avenaceum 3.0 0.57 *** -0.19 2.0 0.32*** -0.20

F. solani 2.5 0.19 -0.13 1.9 0.01 0.05

D. pinodes 2.5 0.44*** -0.21 2.2 0.13 -0.09

P. medicaginis 3.3 0.02 0.06 1.8 0.05 0.19

Significance codes: p < 0.001 ‘***’; p < 0.01 ‘**’; p < 0.05 ‘*’.


56

3.3.2 Effect of temperature on disease severity caused by F. avenaceum and P.

medicaginis

Both pathogens caused moderate to severe disease with DI between 4.5 and 7.4 on Santana

(Fig. 3.8). DIs at 19°/16°C day/night regime were the same as in the first experiment (see

Fig. 3.6). They were generally higher than at the lower temperatures. Again, in contrast to

Santana, disease severity on EFB33 was overall low. Although there were no statistically

significant differences among different temperature regimes, there was a tendency that with

decresing temperature disease severity decreased. The highest DI on EFB33 was 3.0 in the

treatment inoculated with P. medicaginis and grown at the highest tested temperature (Fig.

3.8).

Figure 3.8. Disease index of Santana and EFB33 inoculated with F. avenaceum (Fa) and P.

medicaginis (Pm) and grown under different temperature regimes. O is the non-inoculated control. The horizontal line in the boxplot shows the median, the bottom and tops of the box the 25th and 75th percentiles and the vertical lines the minimum and maximum values; outliers as single points. Mean values of DI are marked with triangles. Different letters within variety and temperature are indicating significant differences in DI at P < 0.05 (Tukey’s HSD test).

b a a b a a c a b

b a a c b a b a a


57

In contrast to DI, plant growth was affected significantly by temperature (Fig. 3.9). In all

treatments both varieties produced the highest biomass at the highest temperature. Infection

caused no statistically significant differences in fresh weights of Santana plants grown at

13°/10°C but at medium and high temperatures. In contrast, fresh weights of EFB 33 were

not affected by infection with pathogens at any of the temperatures (Fig. 3.9).

Figure 3.9. Mean values of fresh weights of Santana and EFB33 plants grown at three temperature regimes and inoculated with F. avenaceum (Fa) and P. medicaginis (Pm). O is the non-inoculated control. Different capital letters within one variety indicate significant differences in fresh weights among temperature regimes, whereas small letters indicate significant differences among treatments within variety and temperature regime at P < 0.05 (Tukey’s HSD test). The error bars represent the ± 1 SD.

3.3.3 Pathogenicity of Fusarium avenaceum

Typical symptoms of F. avenaceum on the eight plant species and two pea varieties included

pre-emergence death, post-emergence damping-off, wilt and stunted growth. Maize and

wheat seedlings were least affected by inoculation with F. avenaceum. Emergence was at

the level of control plants and reduction of fresh weight for both crops and both isolates was

less than 10% (Tab. 3.5). Isolate Fa1, isolated from symptomatic wheat plants, did not cause

negative effects on oat, on the contrary, inoculated plants had 25% higher fresh matter yield

compared to the non-inoculated control. In contrast, isolate Fa4 from pea plants negatively

affected emergence of oats and reduced fresh weight by 40%. This stunted growth was also

observed for black oats, peas (Santana and EFB33), subterranean clover and Indian

mustard. White clover and white mustard exhibited severe wilting so it was not possible to

Santana EFB33

A B B

A B B

a

b b

a

b

b


58

record yield data for these two species. Percentage of wilted plants was close to 80% for

white mustard and 55% for white clover inoculated with Fa4 and 70% for Fa1 (Tab. 3.5).


59

Table 3.5. Emergence, percentage of emerged plants that wilted, fresh weight per pot and plant height of nine plant species inoculated with two F.

avenaceum isolates, (Fa1 – isolate from wheat, Fa4 – isolate from pea).

Emergence (%) Wilted (%) Fresh weight (g) Height (cm)

Species Control Fa1 Fa4 Control Fa1 Fa4 Control Fa1 Fa4 Control Fa1 Fa4

Black oat 88 100 100 0 0 0 3.92 3.38 3.03 35.75 36.94 35.03

Oat 80 88 44 0 0 0 3.57 ab1 4.51 a 2.16 b 25.65 a1 28.19 a 14.46 b

Wheat 96 96 96 0 0 0 4.84 4.77 4.85 38.13 36.32 33.64

Maize 100 100 100 0 0 0 28.31 26.37 27.57 52.07 48.02 51.44

EFB33 97.5 97.5 97.5 0 0 0 13.87 11.98 10.76 36.57 34.72 36.08

Santana 95 95 100 0 13.16 5.00 18.45 a 11.69 b 11.95 b 16.66 a 12.09 b 12.85 b

Subclover 96.7 76.7 88.3 1.72 41.30 35.85 3.52 a 1.53 b 2.08 ab 6.58 a 4.21 b 4.96 b

White clover 96.7 48.3 70 3.45 68.97 54.76 - 2 - - 2.75 a 0.88 c 1.71 b

Indian mustard 91.7 86.7 88.3 0 9.61 22.64 8.98 7.82 6.01 9.41 a 8.30 ab 7.78 b

White mustard 43.33 28.33 30 11.54 76.47 77.77 - 2 - - 4.68 a 1.77 b 2.10 b

1 Different letters withinh one row are indicate significant differences in fresh weights or plant height among treatments within one plant species at P < 0.05 (Tukey’s HSD test).

2 Due to extreme wilting no data available.


60

3.4 Discussion

In sterile sand F. avenaceum was the most aggressive pathogen, followed by P. medicaginis,

D. pinodes, and F. solani. In contrast, in non-sterile field soil, overall aggressiveness of all

pathogens was greatly reduced and F. avenaceum and D. pinodes were similar in their

aggressiveness in that system. Overall, the spring pea variety Santana was considerably

more susceptible than the winter pea variety EFB33 in both systems. Lower temperatures

reduced plant growth and slightly increased disease on winter variety EFB33, with the

highest DI at low temperatures (13/10°C). In contrast, the highest disease severity on

Santana was observed at the highest temperature, 19/16°C. The two isolates of F.

avenaceum originating from wheat and pea caused severe symptoms on roots of all plant

species that were tested, especially on peas, white clover and subterranean clover as well as

mustard and to a lesser degree on Indian mustard. The pea isolate also affected growth of

oats, whereas the isolate from wheat had no negative effect on oats.

Zhang et al. (2013) showed that in steam-sterilized peat-soil potting mix, among nine fungal

species (F. oxysporum, F. graminearum, F. solani, F. avenaceum, F. tricinctum, F.

sporotrichioides, F. equiseti, F. poae and Rhizoctonia solani), F. avenaceum together with F.

graminearum and R. solani, was the most pathogenic on soybean seedlings. Persson et al

(1997) observed that inoculation with F. avenaceum resulted in high disease severity and low

plant weight of peas in sterilized 1:1 peat/sand mix. The observed decrease in

aggressiveness in non-sterilized soil can be explained by the low competitive ability of this

pathogen, that is unable to compete with fast growing soil saprophytes and can be

suppressed by the soil microbiota (Fletcher et al., 1991; Lin and Cook, 1979).

With respect to the rot symptoms, Phoma medicaginis was the second most aggressive

pathogen on Santana and EFB33 and caused moderate disease. P. medicaginis is important

pea pathogen in the Netherlands, Scandinavia, and Germany (Oyarzun and Loon, 1989;

Persson et al., 1997; Pflughöft et al., 2012). It was the most frequently isolated root pathogen

of peas in Sweden in the years 1993-1995 (Persson et al., 1997) and second most frequent

(after Fusarium spp.) in Germany in 2005-2007 (Pflughöft et al., 2012) and most frequent in

Germany from 2008-2012. In a field study that we conducted from 2009-2012 (see Chapter

2) P. medicaginis was the dominant pathogen on EFB33, isolated from 82% of the sampled

plants. It is part of the Ascochyta blight complex, and it can reduce pea yields by 25%

(Wallen, 1974). In our study, infection with P. medicaginis did not reduce fresh weights of

three week old plants, except for Santana in sterile sand. Similar results were reported by

Persson et al. (1997) who found that although the pathogen caused moderate symptoms on

the epicotyl of pea in a pot trial, reduction of plant fresh weight was small. Severity of disease

caused by P. medicaginis in the sterile system did not change under different temperature


61

conditions. Fresh weights of EFB33 plants did not differ from the control plants; in Santana

they were reduced by 35% on average regardless of the temperature applied.

In general, effects of pathogens in our experiments were more visible on the fresh than on

the dry weights. This is likely due to the fact that disease caused by tested pathogens firstly

impeded water uptake and before the intensive accumulation of dry matter in plants

occurred. As the plants were harvested after three weeks of growing effects on dry weights

were therefore expected to be less pronounced.

Fusarium solani and Didymella pinodes caused moderate disease symptoms on Santana

and low symptoms on EFB33. Nevertheless, inoculation with D. pinodes resulted in high

reductions of plant fresh weight of both pea varieties. Plants inoculated with this pathogen

had, after F. avenaceum, the lowest biomass yield. There are few data available about the

role of D. pinodes as a part of foot and root rot in the yield reduction of peas. In contrast to

our findings, (Persson et al., 1997) reported only small yield reductions after inoculation of

pea with D. pinodes in greenhouse trials. High genetic variability in the D. pinodes

populations and existence of different pathotypes can be a reason for this disagreement. As

part of the Ascochyta blight complex, D. pinodes is the most damaging (Boros and Wawer,

2009; Onfroy et al., 1999; Wallen, 1974) causing yield reductions of up to 70% (Le May et al.,

2009). Results obtained in our study strongly support Campbell and Neher (1994)

observation that in the case of root disease in order to observe the full effect of a pathogen,

assessment of severity of disease symptoms on plant roots must be correlated with

measurement of above-ground plant growth parameters (leaf area, weights, heights, leaf

number, respiration intensity, etc).

Fusarium solani has been recognized as one of the most prevalent and important parts of the

foot rot complex on peas (Kraft et al., 1988; Rush and Kraft, 1986). In our study, F. solani

caused the least damage compared to the three other pathogens. Besides a low DI, it did not

affect plant growth. Moreover, fresh weight of both varieties inoculated with F. solani in soil

was higher than in the control plants. However, this difference was not statistically significant.

Lack of external stress and the short time for colonization (3 weeks) of the root system by

this pathogen can be reasons for the low effects of F. solani. Rush and Kraft (1986) observed

that despite of severe rot symptoms at the cotyledon attachment area even 35 days after

emergence of peas, deterioration of roots and reduction of plant growth was still low. The

same rot symptoms caused severe loss of roots and stunting of plant growth 49 days after

emergence (beginning of flowering). Hence, they concluded that one of the most important

predictors of potential effects of F. solani f.sp. pisi is the timing of deterioration of roots and

not the severity of symptoms. Any factor that stresses the host root system will shorten the

time between infections and severe root rot (Thung and Rao, 1999). As F. solani f. sp pisi is


62

a slow colonizer of the roots (Huisman, 1982), it is very likely that three weeks for the

experiments were not long enough for the pathogen to colonize roots and cause more severe

damage. Besides that, in the bioassays done, the only stress factor for pea plants was the

pathogen itself, and this is rarely the situation in field conditions. It is more usual that in the

field plants are exposed to a combination of different environmental stress factors (drought,

frost, soil compaction, etc.).

F. avenaceum is known to cause severe disease on a range of legume species (Feng et al.,

2010; Fletcher et al., 1991; Lamprecht et al., 1986; Persson et al., 1997; Zhang et al., 2013)

as well as on canola (Berkenkamp and Vaartnou, 1972; Hwang et al., 2000), oat, barly and

wheat (Cook, 1980; Hwang et al., 2000; Smiley and Patterson, 1996). The high fresh weight

reduction observed in our study is in agreement with findings of Persson et al. (1997) who

reported biomass reductions of 66% in peas in comparison to the uninoculated control. Our

results suggest a great potential of F. avenaceum to damage some brassicaceous species at

least in a sterile system. Indeed, epidemics of F. avenaceum on Brassica napus have been

reported from Canada (Feng et al., 2010).

The optimal temperature for growth of F. avenaceum is in the range of 20° and 25°C

(Campbell and Lipps, 1998; Pettitt et al., 1996). Brennan et al. (2003) showed that at the

temperature range of 20-25°C F. avenaceum caused the greatest retardation in coleoptile

growth of wheat (> 89.3%). At the same time, in in vitro tests, growing rates of F. avenaceum

culture increased with increasing temperature until 20°C and then decreased. Hwang et al.

(2000) obtained similar results in a controlled-environment study on lentil seedlings. Root rot

symptoms were the most severe when lentil was grown in warmer temperatures from 20°C to

27.5°C. At lower or higher temperatures disease severity on seedlings declined.

Furthermore, Satyaprasad et al. (2000) reported the increase in mean disease scores

caused on wheat and lupines by F. avenaceum with rising temperature.

Our results suggest that due to increases of host growth at increased temperatures the

overall damage may still be less at higher temperatures. An important consideration is that F.

avenaceum is capable of continuous growth in wet and cool soils at a high carbon dioxide

partial pressure which gives it an advantage over other soil microorganisms and enables it to

escape potential antagonism (Forbes and Dickinson, 1977). Thus, warmer and wetter winter

conditions may change the importance of F. avenaceum in the future. We observed a severe

outbreak of F. avenaceum on EFB33 when a frost-free winter period was followed by severe

frost and subsequent higher than normal temperatures (see Chapter 2).


63

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Ali, S.M., Sharma, B., Ambrose, M.J., 1993. Current status and future strategy in breeding pea to improve resistance to biotic and abiotic stresses. Euphytica 73, 115–126.

Bateman, G.L., Gutteridge, R.J., Gherbawy, Y., Thomsett, M.A., Nicholson, P., 2007. Infection of stem bases and grains of winter wheat by Fusarium culmorum and F. graminearum and effects of tillage method and maize-stalk residues. Plant Pathol. 56, 604–615.

Berkenkamp, B., Vaartnou, H., 1972. Fungi associated with rape root rot in Alberta. Can. J. Plant Sci. 52, 973–976.

Blume, M.C., Harman, G.E., 1979. Thielaviopsis basicola: A component of the pea root rot complex in New York State. Phytopathology 69, 785–788.

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Boros, L., Wawer, A., 2009. Garden pea varietal susceptibility to Mycosphaerella pinodes and its effect on yield components of single plants. Veg. Crops Res. Bull. 70.

Bowen, J.K., Lewis, B.G., Matthews, P., 1997. Discovery of the teleomorph of Phoma medicaginis var pinodella in culture. Mycol. Res. 101, 80–84.

Bretag, T., Price, T., Keane, P., 1995. Importance of seed-borne inoculum in the etiology of the Ascochyta blight complex of field peas (Pisum sativum L.) grown in Victoria. Aust. J. Exp. Agric. 35, 525–530.


Campbell, C.L., Neher, D.A., 1994. Estimating disease severity and incidence, in: Epidemiology and Management of Root Diseases. Springer Berlin Heidelberg, pp. 117–147.

Campbell, K.A.G., Lipps, P.E., 1998. Allocation of resources: sources of variation in Fusarium head blight screening nurseries. Phytopathology 88, 1078–1086.

Cook, R.J., 1980. Fusarium foot rot of wheat and its control in the pacific northwest. Plant Dis. 64, 1061–1066.

Corbière, R., Gelie, B., Molinero, V., Spire, D., Agarwal, V.K., 1994. Investigations on seedborne nature of Mycosphaerella pinodes in pea seeds. Seed Res. 22, 26–30.

Cormack, M.W., 1951. Variation in the cultural characteristics and pathogenicity of Fusarium avenaceum and F. arthrosporioides. Can. J. Bot. 29, 32–45.

Feng, J., Hwang, R., Chang, K.F., Hwang, S.F., Strelkov, S.E., Gossen, B.D., Conner, R.L., Turnbull, G.D., 2010. Genetic variation in Fusarium avenaceum causing root rot on field pea: Genetic variation in Fusarium avenaceum. Plant Pathol. 59, 845–852.

Fletcher, J.D., Broadhurst, P.G., Bansal, R.K., 1991. Fusarium avenaceum : A pathogen of lentil in New Zealand. N. Z. J. Crop Hortic. Sci. 19, 207–210.

Forbes, R.S., Dickinson, C.H., 1977. Behaviour of Fusarium avenaceum in soil growth analysis plates. Trans. Br. Mycol. Soc. 69, 197–205.

Foroud, N.A., Chatterton, S., Reid, L.M., Turkington, T.K., Tittlemier, S.A., Gräfenhan, T., 2014. Fusarium diseases of Canadian grain crops: Impact and disease management strategies, in: Goyal, A., Manoharachary, C. (Eds.), Future Challenges in Crop Protection Against Fungal Pathogens, Fungal Biology. Springer New York, pp. 267–316.


Gossen, B.D., McDonald, M.R., Conner, R.L., Hwang, S.F., Chang, K.F., 2010. Significance of seed infection on epidemics of Mycosphaerella blight in field pea. Can. J. Plant Pathol. 32, 458–467.


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Huisman, O.C., 1982. Interrelations of root growth dynamics to epidemiology of root-invading fungi. Annu. Rev. Phytopathol. 20, 303–327.

Hwang, S.F., Gossen, B.D., Turnbull, G.D., Chang, K.F., Howard, R.J., Thomas, A.G., 2000. Effect of temperature, seeding date, fungicide seed treatment and inoculation with Fusarium avenaceum on seedling survival, root rot severity and yield of lentil. Can. J. Plant Sci. 80, 899–907.

Jensen, B., Bødker, L., Larsen, J., Knudsen, J.C., Jørnsgaard, B., 2004. Specificity of soil-borne pathogens on grain legumes.

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Kraft, J.M., Pfleger, F.L., 2001. Compendium of pea diseases and pests. APS Press. Lamprecht, S.C., Marasas, W.F.O., Thiel, P.G., Schneider, D.J., Knox-Davies, P.S., 1986.

Incidence and Toxigenicity of seedborne Fusarium species from annual Medicago species in south Africa. Phytopathology 76, 1040–1042.

Le May, C., Jumel, S., Schoney, A., Tivoli, B., 2009. Ascochyta blight development on a new winter pea genotype highly reactive to photoperiod under field conditions. Field Crops Res. 111, 32–38.


Lin, Y.S., Cook, R.J., 1979. Supression of Fusarium roseum “avenaceum” by soil microorganisms. Phytopathology 69, 384–388.

Moussart, A., Tivoli, B., Lemarchand, E., Deneufbourg, F., Roi, S., Sicard, G., 1998. Role of seed infection by the Ascochyta blight pathogen of dried pea (Mycosphaerella pinodes) in seedling emergence, early disease development and transmission of the disease to aerial plant parts. Eur. J. Plant Pathol. 104, 93–102.

Nasir, M., Hoppe, H.-H., Ebrahim-Nesbat, F., 1992. The development of different pathotype groups of Mycosphaerella pinocles in susceptible and partially resistant pea leaves. Plant Pathol. 41, 187–194.

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Oyarzun, P., Loon, J. van, 1989. Aphanomyces euteiches as a component of the complex of foot and root pathogens of peas in Dutch soils. Neth. J. Plant Pathol. 95, 259–264.


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DISEASE SUPPRESSION WITH COMPOST APPLICATION

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4. EFFECTS OF COMPOST APPLICATION ON FOOT AND ROOT ROT OF SPRING AND WINTER PEA VARIETIES CAUSED BY FUSARIUM AVENACEUM, FUSARIUM SOLANI, PHOMA

MEDICAGINIS AND DIDYMELLA PINODES

Abstract

In order to evaluate the potential of yard waste compost (YWC) to suppress foot rot disease of pea caused by Didymella pinodes, Phoma medicaginis, Fusarium solani f. sp pisi and F.

avenaceum, pot experiments were carried out under controlled conditions in sterile sand and non-sterilized field soil. In addition, effects of application rate of YWC and of reduced temperatures on the disease suppression were assessed. The spring pea Santana and winter pea EFB33 were inoculated with spore suspensions of the pathogens at sowing or left uninoculated, and grown in sterile sand and field soil amended with and without YWC (20% v/v). Part of the experiments included γ irradiated compost as additional controls. After three weeks disease severity was assessed and yield parameters measured. Application of compost to sterile sand significantly reduced the disease index (DI) of Santana caused by F.

avenaceum, P. medicaginis and D. pinodes. Disease caused by F. solani was low (mean DI 3.0) and no effect of compost application was observed. The suppressiveness of compost was reduced through γ irradiation for D. pinodes and P. medicaginis, while disease symptoms of F. avenaceum and F. solani were even higher than in inoculated control without compost. When plants were grown in non-sterilized soil symptoms due to F. avenaceum, P.

medicaginis and D. pinodes were lower than in the sterile sand while for F. solani they were on the same level. Compost application to soil did not have an effect on disease severity. However, when sterile compost was applied disease severity due to all pathogens was significantly increased compared to normal compost in soil. With the exception of the treatment inoculated with F. avenaceum, application of non- and sterilized compost had positive effects on the plant weight increasing it between 25 % and 60 % depending on pathogen and growing substrate. When lower rates of compost were applied (3.5% v/v) to sand, disease symptoms were significantly reduced. Temperature did not have significant effects on level of disease suppression by compost. The YWC used in the trials could suppress disease caused by the tested pathogens and the intensity of suppression depended on variety, pathogens and growing media. Results indicate that it is likely that suppression of disease caused by Fusarium spp. is biological in origin and for D. pinodes

and P. medicaginis chemical and physical properties of compost are also contributing to the suppression of foot and root rot disease.

Keywords: compost, F. avenaceum, P. medicaginis, D. pinodes, F. solani, foot rot, peas


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4.1 Introduction

It is widely recognized that application of high quality compost can effectively suppress

diseases caused by a range of soil borne pathogens on a range of different crops (Aviles et

al., 2011; Bonanomi et al., 2007; Hadar, 2011; Hoitink, 2006; Hoitink et al., 1997; Hoitink and

Fahy, 1986; Noble, 2011; Noble and Coventry, 2005; Termorshuizen et al., 2006; Yogev et

al., 2006). Success of compost in disease suppression depends on the type of substrate,

maturity, and the composting process used (Hoitink and Fahy, 1986). Inconsistency in the

level of disease suppression between pot and field experiments is often reported (Hoitink et

al., 1997; Litterick et al., 2004; Noble and Roberts, 2003).

Suppressive effects of composts are mainly due to beneficial microorganisms (Hoitink and

Boehm, 1999) which has been supported by loss of suppressivness after heat treatment, e.g.

pasteurization or sterilization. Hoitink and Boehm (1999) described four mechanisms

involved in compost induced microbial disease suppression: competition for nutrients

between compost inhabiting microbes and pathogens, antibiosis due to production of

different metabolites by beneficial microorganisms, parasitism and predation of pathogens by

beneficial nematodes, mites or fungi, and induced resistance. Chemical and physical

properties as affected by compost age and level of organic matter decomposition, C:N ratio,

cellulose and lignin content, nitrogen content and form, pH, electrical conductivity (EC),

organic chemicals released by compost, etc., are also contributing to disease suppression

(Bonanomi et al., 2007; Hoitink, 2006; Hoitink et al., 1997; Hoitink and Fahy, 1986; Recycled

Organics Unit, 2006). Besides direct suppressive effects, compost application also improves

soil quality by adding organic matter and improving various physical soil properties.

Depending if the suppression is caused by the action of a specific organism or the total

microbial community two categories of suppression can be distinguished - specific and

general (Hoitink, 2006; Hoitink et al., 1997). These two types do not exclude each other.

General suppression is the result of the activity of overall microbial biomass (Recycled

Organics Unit, 2006), and it cannot be transferred. In contrast, in specific suppression one or

several groups of organisms are responsible for suppression and it can be transferred to

other substrates/soils by adding a small amount of suppressive substrate/soil (Aviles et al.,

2011).

Peas are strongly affected by root and foot rots that limit their production worldwide (Ali et al.,

1993; Bretag and Ramsey, 2001). A complex of more than 20 different species of soil-borne

fungal pathogens can cause foot and root rot of peas (Blume and Harman, 1979; Bodker et

al., 1993; Kraft et al., 1988; Olofsson, 1967; Persson et al., 1997). The most important

species causing foot and root rot on peas are Didymella pinodes (syn Mycospharella


68

pinodes), Phoma medicaginis, Fusarium spp. (F. solani f. sp pisi, F. oxysporum f.sp pisi, F.

avenaceum, F. culmorum), Pythium spp., Thielaviopsis basicola, Aphanomyces euteiches,

and Rhizoctonia solani (Feng et al., 2010; Kraft et al., 1988; Nasir et al., 1992; Oyarzun,

1993; Persson et al., 1997; Pflughöft et al., 2012). Prevalence, dominance and importance of

the single pathogens vary greatly depending on location, climate, and agricultural practice

(Gaurilckiene and Cesnuleviciene, 2013; Jensen et al., 2004; Persson et al., 1997).

Especially impaired soil structure through compaction and inadequate drainage play a crucial

role for pea health (Solaiman et al., 2007).

Regular applications of organic amendments, especially high quality composts, could help

improve soil structure and supressiveness against pathogen. However, suppressiveness of

compost against one pathogen does not necessarily mean that it will suppress another

pathogen. This variability in the disease suppressiveness has been reported also for

composts made of similar substrates and under similar conditions (Noble and Coventry,

2005). Given the high variation in the response Termorshuizen et al. (2006) emphasize that

prediction of potential suppression of disease should be made based on specific

pathosystems only. Also, in pot trials successful suppression is usually achieved with

application rates of 30 to 50% v/v of compost to the potting mixes (Noble and Coventry,

2005; Termorshuizen et al., 2006). However, in organic farming in Germany application rate

of externally brought compost is limited by law to only 5 t dry matter ha -1 per year. Even at

this low rate application of high quality composts have been successful in reducing R. solani

in potatoes in the field (Bohne et al., 2013; Schulte-Geldermann et al., 2009). Also, raw

application together with pea seeds was successful in suppressing P. medicaginis. The

research presented here was conducted to determine if a locally produced Yard Waste

Compost (YWC) can suppress root and foot rot disease of peas caused by the four

pathogens Fusarium avenaceum, Fusarium solani f. sp. pisi, Phoma medicaginis var.

pinodella, and Didymella pinodes. This was assessed in two growing substrates, sterile sand

and non-sterilized field soil on a spring and winter pea variety. In additional experiments the

effects of application rate of YWC and of reduced temperatures on the disease suppression

by compost application were assessed.


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4.2 Material and Methods All experiments were replicated five times in a completely randomized design.

4.2.1 Potential of yard waste compost to suppress foot and root rot of peas caused by

F. avenaceum, F. solani, P. medicaginis and D. pinodes

The winter pea variety EFB33 and spring variety Santana (KWS LOCHOW, Gmbh) were

used for the experiments. EFB33 is a regularly leafed, small seeded variety with thousand

seed weight of about 130 g and violet flowers. Santana is a spring pea widely grown in

Germany. It is semi-leafless, white flowering with white seed coat and a thousand seed

weight of 250 to 300g. Prior to use, pea seeds were surface disinfected with 70% ethanol for

five minutes, rinsed with running distilled water and air dried under a laminar flow cabinet. All

defective and damaged seeds were discarded.

Sterilized yellow sand and non-sterilized field soil, without or amended with non-sterilized

and sterilized compost were used as growing media (Tab 4.1). Sand was autoclaved for 20

minutes at 121°C and stored in a closed container until the beginning of the trial. The field

soil for the trial was collected from the experimental field of the University of Kassel at the

Neu-Eichenberg. The soil type is a deep Haplic Luvisol from loess with 78 soil points

(Wildhagen, 1998). Locally produced mature YWC made of garden trimmings was used in

the trial. In order to determine whether the effects of YWC were biological in origin, half of the

compost used was gamma irradiated with 31.5 kGy one week prior to the experiment and

stored at 4°C until the beginning of the trial.

At the day of sowing, compost was thoroughly mixed with sand or soil at the rate of 20% v/v.

Approximately 1.3 kg of substrate was filled in square pots of 1 L (12 x 12 x 11 cm) and eight

pea seeds per pot were sown at 2 cm depth. Following sowing, pots were inoculated with

spore suspensions of F. avenaceum, F. solani, P. medicaginis and D. pinodes at a rate of

104 spores g-1 of substrate. Five isolates for each pathogen (Tab. 3.1, Chapter 3.2.1) were

used in a mixture. Preparation of inoculum was as described in section 3.2.

Pots were watered every second day and treatments with sole sand as a substrate were

fertilized with the complex N:P:K fertilizer Wuxal Super (8:8:6 + trace elements) in the

amount corresponding to a total of 100 mg of N L-1 substrate. Plants were grown for 21 days

with a day/night temperature regime of 19/16°C.


70

Table 4.1. Chemical properties of substrates and composts used in the experiment.

pH

EC

(µS/cm) % N % C

C/N

ratio

Mg

(mg/kg)

K

(mg/kg)

P

(mg/kg)

Sand 5.70 7.8 0.002 0.01 6.53 32.85 8.26 2.55

Field soil 5.86 33.8 0.127 1.17 9.19 139.35 103 35.37

Compost 7.50 498 1.81 29.01 16.04 454.15 3104 541.76

Sterilized

compost 6.92 462 1.61 25.65 15.84 515.25 3457 732.51

4.2.2 Effect of compost application rate on disease suppression

Autoclaved yellow sand was used as substrate, sole or amended with two levels of YWC, low

- 3.5% v/v corresponding to about 5 t DM ha-1 applied in the field and high – 20% v/v.

Preparation of seeds, sowing, fertilization, growth conditions, watering and duration of the

experiment were as described above (see 4.2.1). As the previous experiment (see 4.2.1) had

shown that inoculation with 104 spores g-1 of substrate did not cause disease on the resistant

variety EFB33, the concentration of inoculum in this experiment was increased to 105 spores

g-1 of substrate. Isolates Fa1, Pm1, Dp5 and Fs5 (Tab. 3.1 Chapter 3.2.1) were used in this

trial.

4.2.3 Effect of reduced temperature on compost induced disease suppression

The effect of reducing temperature from 19°/16°C to 16°/12°C was tested for compost

induced disease suppression caused by P. medicaginis and F. avenaceum on Santana and

EFB33, in sterile sand with 20% v/v of YWC. Isolates Pm1 and Fa1 (Tab. 3.1, Chapter 3.2.1)

at the rate of 105 spores g-1 of substrate were used. Experimental conditions as well as

duration were as described above (see 4.2.1).

4.2.4 Measurement of plant biomass and assessment of disease Plants were harvested after 21 days by carefully pulling them out of the substrate. Roots

were cut off, washed with running tap water, and stored at 4°C until use. Above ground plant

parts were weighed and dried at 105°C until constant weight. Root disease severity was

assessed according to the key of Pflughöft (2008) (Tab. 2.4, Chapter 2). Assessments were

always performed within 48 hours following harvest. Symptoms on the external plant tissue

were evaluated and followed by the assessment of damage of the internal tissue. Root

sections of two plants per replication were plated on Coon’s agar for reisolation of pathogens

in order to follow Koch's postulates.


71

4.2.5 Data processing and analysis

Disease index (DI) for each plant was calculated as the average of the external and internal

lesion score. As all plants in sand controls emerged, missing plants in the treatments were

considered dead and score 9 was given for both external and internal tissue. The mean DI of

all plants in each pot was calculated to present one replication, and further analyses were

done on these values. Biomass production was expressed as fresh and/or dry weight per

plant. Weights of all plants in one pot were measured together and then divided with the

number of plants per pot and represents one replication.

Analyses of variance (ANOVA) were applied in order to analyze the effect of the pathogens,

substrate and temperature on the DI, plant weights and survival rates. Prior to ANOVA, data

were checked for normality using Shapiro-Wilks-W-Test. Mean separations were made by

Tukey honest significant differences (HSD) test at the P < 0.05. All statistical analyses were

conducted with statistical program R version 2.15.2.


72

4.3 Results

4.3.1 Potential of yard waste compost to suppress foot and root rot of peas caused by

F. avenaceum, F. solani, P. medicaginis and D. pinodes

Plants grown in non-inoculated sand control with and without compost were healthy and had

mean DI below 2.5. In the treatment with γ irradiated compost, DI of the control plants was

significantly higher, however still below 3. In contrast, plants grown in the non-inoculated soil

controls had moderate to severe disease symptoms in treatments with and without compost

(Tab. 4.2).

Application of compost to sterile sand significantly reduced the DI of Santana plants

inoculated with F. avenaceum, P. medicaginis and D. pinodes (Tab. 4.2). In the treatment

inoculated with F. solani, disease severity was low (mean DI 3.0) and no effect of compost

application (mean DI 2.5) was observed. The disease suppressiveness of compost was

significantly reduced or lost through γ irradiation for D. pinodes and P. medicaginis,

respectively while disease symptoms of F. avenaceum and F. solani were significantly higher

than in the non-amended sand.

When non-sterilized soil was used as growing medium, the non-inoculated plants showed

moderate symptoms with a mean DI of 4.8. This was not affected by the compost application.

Symptoms due to F. avenaceum, P. medicaginis and D. pinodes were lower than in the

sterile sand while for F. solani the DI was 3.9 in soil compared to 3.0 in sand (Tab. 4.2).

When sterile compost was used, disease severity due to all pathogens was significantly

increased compared to normal compost in soil. Compared to sand, symptoms caused by F.

avenaceum were less severe in soil with sterile compost while for F. solani, P. medicaginis

and D. pinodes severities were higher in soil than in sand.


73

Table 4.2. Disease Index (DI) of Santana in sterile sand and non-sterilized field soil in non-inoculated control and treatments inoculated with F. avenaceum, F. solani, P. medicaginis and D. pinodes and amended with compost (no-without, N-not sterilized and S-sterilized compost). Different letters within one pathogen and one substrate indicate significant differences in DI among compost treatments at P < 0.05 (Tukey’s HSD test).

Pathogen Compost Sand Soil Control no 1.5 b 4.8

N 2.0 b 4.5 S 3.0 a 6.0

F. avenaceum no 5.1 b 3.8 b N 2.9 c 3.7 b S 8.3 a 5.9 a

F. solani no 3.0 b 3.9 ab N 2.5 b 3.0 b S 5.3 a 5.4 a

P. medicaginis no 4.7 a 3.6 b N 3.2 b 3.5 b S 4.4 a 6.8 a

D. pinodes no 4.6 a 4.2 ab N 2.9 c 3.4 b S 3.6 b 5.5 a

With the exception of the treatment inoculated with F. avenaceum, application of compost,

regardless if it was sterilized or not, had positive effects on the plant weights of Santana in

sand (Fig. 4.1). The fresh weight increases ranged between 35% for F. solani and 55% for P.

medicaginis and D. pinodes. Application of non-sterilized compost in the treatment with F.

avenaceum significantly increased fresh weight (50%) while with γ irradiated compost

biomass of plants was reduced by 60%. In soil, significant increases of the fresh weight

following non-sterile compost application were only observed in the non-inoculated controls

and when inoculted F. solani (Fig. 4.1). Sterilized compost had no significant effect on plant

weights. Disease severity of EFB33 was overall low (DI between 2 and 3) and was not

affected by compost application, regardless if sand or soil were used as growing substrate

(see Appendix Fig. A. 4.1). Both types of compost positively affected plant weights (see

Appendix Fig. A. 4.2). Increases in fresh weights were in the range between 25% and 50%,

depending on the pathogen and growing substrate. Dry weights of both varieties followed

similar pattern as the fresh weights (see Appendix Fig. A. 4.3 and A. 4.4).


74

Figure 4.1. Effect of compost application (no-without, N-not sterilized and S-sterilized compost) on fresh weight per plant of Santana in sand and soil inoculated with F. avenaceum (Fa), F. solani (Fs), P. medicaginis (Pm) and D. pinodes (Dp). Different letters within one substrate and one pathogen indicate significant differences in fresh weights among compost treatments at P < 0.05 (Tukey’s HSD test). The error bars represent the ± 1 SD.

b

b

a

b

a a

b

a

a

b

a

a

c

b

a b

a ab

b

a

b


75

4.3.2 Effect of compost application rate on disease suppression

Increasing inoculum concentration to 105 spores g-1 of substrate resulted in slightly higher DI

for both varieties. All DIs of Santana were lower in the compost treatments than in the

controls (Fig. 4.2). The reductions were significant for P. medicaginis and the high amount of

compost, and for D. pinodes at both compost rates. Application of 20% v/v compost in the

system with F. avenaceum and EFB33 significantly reduced disease severity, while both

compost rates reduced P. medicaginis.

Figure 4.2. Disease index of Santana and EFB33 inoculated with F. avenaceum (Fa), F. solani (Fs), P. medicaginis (Pm) and D. pinodes (Dp) and amended with compost (no-without, low- 3.5% v/v and high- 20% v/v). O is the non-inoculated control. The horizontal line in the boxplot shows the median, the bottom and tops of the box the 25th and 75th percentiles and the vertical lines the minimum and maximum values; outliers as single points. Mean values of DI are marked with triangles. Different letters within variety and pathogen are indicating significant differences in DI at P < 0.05 (Tukey’s HSD test).

Compost application positively affected biomass of Santana plants, except when inoculated

with F. solani, where compost had no effect (Fig. 4.3). For treatments inoculated with F.

avenaceum and P. medicaginis increase in biomass was significant when compost was

applied at the high rate, and for D. pinodes at both compost treatments. In contrast to

Santana, weights of EFB33 were not affected by compost application (Fig. 4.3).

a ab b a b b

ab a b a b b


76

Figure 4.3. Effect of compost application rate (no-without, low- 3.5% v/v and high- 20% v/v) on the fresh weight of (A) Santana and (B) EFB33 plants inoculated with 105 spores g-1 of substrate

of F. avenaceum (Fa), F. solani (Fs), P. medicaginis (Pm) and D. pinodes (Dp). Different letters within pathogen are indicating significant differences in fresh weights at P < 0.05 (Tukey’s HSD test). The error bars represent the ± 1 SD.

b

b

a ab

a

b ab

a

b

a

b

a

A)

B)


77

4.3.3 Temperature effect on compost induced disease suppression

Reducing temperatures from 19°/16°C to 16°/12°C did not affect the symptoms on the

controls. However, it reduced DI caused by F. avenaceum on both pea varieties, whereas

disease caused by P. medicaginis was not affected. Compost application reduced the

symptoms caused by both tested pathogens under both temperature regimes on Santana

and EFB33 (Tab. 4.3). However, the level of disease suppression varied in the two

pathosystems with a significant interaction of temperature, variety and pathogen due to the

low effect of compost on F. avenaceum severity on EFB33 at the low temperature.

Table 4.3. Disease index of Santana and EFB33 inoculated with F. avenaceum and P.

medicaginis, amended with compost (no-without, compost - 20% v/v) and grown under two temperature regimes (low - 16/12°C and high - 19/16°C). Different letters within one variety, temperature and pathogen are indicating significant differences in DI among compost treatments at P < 0.05 (Tukey’s HSD test). Percent change presents difference in DI in low temperature treatment compared to high.

Santana EFB33 Pathogen Compost 19/16 16/12 % change 19/16 16/12 % change Control no 2.1 2.5 + 19 1.4 1.6 + 14 Control compost 1.8 2.3 + 28 1.9 1.8 - 5 F. avenaceum no 8.0 a 6.4 a - 20 5.2 a 3.4 - 35 F. avenaceum compost 6.0 b 4.5 b - 25 2.3 b 2.3 0 P. medicaginis no 6.7 a 6.3 a - 6 5.1 a 5.0 a - 2 P. medicaginis compost 4.1 b 3.3 b -19 2.8 b 2.0 b - 29

Suppression of disease due to inoculation with P. medicaginis with compost was usually

greater than the suppression of F. avenaceum at both temperatures in both varieties (Tab.

4.3). The lower apparent effectiveness of compost against F. avenaceum in EFB33 at the

low temperature was due to reduced aggressiveness of F. avenaceum resulting in low

disease on EFB33 in absence of compost.

Compost application significantly increased the fresh weight of Santana plants inoculated

with both pathogens regardless of the growing temperature (Fig. 4.4A). For EFB33 significant

increase of fresh weight was measured only for the plants inoculated with F. avenaceum

under both temperature regimes (Fig. 4.4B).


78

Figure 4.4. Effect of compost application (no-without, compost- 20% v/v) on the fresh weights of Santana (A) and EFB33 (B) plants inoculated with F. avenaceum (Fa) and P. medicaginis (Pm) and grown under different temperature regimes (low - 16/12°C and high - 19/16°C). Asterisk indicates significant differences at P < 0.05 among treatments with and without compost within one temperature regime and pathogen. The error bars represent the ± 1 SD.

A

B

*

*

*

*

*

*

*


79

4.4 Discussion The Yard Waste Compost (YWC) we used could suppress root and foot rot of peas caused

by Fusarium avenaceum, Fusarium solani f. sp. pisi, Phoma medicaginis var. pinodella, and

Didymella pinodes. However, the intensity of suppression depended on pea varieties,

pathogens and growing media used. Disease severity on EFB33 was low and no effect of

compost application was observed. An increase of the inoculum concentration in the trials on

compost amount and temperature only slightly increased the severity of disease. A

temperature effect was observed only in the system with EFB33 and F. avenaceum where

the disease suppression through compost application was lower at the reduced temperature

because the overall aggressiveness of F. avenaceum was also low.

After γ sterilization of the compost, the suppressive effect was lost in the pathosystems with

Fusarium spp. suggesting that suppression was biological in origin. Moreover, severity of

symptoms caused by F. avenaceum and F. solani increased after incorporation of sterilized

compost. In contrast, part of the suppressive effects against P. medicaginis and D. pinodes

apparently remained even after γ sterilization.

In the case of root disease in order to observe the full effect of a pathogen, assessment of

severity of disease symptoms on plant roots and measurements of above-ground plant

growth parameters have to be considered (Campbell and Neher, 1994). Our results show

that when sterilized sand was used as growing substrate compost application successfully

reduced disease caused by F. avenaceum, P. medicaginis, and D. pinodes on Santana.

Furthermore, fresh weight was significantly increased with compost application in all

treatments including controls. While the overall effect of all three pathogens on the

susceptible pea variety was minimized through compost application, clearly, the mineral

fertilization could not compensate for additional beneficial effects of compost.

The increased disease caused by Fusarium spp. in sand amended with γ-irradiated compost

in our trial could be due to active colonization of the nutrient rich sterile compost by the

pathogens enhancing their activity and population buildup leading to more severe symptoms

in comparison to the inoculated control. The fact that in the systems with D. pinodes

sterilized compost decreased DI and the trend was similar with P. medicaginis suggests that

different mechanisms of suppression may be involved and that the observed reduction in

symptoms caused by Fusarium spp. is biological in origin, whereas chemical and physical

properties of compost are playing an additional role in the suppression of disease caused by

D. pinodes and P. medicaginis on peas. Physical and chemical characteristics are known to

affect the suppressive effects of composts. Incorporation of composts in growing substrates

increases air capacity and in addition to positively affecting plant growth, it also reduces

severity of root rots (Aviles et al., 2011).


80

When Santana was grown in soil, compost application did not result in significant reduction of

DI, and in the treatments with γ-irradiated compost disease symptoms were even more

severe than without compost application for all four pathogens. Serra-Wittling et al. (1996)

reported a higher decrease in the population of F. oxysporum f. sp lini in soil treated with

control compost compared with heat treated compost, however, intensity of disease

suppression was at the same level for both composts. In contrast to our results, Filippi and

Pera (1989) observed suppression of Fusarium wilt in carnations caused by Fusarium

oxysporum f. sp. dianthi when sterilized compost was incorporated in the soil. The disease

reduction was attributed to the activity of the fraction of native soil microorganisms that was

more competitive for nutrients than the pathogen population and acted as antagonists, and

not to the activity of the microflora present in compost. In our trial, relatively high severity of

disease symptoms in the non-inoculated control suggest that soil borne inoculum was

present in the field soil used in the trial. Fusarium solani was mainly isolated from

symptomatic plants grown in the non-inoculated soil. An explanation for increased disease in

soil amended with sterilized compost can be that the level of native microflora in the field soil

used in our trial was not high enough to compete with the existing inoculum and the

introduced pathogens for the available food source provided by sterilized compost.

Pathogens were most likely more efficient in the colonization of compost, which resulted in

faster population growth and consequently an increase in disease severity.

An additional factor that could have contributed to the loss of suppressiveness through γ-

irradiation are changes in the chemical composition and pH of the compost. The combination

of higher amounts of available Mg, K, and P in the sterilized compost, a lower pH, and

microbial-free organic matter could have increased the advantage of pathogens even more

and may thus have resulted in increased disease severity. Borrero et al. (2009, 2004)

showed a strong correlation between pH and disease suppression of Fusarium wilt in tomato

(F. oxysporum f. sp. lycopersici) and carnations (F. oxysporum f. sp dianthi). Namely,

substrates with low pH were conducive to wilt, whereas adding compost increased the pH of

the substrate and disease was successfully suppressed.

The disease suppressive effect of compost generally increased with rate of application, and

rates of at least 20% (v/v) are usually recommended to consistently obtain a disease

suppressive effect, especially when inert material like peat is used as growing substrate

(Noble and Coventry, 2005). Although we observed a tendency of lower DI in systems with

20% v/v of compost, there were no significant differences in the DI between treatments

amended with the two levels of compost. Lumsden et al. (1983) reported significant reduction

in the disease severity of Fusarium wilt in melons (Fusarium oxysporum f. sp. melonis),

crown rot (Phytophtora capsici) in peppers, blights (Pythium myriotylum) on bean and lettuce


81

drop caused by Sclerotinia minor after amending soil with 10% v/v composted sewage

sludge in a pot trial. In contrast, in the pea - Fusarium solani f. sp pisi pathosystem foot rot

increased by 76% compared to the control without compost application. Schüler et al. (1993)

showed that foot rot caused by D. pinodes could be suppressed by amending sand and also

field soil with 30% of compost, whereas an amount of 10% did not affect the disease.

However, an important difference to our study is that they introduced the pathogen in the

system by soaking pea seeds in the inoculum suspension. This led to seed borne inoculum

and infection may have started earlier compared to our study. Watering the substrate with

the spore suspension after sowing in our system gave more time for the physical, chemical

and microbial changes resulting from compost incorporation, potentially hindering infection or

at least slowing down the pathogens. This gave the compost a chance to suppress the

pathogens before infection occurred.

Our results show that the YW compost we used can suppress foot disease on the

susceptible variety Santana caused by F. avenaceum, P. medicaginis, and D. pinodes, by

reducing the intensity of disease symptoms and promoting plant growth. Although high

amounts of compost (30-50% v/v) are recommended for successful disease suppression,

application of 3.5% compost in sterile sand was enough to achieve suppression of disease

symptoms for all four pathogens. Moreover, results we obtained indicate that different

mechanisms of suppression may be involved in the reduction of symptoms caused by

Fusarium spp. and by D. pinodes and P. medicaginis on peas. It is likely that suppression of

disease caused by Fusarium spp. is biological in origin and for D. pinodes and P.

medicaginis in addition chemical and physical properties of compost were contributing to the

suppression of foot and root rot disease.


82

4.5 References Ali, S.M., Sharma, B., Ambrose, M.J., 1993. Current status and future strategy in breeding

pea to improve resistance to biotic and abiotic stresses. Euphytica 73, 115–126. Aviles, M., Borrero, M., Trillas, M.I., 2011. Review on compost as inducer of disease

suppression in plants grown in soilless culture. Dyn. Soil Dyn. Plant 5, 1–11. Blume, M.C., Harman, G.E., 1979. Thielaviopsis basicola : A component of the pea root rot

complex in New York State. Phytopathology 69, 785–788. Bodker, L., Leroul, N., Smedegaard-Petersen, V., 1993. Influence of pea cropping history on

disease severity and yield depression. Plant Dis. 77, 896–900. Bohne, B., Hensel, O., Bruns, C., 2013. Reihenapplikation von Komposten zur Kontrolle

bodenbürtiger Krankheiten – technische Lösungen für Kartoffeln und Körnerleguminosen.

Bonanomi, G., Antignani, V., Pane, C., Scala, F., 2007. Suppression of soilborne fungal diseases with organic amendments. J. Plant Pathol. 89, 311–324.

Borrero, C., Trillas, I., Aviles, M., 2009. Carnation Fusarium wilt suppression in four composts. Eur. J. Plant Pathol. 123, 425–433.

Borrero, C., Trillas, M.I., Ordovás, J., Tello, J.C., Avilés, M., 2004. Predictive factors for the suppression of Fusarium wilt of tomato in plant growth media. Phytopathology 94, 1094–1101.


Campbell, C.L., Neher, D.A., 1994. Estimating disease severity and incidence, in: Epidemiology and Management of Root Diseases. Springer Berlin Heidelberg, pp. 117–147.

Chen, W., Hoitink, H.A.J., Schmitthenner, A.F., Touvinen, O.H., 1988. The role of microbial activity in suppression of damping-off caused by Pythium ultimum. Phytopathology 78, 314–322.

Feng, J., Hwang, R., Chang, K.F., Hwang, S.F., Strelkov, S.E., Gossen, B.D., Conner, R.L., Turnbull, G.D., 2010. Genetic variation in Fusarium avenaceum causing root rot on field pea: Genetic variation in Fusarium avenaceum. Plant Pathol. 59, 845–852.

Filippi, C., Pera, A., 1989. The role of telluric microflora in the control of Fusarium wilt in carnations grown in soils with bark compost. Biol. Wastes 27, 271–279.


Hadar, Y., 2011. Suppressive compost: when plant pathology met microbial ecology. Phytoparasitica 39, 311–314.

Hoitink, H.A., 2006. Compost use for disease suppression, in: Presentation at “Washington Organic Recycling Council 2006 Meeting”. Seattle, WA.

Hoitink, H.A.J., Boehm, M.J., 1999. Biocontrol within the context of soil microbial communities: a substrate-dependent phenomenon. Annu. Rev. Phytopathol. 37, 427–446.

Hoitink, H.A.J., Fahy, P.C., 1986. Basis for the control of soilborne plant pathogens with compost. Annu. Rev. Phytopathol. 24, 93–114.


Jensen, B., Bødker, L., Larsen, J., Knudsen, J.C., Jørnsgaard, B., 2004. Specificity of soil-borne pathogens on grain legumes.


Kuter, G.A., Nelson, E.B., Hoitink, H.A.J., Madden, L.V., 1983. Fungal populations in container media amended with composted hardwood bark suppressive and conducive to Rhizoctonia damping-off. Phytopathology 73, 1450–1456.


83


Lumsden, R.D., Lewis, B.G., Millner, P.D., 1983. Effect of composted sewage sludge on several soilborne pathogens and diseases. Phytopathology 73, 1543–1548.

Nasir, M., Hoppe, H.-H., Ebrahim-Nesbat, F., 1992. The development of different pathotype groups of Mycosphaerella pinocles in susceptible and partially resistant pea leaves. Plant Pathol. 41, 187–194.

Noble, R., 2011. Risks and benefits of soil amendment with composts in relation to plant pathogens. Australas. Plant Pathol. 40, 157–167.


Noble, R., Roberts, S.J., 2003. A review of the literature on eradication of plant pathogens and nematodes during composting, disease suppression and detection of plant pathogens in compost. Published by: The Waste and Resources Action Programme, Banbury, Oxon, UK. 42pp. Oxon-UK Wastes Resour. Action Programme WRAP.

Olofsson, J., 1967. Root rot of canning and freezing peas in Sweden. Acta Agric. Scand. 17, 101–107.



Pflughöft, O., Merker, C., von Tiedemann, A., Schäfer, B.C., 2012. Zur Verbreitung und Bedeutung von Pilzkrankheiten in Körnerfuttererbsen (Pisum sativum L.) in Deutschland. Gesunde Pflanz. 64, 39–48.

Recycled Organics Unit, 2006. Compost use for pest and disease suppression in NSW. The University of New South Wales, Australia.

Schüler, C., Pikny, J., Nasir, M., Vogtmann, H., 1993. Effects of composted organic kitchen and garden waste on Mycosphaerella pinodes (Berk. et blox) Vestergr., causal organism of foot rot on peas (Pisum sativum L.). Biol. Agric. Hortic. 9, 353–360.

Schulte-Geldermann, E., Bruns, C., Hess, J., Finckh, M.R., 2009. Einfluss von ligninhaltigen Komposten und Pflanzgutgesundheit auf den Befall mit Rhizoctonia solani bei Kartoffeln.

Serra-Wittling, C., Houot, S., Alabouvette, C., 1996. Increased soil suppressiveness to Fusarium wilt of flax after addition of municipal solid waste compost. Soil Biol. Biochem. 28, 1207–1214.

Solaiman, Z., Colmer, T.D., Loss, S.P., Thomson, B.D., Siddique, K.H.M., 2007. Growth responses of cool-season grain legumes to transient waterlogging. Aust. J. Agric. Res. 58, 406–412.


Yogev, A., Raviv, M., Hadar, Y., Cohen, R., Katan, J., 2006. Plant waste-based composts suppressive to diseases caused by pathogenic Fusarium oxysporum. Eur. J. Plant Pathol. 116, 267–278.

GENERAL DISCUSSION

84

5. GENERAL DISCUSSION

Seasonal climatic conditions strongly affected overall crop performance of peas, maize and

wheat in the field trials (Chapter 2). The dry summer of 2011 resulted in low maize yields

compared with the two other experimental years. The sudden drop of the temperature in the

first two weeks of February 2012 followed by a warm and dry March resulted in the failure of

the pea crop in 2011/12. The extreme weather events led to enhanced mineralization and

resulted in the relatively high amounts of about 55 kg ha-1 mineral nitrogen in the soil in

spring 2012 available for maize despite the failure of the peas. N inputs in organic systems

are low and catch crops are often used, N leaching is minimized in comparison to

conventional agricultural systems (Hansen et al., 2000). However, in the field trial between

2009 and 2013 N min residues in fall before wheat ranged between 40 and 55 kg ha-1. As

winter wheat does not require more than 17 – 30 kg N ha-1 before spring (Alley et al., 2009),

this means that there was a potential for N-leaching risks leading to N loses in the winter

wheat.

Field application of 5 t dry matter ha-1 of compost did not have any statistically significant

effects on yields of any of the three crops in the rotation (Chapter 2). An exception occurred

in main plot 24 (see Figure 2.7) which is located at the lower end of the experimental field.

Due to the very wet winter temporary water logging occurred in this plot. Field peas are

adversely affected by waterlogging and respond with a decrease of root growth and a decline

in nitrogen fixation (Solaiman et al., 2007). In this plot overall fresh matter production of peas

was low with about 250 g m-2 in the treatment inoculated with Phoma medicaginis and 900 g

m-2 in the non-inoculated control. Compost application resulted in strong increases in yield to

750 g m-2 in inoculated and 1100 g m-2 in the non-inoculated treatments (see Figure 2.7).

This suggests that application of high quality composts was able to minimize the negative

effects of the soil conditions on the pea performance and stabilize the yield.

A complex of different soil borne pathogens was isolated from peas and among them Phoma

medicaginis was dominating. Its incidence was not affected by inoculation, compost

application or seasonal climatic conditions (see Figure 2.10). P. medicaginis has been

reported as an important and commonly found pathogen causing foot rot of peas in Germany

(Pflughöft et al., 2012). In addition, Didymella pinodes, Fusarium oxysporum, Fusarium

solani and Fusarium avenaceum were commonly found on peas. The most aggressive pea

pathogen was F. avenaceum, followed by P. medicaginis, D. pinodes and F. solani. F.

avenaceum was also the pathogen that caused the highest reduction in fresh weights, 65%

compared to the non-inoculated control in pot experiments. These findings are in agreement

GENERAL DISCUSSION

85

with Persson et al. (1997) who reported reduction in pea biomass by 66% in greenhouse

trials. The violet flowering winter pea variety EFB33 used in the field trials usually had only

moderate disease symptoms both in the field and under controlled conditions. An exception

was the field in 2011/12 when the few plants that had survived the frost in February all were

severelly affected by root rot. In contrast, the white flowering spring variety Santana was

highly susceptible to all tested foot rot pathogens under controlled conditions (Chapter 3).

F. avenaceum is a wide host range, opportunistic pathogen that causes damage to plants

weakened by stress (Leslie et al., 1990). In healthy and active soils it is usually suppressed

by the native soil microbiota (Fletcher et al., 1991; Lin and Cook, 1979). F. avenaceum does

not produce chlamidospores and survives in the soil on crop residues (Leslie and Summerell,

2006). Agricultural practices such as reduced tillage, increased glyphosate application,

rotation of susceptible hosts in combination with changing climate and general reductions of

SOM have likely contributed to increased occurrence of F. avenaceum and damaging levels

in Northern Europe and Canada in recent years (Fernandez, 2007; Fernandez et al., 2008;

Foroud et al., 2014; Pflughöft et al., 2012). Moreover, due its genetic and ecological diversity

it can occupy several ecological niches allowing selection for different fitness traits for both

saprophytic and pathogenic phases of its life cycle (Foroud et al., 2014; Holtz et al., 2011).

Forbes and Dickinson (1977) reported that this pathogen is capable of continuous growth in

wet and cool soils which gives it an advantage over other soil microorganisms. It can also

thrive at relatively high partial pressures of carbon dioxide and reduced oxygen leveIs in the

wet soils. Incidence of F. avenaceum in the field in the season 2011/12 was higher than in

the other two years. This was most likely due to the late frost. After the prolonged frost

surface soil layers melted before subsoil resulting in water logging in the upper soil creating

unfavorable conditions for root growth relative to F. aveanceum.

D. pinodes also strongly affected growth of peas. Although it caused only moderate disease

symptoms with mean DI of 4.8, reduction of fresh weight was on average 34% compared to

mean DI of 5.7 due to P. medicaginis causing fresh weight reduction of 20%. Clearly, plant

growth parameters and disease severity need to be considered together in order to correctly

judge the damage potential of pathogens. Fusarium solani has been recognized as one of

the most prevalent and important pathogen of the foot rot complex on peas (Kraft et al.,

1988b; Rush and Kraft, 1986). This pathogen also had a high damage potential in the field

used in these studies as demonstrated by high DI in non-inoculated controls in Chapter 3

and 4. The reisolation from the control plants grown in field soil were dominated by F. solani.

However, under the testing conditions in Chapter 3 and 4, F. solani was the least damaging

pathogen causing low DI and no adverse effects on plant growth. F. solani f. sp pisi is a slow

colonizer of the roots (Huisman, 1982). After penetrating the epidermis, F. solani grows

GENERAL DISCUSSION

86

through the cortex until the Casparian strip and then degradation of the vascular system

starts (Foroud et al., 2014). Any factor that stresses the host root system will shorten the time

between infection and symptom development (Thung and Rao, 1999). The short time of

growing (21 days) and lack of external stress are likely reasons for the lack of symptoms on

peas and adverse effects due to F. solani in the greenhouse tests.

The most important Fusarium spp. isolated from maize and wheat were F. graminearum and

F. culmorum, however they were isolated at much lower frequencies from wheat seedlings. It

is common that many different Fusarium species occur simultaneously in plants and overall

diversity is affected by cultural practices, plant cultivars and weather patterns (Gatch and

Munkvold, 2002; Munkvold, 2003; Osunlaja, 1990; Scauflaire et al., 2011). Besides the

above mentioned F. avenaceum, F. gramineraum and F. culmorum, F. oxysporum, F.

equiseti, F. sambucinum and F. crookwellense were commonly isolated from maize and

wheat. Although these two crops shared a range of Fusarium species, there was no

correlation between isolation frequencies of different Fusarium spp. among them. Therefore,

no conclusion about the risk of increased Fusarium infestation by growing maize before

wheat can be made. However, the high diversity of species isolated from both crops

combined with the fact that Fusarium spp. are soil - and residue - borne pathogens able to

survive for a long time, clearly indicate the potential of the maize – wheat rotation to

contribute to an increase in the diversity and size of the soil borne Fusarium community. On

the long run, considering the changing climate with more frequent extreme weather events

predicted, this can lead to a comparative advantage of the Fusarium community over the

plants in agricultural systems leading to increased disease risk and mycotoxin contamination.

When Santana and EFB33 were grown in non-sterilized field soil, overall disease caused by

the pathogens tested was lower compared to sterilized sand. However, low to moderate

disease symptoms occurred on the non-inoculated soil controls suggesting that natural

inoculum of F. solani was present in the soil used as confirmed by reisolations. Soils used in

the greenhouse trials were collected from the control plots of the field experiments (no

compost application and no inoculation with P. medicaginis) during the early spring while

winter peas were grown. Peas had not been grown in these plots in the last five years prior to

the experiments; however, clover in the mixture with grass was grown for two years in a row,

a year before the field experiments. This could have contributed to the buildup of natural

inoculum in the soil as most of the root rot pathogens of pea are also pathogenic to clovers

(Farr and Rossman, 2015). When EFB33 was grown in the non-sterilized field soil in the

greenhouse trial, disease severity was considerably lower than on Santana just like in all

inoculation trials. This confirms the broad resistance of EFB33 towards wide range of

pathogens from the root and foot rot complex. Violet flowering pea varieties have been

GENERAL DISCUSSION

87

reported to be more resistant to soil-borne diseases, compared to the white flowering

varieties (Vogt-Kaute et al., 2013). However, Vogt-Kaute et al. (2013) could not find a

statistically significant correlation between flower color and susceptibility to foot and root rot

of twenty four pea lines and varieties. From the results it can be concluded that EFB33 could

be a good alternative to the commonly grown spring variety Santana with respect to

resistance foot and root rot. EFB33 should be included in pea breeding programs as it can

bring valuable insights in the pathogen-host interaction and resistance mechanisms.

Inconsistency in the level of compost induced disease suppression between pot and field

experiments has often been reported (Hoitink et al., 1997; Litterick et al., 2004; Noble and

Roberts, 2003) and was also observed in this work (Chapter 2 and 4). Application of yard

waste compost (YWC) did not reduce the severity of foot rot of winter peas or wheat in the

field (Chapter 2); however, different levels of suppression of foot rot of peas were observed

in the greenhouse experiments (Chapter 4). The YWC used suppressed foot disease

caused by F. avenaceum, P. medicaginis, and D. pinodes on the susceptible variety Santana

by reducing the intensity of disease symptoms and promoting plant growth. Although high

amount of compost (30-50% v/v) are recommended for successful disease suppression,

application of 3.5% compost in sterile sand was high enough to achieve suppression of

disease symptoms of all four pathogens. When non-sterilized field soil was used as growing

substrate compost application did not result in significant reduction. The complexity of

disease suppressiveness can be reflected by the various and complex interactions between

pathogens and antagonistic microflora, and between soil abiotic factors and microflora

(Pérez-Piqueres et al., 2006). Complexity of the system is the main reason why it is difficult

to clearly show the beneficial effect of short term compost application on the health and

disease reduction in field grown crops although bioassays under the controlled conditions

have been successful in reducing the disease of the numerous crops caused by the range of

soil-borne pathogens (Bailey and Lazarovits, 2003; Hoitink, 2006; Litterick et al., 2004; Noble

and Coventry, 2005; Termorshuizen et al., 2006). Although we did not observe reduction of

the severity of foot rot of winter peas or wheat in the field trial, a long term field application of

high quality composts might have multiple beneficial effects on overall soil quality that could

help in buildup of soil suppressiveness towards soil-borne diseases. Addition of composts

increases the level of soil organic matter (SOM) which is considered helpful for developing

soil suppressiveness for soil borne diseases as it supports greater microbial biomass and

activity (Stone et al., 2003). In addition, improvement in physical soil characteristics like

water holding capacity, aggregate stability and soil porosity allows plants to develop deep

and well developed root systems that can minimize detrimental effects of the infections with

soil-borne pathogens.

GENERAL DISCUSSION

88

Different mechanisms of suppression were possibly involved in reduction of symptoms

caused by Fusarium spp. and by D. pinodes and P. medicaginis on peas. After γ sterilization

of compost severity of symptoms caused by F. avenaceum and F. solani increased, whereas

in the systems with D. pinodes and P. medicaginis even sterilized compost reduced disease

to some extent. It is likely that suppression of disease caused by Fusarium spp. is biological

in origin and for D. pinodes and P. medicaginis in addition chemical and physical properties

of compost are contributing to the suppression of disease. The disease increase after

application of sterilized compost to field soil was likely due to a low level of native beneficial

microflora in the field soil that was not able to compete with the natural inoculum and the

introduced pathogens for the available food source provided by the sterilized compost.

Pathogens were most likely more efficient in colonizing the sterile compost which resulted in

faster population growth and consequently increase in disease severity.

Due to the complex interactions between site specific factors affecting agricultural production

it is difficult to predict the direct effects of changing climate on the agricultural systems. For

that reason the goal should be to aim at building sustainable agricultural systems that are

resilient to environmental stress. This should be achieved by maintaining active and healthy

soils, increasing above and belowground biodiversity through the long term application of

organic amendments such as compost, green manures or manures in the production system

and introduction of cover crops, living or dead mulches (Abawi and Widmer, 2000).

GENERAL DISCUSSION

89

5.1 References

Abawi, G.S., Widmer, T.L., 2000. Impact of soil health management practices on soilborne pathogens, nematodes and root diseases of vegetable crops. Appl. Soil Ecol. 15, 37–47.

Alley, M.M., Scharf, P., Brann, D.E., Hammons, J.L., 2009. Nitrogen management for winter wheat: principles and recommendations. Va. Coop. Ext. 424, 1–6.


Fernandez, M.R., 2007. Fusarium populations in roots of oilseed and pulse crops grown in eastern Saskatchewan. Can. J. Plant Sci. 87, 945–952.

Fernandez, M.R., Huber, D., Basnyat, P., Zentner, R.P., 2008. Impact of agronomic practices on populations of Fusarium and other fungi in cereal and noncereal crop residues on the Canadian Prairies. Soil Tillage Res. 100, 60–71.

Fletcher, J.D., Broadhurst, P.G., Bansal, R.K., 1991. Fusarium avenaceum : A pathogen of lentil in New Zealand. N. Z. J. Crop Hortic. Sci. 19, 207–210.

Forbes, R.S., Dickinson, C.H., 1977. Behaviour of Fusarium avenaceum in soil growth analysis plates. Trans. Br. Mycol. Soc. 69, 197–205.

Foroud, N.A., Chatterton, S., Reid, L.M., Turkington, T.K., Tittlemier, S.A., Gräfenhan, T., 2014. Fusarium diseases of Canadian grain crops: Impact and disease management strategies, in: Goyal, A., Manoharachary, C. (Eds.), Future Challenges in Crop Protection Against Fungal Pathogens, Fungal Biology. Springer New York, pp. 267–316.

Gatch, E.W., Munkvold, G.P., 2002. Fungal species composition in maize stalks in relation to European corn borer injury and transgenic insect protection. Plant Dis. 86, 1156–1162.

Hansen, B., Kristensen, E.S., Grant, R., Høgh-Jensen, H., Simmelsgaard, S.E., Olesen, J.E., 2000. Nitrogen leaching from conventional versus organic farming systems — a systems modelling approach. Eur. J. Agron. 13, 65–82.

Hoitink, H.A., 2006. Compost use for disease suppression, in: Presentation at “Washington Organic Recycling Council 2006 Meeting”. Seattle, WA.


Holtz, M.D., Chang, K.F., Hwang, S.F., Gossen, B.D., Strelkov, S.E., 2011. Characterization of Fusarium avenaceum from lupin in central Alberta: genetic diversity, mating type and aggressiveness. Can. J. Plant Pathol. 33, 61–76.

Huisman, O.C., 1982. Interrelations of root growth dynamics to epidemiology of root-invading fungi. Annu. Rev. Phytopathol. 20, 303–327.


Leslie, J.F., Pearson, C.A.S., Nelson, P.E., Toussoun, T.A., 1990. Fusarium spp. from Corn, Sorghum, and Soybean Fields in the Central and Eastern United States. Phytopathology 80, 343–350.


Lin, Y.S., Cook, R.J., 1979. Supression of Fusarium roseum “avenaceum” by soil microorganisms. Phytopathology 69, 384–388.


Munkvold, G.., 2003. Epidemiology of Fusarium diseases and their mycotoxins in maize ears. Eur. J. Plant Pathol. 109, 705–713.


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Noble, R., Roberts, S.J., 2003. A review of the literature on eradication of plant pathogens and nematodes during composting, disease suppression and detection of plant pathogens in compost. Published by: The Waste and Resources Action Programme, Banbury, Oxon, UK. 42pp. Oxon-UK Wastes Resour. Action Programme WRAP.

Osunlaja, S.O., 1990. Effect of organic soil amendments on the incidence of stalk rot of maize. Plant Soil 127, 237–241.

Pérez-Piqueres, A., Edel-Hermann, V., Alabouvette, C., Steinberg, C., 2006. Response of soil microbial communities to compost amendments. Soil Biol. Biochem. 38, 460–470.


Pflughöft, O., Merker, C., von Tiedemann, A., Schäfer, B.C., 2012. Zur Verbreitung und Bedeutung von Pilzkrankheiten in Körnerfuttererbsen (Pisum sativumL.) in Deutschland. Gesunde Pflanz. 64, 39–48.

Rush, C.M., Kraft, J.M., 1986. Effects of inoculum density and placement on Fusarium root rot of peas. Phytopathology 76, 1325–1329.

Scauflaire, J., Mahieu, O., Louvieaux, J., Foucart, G., Renard, F., Munaut, F., 2011. Biodiversity of Fusarium species in ears and stalks of maize plants in Belgium. Eur. J. Plant Pathol. 131, 59–66.

Solaiman, Z., Colmer, T.D., Loss, S.P., Thomson, B.D., Siddique, K.H.M., 2007. Growth responses of cool-season grain legumes to transient waterlogging. Aust. J. Agric. Res. 58, 406–412.

Stone, A.G., Vallad, G.E., Cooperband, L.R., Rotenberg, D., Darby, H.M., James, R.V., Stevenson, W.R., Goodman, R.M., 2003. Effect of organic amendments on soilborne and foliar diseases in field-grown snap bean and cucumber. Plant Dis. 87, 1037–1042.


Thung, M., Rao, I.M., 1999. Integrated management of abiotic stresses, in: Singh, S.P. (Ed.), Common Bean Improvement in the Twenty-First Century, Developments in Plant Breeding. Springer Netherlands, pp. 331–370.

Vogt-Kaute, W., Jorek, B., Tilcher, R., 2013. Gibt es bei Körnererbsen Sortenunterschiede in der Anfälligkeit gegenüber bodenbürtigen Krankheiten?

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APPENDIX

APPENDIX

92

Chapter 2

INCIDENCE OF FUSARIUM SPP. IN THE CROP ROTATION WINTER PEA – MAIZE – WINTER WHEAT AND THE POTENTIAL OF YARD WASTE COMPOST TO SUPPRESS FOOT AND ROOT ROT OF PEAS UNDER FIELD CONDITIONS

Figure A. 2.1. Effect of treatments on pea emergence one month after sowing presented as percentage of sowing densities. The error bars represent the ± 1 SD.

Figure A. 2.2. Emergence (Autumn) and winter survival (Spring) of peas as a percentage of sown plants in 2011/12. The error bars represent the ± 1 SD.

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93

Table A. 2.1. Mineral nitrogen (Nmin) content in the soil at 0-60 cm depth. O=Control, I=Inoculated, C=Compost, I+C=Inoculated+Compost.

Peas Before Maize After Maize After Wheat

Rotation I 19.03.2010 26.05.2010 - 02.08.2011

O 35.07 16.90 6.21

I 37.25 25.01 7.53

C 37.06 16.37 7.37

I+C 39.38 15.24 8.92

Rotation II 05.04.2011 16.05.2011 15.10.2011 17.08.2012

O 41.50 32.82 59.79 28.75

I 52.11 34.32 57.89 35.02

C 44.95 28.23 55.40 29.64

I+C 44.96 32.49 55.65 33.22

Rotation III - 26.04.2012 04.10.2012 22.08.2013

O 56.84 43.67 33.13

I 58.57 32.57 31.64

C 49.96 37.06 34.56

I+C 57.38 41.18 31.88

Table A. 2.2. Fresh and dry weights of pea and weeds in 2010 and 2011. O = Control, I = Inoculated, C = Compost, I+C = Inoculated + Compost

Year Fresh weight

pea

Fresh weight weeds Dry weight pea Dry weight

weeds

g m-1 SD g m-1 SD % of total FW g m-1

SD g m-1 SD

2010 O 1410 459 191 75 11 169 45 27 8

I 979 618 203 61 17 124 69 27 7

C 1395 454 170 70 12 173 49 25 10

I+C 1296 437 183 81 12 160 48 26 14

2011 O 914 222 175 86 17 127 29 27 14

I 962 284 169 63 15 115 31 36 17

C 982 276 202 57 15 130 34 32 11

I+C 1025 238 175 90 16 123 25 40 20

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94

Table A. 2.3. F-values for dry matter yields of peas and maize, and grain yield of wheat in field experiment from 2009-2013.

Factor Peas Maize Wheat

Df F - value Df F - value Df F - value

Treatment (T) 3 2.60 1 0.10 1 0.00

Year (Y) 1 13.54*** 2 29.81*** 2 17.90***

Block (B) 3 2.59 3 0.58 3 4.87**

TxY 3 0.83 2 0.39 2 0.27

Significance codes: p < 0.001 ‘***’; p < 0.01 ‘**’; p < 0.05 ‘*’

Figure A. 2.3. Fresh weight per plant of maize in 2010, 2011 and 2012. Different letters indicate significant differences at P < 0.05 (Tukey’s HSD test). The error bars represent the ± 1 SD.

Figure A. 2.4. Thousand grain weights (g) (A) and number of the heads per m- 2 (B) of wheat in treatments with (+ compost) and without compost (- compost) in three experimental years (2011 - 2013). Different letters indicate significant differences at P < 0.05 (Tukey’s HSD test). The error bars represent the ± 1 SD.

a b c

a

b b

A) B)

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95

Table A. 2.4. F-values for the disease index (DI) for root and foot rot of peas in 2010 and 2011.

Factor Df F-values

Treatment (T) 3 0.80

Year (Y) 1 40.97***

Time (Ti) 1 58.78***

Block (B) 3 2.01

TxY 3 1.94

TxTi 3 0.10

YxTi 1 4.85*

TxYxTi 3 0.67


Figure A. 2.5. Incidence (%) of pathogens isolated from pea in different treatments in March 2010 and 2011. O = Control; I = Inoculated with P. medicaginis; C= Compost; I+C= Inoculated with P.

medicaginis + Compost.

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96

Figure A. 2.6. Incidence (%) of pathogens isolated from pea in different treatments in May of three experimental years. O = Control; I = Inoculated with P. medicaginis; C= Compost; I+C= Inoculated with P. medicaginis + Compost.

Figure A. 2.7. Pathogen incidence on pea in March and May of 2010 and 2011. Pm = P.

medicaginis, Dp = D. pinodes, Fs = F. solani and Fo = F. oxysporum.

APPENDIX

97

Figure A. 2.8. External (A) and Internal (B) disease scores of pea in May of all three experimental years. Score 1 stands for healthy plant; score 9 for dead plant. O = Control; I = Inoculated with P. medicaginis; C= Compost; I+C= Inoculated with P. medicaginis + Compost.

A)

B)

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98

Figure A. 2.9. External (A) and Internal (B) disease scores of pea in March and May 2010. Score 1 stands for healthy plant; score 9 for dead. O = Control; I = Inoculated with P. medicaginis; C= Compost; I+C= Inoculated with P. medicaginis + Compost.

A)

B)

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99

Figure A. 2.10. External (A) and Internal (B) disease scores of pea in March and May 2011. Score 1 stands for healthy plant; score 9 for dead plant. O = Control; I = Inoculated with P.

medicaginis; C= Compost; I+C= Inoculated with P. medicaginis + Compost.

B)

A)

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100

Figure A. 2.11. Incidence of different Fusarium spp. isolated from maize (A) and wheat (B) in treatments with (+ compost) and without compost (- compost) from 2009-2013. For wheat n=30 in 2011 and n=20 in 2012 and 2013. For maize n=60 for all three years.

B)

A)

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101

Chapter 3

AGGRESSIVENESS OF FOUR PATHOGENS CAUSING FOOT AND ROOT ROT

OF PEA (PISUM SATIVUM L.) ON A SPRING AND A WINTER PEA CULTIVAR

Table A. 3.1. F-values for DI for isolate comparison of four pathogens in three-way ANOVA.

Factor Df F. avenaceum F. solani P. medicaginis D. pinodes

Variety (V) 1 193.3*** 127.7*** 169.5*** 140.4***

Substrate (S) 1 100.7*** 2.5 235.7*** 5.8**

Isolate (I) 4 4.1*** 0.4 3.2* 5.6***

VxS 1 30.0*** 0.8 15.6*** 0.4

VxI 4 1.5 0.8 0.6 3.3**

SxI 4 1.8 0.4 1.4 1.1

VxSxI 4 0.8 0.4 0.3 0.8

Table A. 3.2. F-values for DI of Santana and EFB33 inoculated with five isolates of four pathogens in sand and soil.


Sand

Variety (V) 1 193.0*** 57.95*** 124.5*** 87.96***

Isolate (I) 4 4.68** 0.7 3.35* 2.48

VxI 4 1.50 0.9 0.5 1.33

Soil

Variety (V) 1 34.57*** 69.73*** 48.77*** 56.79***

Isolate (I) 4 1.36 0.1 0.9 4.02**

VxI 4 1.07 0.4 0.4 2.70*

Table A. 3.3. F-values for fresh weights of Santana and EFB33 grown in sand and soil and inoculated with five isolates of four pathogens.



Sand

Variety (V) 1 19.51*** 86.98*** 41.65*** 16.06***

Isolate (I) 4 21.62*** 1.39 4.04*** 1.25

V x I 4 25.17*** 1.00 1.27 1.09

Soil

Variety (V) 1 31.31*** 53.38*** 15.42*** 24.49***

Isolate (I) 4 0.38 0.97 1.39 0.80

V x I 4 1.22 0.73 0.54 0.90

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102

Table A. 3.4. F-values for dry weights of Santana and EFB33 grown in sand and soil and inoculated with five isolates of four pathogens.



Sand

Variety (V) 1 25.68*** 10.06*** 1.25 0.08

Isolate (I) 4 6.83*** 1.61 9.25*** 5.29***

V x I 4 10.45*** 1.83 2.05* 1.91

Soil

Variety (V) 1 2.32 9.29*** 1.86 0.004

Isolate (I) 4 1.67 1.37 2.55** 3.45***

V x I 4 0.71 2.64** 0.77 0.67

APPENDIX

103

Figure A. 3.1. External lesion scores for all of the isolates of all tested pathogens on Santana.

APPENDIX

104

Figure A. 3.2. External lesion scores for all of the isolates of all tested pathogens on EFB33.

APPENDIX

105

Figure A. 3.3. Fresh weight of Santana and EFB33 plants inoculated with five isolates of F.

solani and grown in sterile sand or non-sterilized field soil. The error bars represent the ± 1 SD.

Figure A. 3.4. Fresh weight of Santana and EFB33 plants inoculated with five isolates of D.

pinodes and grown in sterile sand or non-sterilized field soil. The error bars represent the ± 1 SD.

Santana EFB33

Santana EFB33

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106

Table A. 3.5. F-values for the DI of pea inoculated with four different pathogens in three-way

ANOVA.

Factor Df F-values

Pathogen (P) 3 9.94***

Variety (V) 1 435.77***

Substrate (S) 1 125.25***

PxV 3 6.38***

PxS 3 10.68***

VxS 1 0.03

PxVxS 3 1.32


Table A. 3.6. F-values for DI for pathogen comparison for Santana and EFB33 in sterile sand

and non-sterilized field soil.

Factor Df Santana EFB33

Substrate (S) 1 111.00*** 34.18 ***

Pathogen (P) 3 24.06*** 5.16 **

SxP 3 22.95*** 2.32


Table A. 3.7. ANOVA table and F values for fresh and dry weights per plant of Santana and

EFB33 inoculated with four different pathogens and grown in sterile sand and non-sterilized

field soil.

Df Fresh weight Dry weight

Santana

Pathogen (P) 4 35.09*** 24.39***

Substrate (S) 1 185.47*** 122.68***

PxS 4 12.66*** 8.41***

EFB33

Pathogen (P) 4 13.67*** 11.51***

Substrate (S) 1 166.09*** 129.74***

PxS 4 11.40*** 7.83***

Significance codes: p < 0.001 ‘***’; p < 0.01 ‘**’; p < 0.05 ‘

APPENDIX

107

Table A. 3.8. F-values for DI for Santana and EFB33 inoculated with P. medicaginis and F.

avenaceum and grown under three different temperature regimes.

Df DI

Santana

Pathogen (P) 2 0.012

Temperature (T) 2 6.32**

PxT 2 1.17

EFB33

Pathogen (P) 2 11.69**

Temperature (T) 2 0.27

PxT 2 0.78


Table A. 3.9. F-values for fresh and dry weights for Santana and EFB33 inoculated with P.

medicaginis and F. avenaceum and grown under three different temperature regimes.


Santana

Pathogen (P) 2 29.89*** 36.56***

Temperature (T) 2 72.35*** 26.05***

PxT 2 1.30 1.69

EFB33

Pathogen (P) 2 0.79ns 0.24

Temperature (T) 2 29.06*** 6.74***

PxT 2 1.93 1.54


APPENDIX

108

Figure A. 3.5. Correlation between fresh weight and disease severity of Santana grown in

sterile sand (A) and non-sterilized field soil (B) and inoculated with different pathogens. Only significant regressions are indicated with regression lines.

A)

B)

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109

Figure A. 3.6. Correlation between fresh weight and disease severity of EFB33 grown in sterile

sand (A) and non-sterilized field soil (B) and inoculated with different pathogens. Only significant regressions are indicated with regression lines.

B)

A)

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110

Chapter 4

EFFECTS OF COMPOST APPLICATION ON FOOT AND ROOT ROT OF SPRING AND WINTER PEA VARIETIES CAUSED BY FUSARIUM AVENACEUM, FUSARIUM SOLANI, PHOMA MEDICAGINIS AND DIDYMELLA PINODES

Table A. 4.1. F-values for fresh and dry weights and DI for Santana and EFB33 inoculated with F. avenaceum, F. solani, P. medicaginis and D. pinodes and amended with 20% v/v of non-sterilized and γ sterilized YWC and grown in sterile sand and non-sterilized field soil.

Factor Df Fresh weight Dry weight DI

Pathogen (P) 4 7.89*** 5.98*** 19.21***

Compost (C) 2 148.30*** 71.55*** 12.71***

Substrate (S) 1 17.69*** 0.43 28.24***

Variety (V) 1 90.77*** 6.97*** 175.64***

PxC 4 3.88*** 3.67*** 1.27

PxS 4 12.09*** 4.50*** 21.19***

CxS 2 16.55*** 4.32** 0.00

PxV 4 15.47*** 7.30*** 2.56*

CxV 2 11.61*** 7.78*** 18.16***

SxV 1 1.04 4.34** 0.54

PxCxS 4 3.23*** 2.25** 2.03*

PxCxV 4 3.74*** 3.72*** 1.21

PxSxV 4 11.84*** 8.28*** 2.56*

CxSxV 2 5.55*** 1.55 0.05

PxCxSxV 4 3.19*** 1.16 1.37

Significance codes: p < 0.001 ‘***’; p < 0.01 ‘**’; p < 0.05 ‘*.’

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Figure A. 4.1. Disease index of EFB33 in sand and soil inoculated with F. avenaceum (Fa), F. solani (Fs), P. medicaginis (Pm) and D. pinodes (Dp) and amended with compost (no-without, N-not sterilized and S-sterilized compost). The horizontal line in the boxplot shows the median, the bottom and tops of the box the 25th and 75th percentiles and the vertical lines the minimum and maximum values; outliers as single points. Mean values of DI are marked with triangles.

Figure A. 4.2. Effect of compost application (no-without, N-not sterilized and S-sterilized compost) on fresh weight per plant of EFB33 in sterile sand and non-sterilized field soil inoculated with F. avenaceum (Fa), F. solani (Fs), P. medicaginis (Pm) and D. pinodes (Dp). Different letters within one substrate and one pathogen indicate significant differences in fresh weights

among compost treatments at P < 0.05 (Tukey’s HSD test). The error bars represent the ± 1 SD.

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Figure A. 4.3. Effect of compost application (no-without, N-not sterilized and S-sterilized compost) on dry weight per plant of Santana in sterile sand and non-sterilized field soil inoculated with F. avenaceum (Fa), F. solani (Fs), P. medicaginis (Pm) and D. pinodes (Dp). Different letters within one substrate and one pathogen indicate significant differences in fresh weights


Figure A. 4.4. Effect of compost application (no-without, N-not sterilized and S-sterilized compost) on dry weight per plant of EFB33 in sterile sand and non-sterilized field soil inoculated with F. avenaceum (Fa), F. solani (Fs), P. medicaginis (Pm) and D. pinodes (Dp). Different letters within one substrate and one pathogen indicate significant differences in fresh weights


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Table A. 4.2. F-values for DI, fresh and dry weights of Santana and EFB33 inoculated with four pathogens and amended with two rates of compost (low – 3.5% v/v and high – 20% v/v) and grown in sterile sand in three-way ANOVA.

Factor Df DI Fresh weight Dry weight

Variety (V) 1 143.74*** 55.61*** 5.89***

Pathogen (P) 4 5.08*** 21.51*** 55.43***

Compost (C) 2 28.84*** 8.12*** 3.99**

VxP 4 14.68*** 9.55*** 2.70

VxC 2 3.33** 3.85** 3.79**

PxC 4 2.11 3.73*** 1.06

VxPxC 4 1.68 2.46** 2.16


Table A. 4.3. F-values for DI, fresh and dry weights of Santana and EFB33 inoculated with four pathogens and amended with two rates of compost (low – 3.5% v/v and high – 20% v/v) and grown in sterile sand.


Santana

Pathogen (P) 2 20.12*** 19.67***

Compost (C) 2 13.64*** 9.38***

PxC 2 2.27* 1.34

EFB33

Pathogen (P) 2 5.30*** 12.67***

Compost (C) 2 0.61 0.32

PxC 2 1.57 0.37


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Figure A. 4.5. Effect of compost application rate (no-without, low- 3.5% v/v and high- 20% v/v) on the dry weight per plant of Santana and EFB33 plants inoculated with F. avenaceum (Fa), F.

solani (Fs), P. medicaginis (Pm) and D. pinodes (Dp). Different letters within one variety and one

pathogen indicate significant differences in fresh weights among compost treatments at P < 0.05

(Tukey’s HSD test). The error bars represent the ± 1 SD.

Table A. 4.4. F-values for DI, fresh and dry weights of Santana and EFB33 inoculated with F.

avenaceum and P. medicaginis, amended with 20% v/v YWC and grown under two temperature regimes (low - 16/12°C and high – 19/16°C) in sterile sand in three-way ANOVA.

Factor Df DI Fresh weight Dry weight

Variety (V) 1 139.77*** 22.08*** 2.57

Temperature (T) 1 12.74*** 40.72*** 10.90***

Pathogen (P) 2 188.08*** 53.79*** 21.69***

Compost (C) 1 141.65** 109.60*** 19.80***

VxT 1 0.09 19.20*** 2.62

VxP 2 31.58*** 57.12*** 17.88***

TxP 2 11.93*** 4.23** 0.94

VxC 1 1.79 60.72*** 14.62***

TxC 1 0.37 0.93 0.22

PxC 2 36.59*** 14.23*** 6.77***

VxTxP 2 1.75 5.78*** 0.33

VxTxC 1 0.21 0.37 0.03

VxPxC 2 0.95 3.38** 0.72

TxPxC 2 3.05* 6.88*** 1.98

VxTxPxC 2 1.89 2.61* 0.10


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Figure A. 4.6. Effect of compost application (no-without, compost- 20% v/v) on the dry weights of Santana (A) and EFB33 (B) plants inoculated with F. avenaceum (Fa) and P. medicaginis (Pm) and grown under different temperature regimes (low – 12/16°C, high – 16/19°C). Asterisk

indicates significant differences in fresh weights between two compost treatments within one

pathogen.

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SUMMARY

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Summary

An alternative way to suppress soil borne diseases is the enhancement of microbial activity

e.g. through the use of organic amendments. Composts are the most effective organic

amendment for this purpose. In pot trials successful disease suppression usually follows

application of 30 to 50% v/v of compost to the potting mixes. In contrast to greenhouse trials,

the amounts of compost that can be applied in the field are considerably lower. In organic

farming field application of off-farm compost is limited to 5 t dry matter (DM) ha-1 and year.

This is equivalent to an application of 3.5% v/v in the top 10 cm of the soil. The work

presented was conducted as part of the “Climate change and production of healthy crops -

processes and adaptation strategies by the year 2030” in the frame of the research project

“KLIFF - Climate Impact Research in Lower Saxony". The overall aim was to evaluate soil

health management with a specific focus on soil borne diseases of peas. For that purpose

field, greenhouse, and laboratory experiments were conducted (i) to explore the disease

dynamics in an organic rotation encompassing winter peas as green manure crop followed

by maize and wheat and the potential of improving system health with a repeated compost

application. (ii) To study in detail the pathogens occurring in the field and (iii) to study the

interactions of these pathogens with a suppressive yard waste compost.

The rotation was followed three times encompassing each time two years starting in fall 2009

to 2011 and ending with the wheat harvest 2011 to 2013, respectively. The winter pea variety

EFB33, Maize Fabregas and winter wheat Achat were used. There were four treatments for

the peas. Two were left uninoculated and two were inoculated by distributing oats colonized

with a local isolate of Phoma medicaginis at sowing with the seed (4 kg 150 m-2). In addition,

two treatments received 5 t dry matter (DM) ha-1 of yard waste compost at sowing resulting in

a complete two-factorial. All field experiments were set up as randomized complete blocks

with four replications. Peas were mulched and incorporated in May followed by Maize. Before

sowing the winter wheat the same plots that had received compost before received the same

amount of compost again.

Overall crop performance of peas, maize and wheat was strongly affected by seasonal

climatic conditions but not by experimental treatments. Application of yard waste compost

overall did not reduce the severity of foot rot of peas or wheat. However, it had a positive

effect on the pea biomass in plots affected by water logging in 2010. Phoma medicaginis was

dominating the root rot pathogen complex on peas and its incidence was not affected by

compost application or year. Besides P. medicaginis, D. pinodes, F. oxysporum, F. solani

and F. avenaceum were isolated from peas. Severe foot rot occurred in 2012 after the frost

SUMMARY

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in early February with a 40% incidence of F. avenacuem compared to <10 % in the previous

years.

Pathogens occurring on all three crops were sampled and identified microscopically in all

experimental years. Phoma medicaginis var pinodella, Didymella pinodes, Fusarium solani f.

sp. pisi and Fusarium avenaceum were identified as the most important pathogens of the

root and foot rot complex on peas in the field experiment. More than fifteen Fusarium species

were isolated from maize and wheat root and/or stem bases. The incidence of different

species of the complex differed among years while the spectrum of species did not. In all

three seasons F. graminearum and F. culmorum dominated the pathogen complex on maize

and wheat. F. sambucinum, F. crookwellense, F. equisety, F. proliferatum and F.

verticilioides were commonly found on maize, whereas F. avenaceum, F. oxysporum, F.

equiseti and M. nivale were common on wheat seedlings. There was no obvious relationship

between incidence and frequencies of the Fusarium spp. in peas, maize and wheat either

over the course of a given rotation or within year suggesting that the factors influencing

occurrence of the species may not have been connected to the soil, seed, or plant species

but rather to climatic and potentially other unknown environmental conditions.

P. medicaginis var pinodella, D. pinodes, F. solani f. sp. pisi and F. avenaceum were

assessed for their aggressiveness on the spring pea variety Santana and winter pea EFB33

under controlled conditions. Furthermore, in addition to peas, the pathogenicity of two

isolates of F. avenaceum originating from wheat and pea was determined on eight different

plant species commonly grown in Germany: Maize (Zea mays), Winter wheat (Triticum

aestivum), Oat (Avena sativa), Black oat (Avena strigosa), Subterranean clover (Trifolium

subterraneum), White clover (Trifolium repens), Indian mustard (Brassica juncea) and White

mustard (Sinapis alba). In addition, the potential of Yard Waste Compost (YWC) to suppress

root and foot rot of peas caused by P. medicaginis, D. pinodes, F. solani and F. avenaceum

was studied in greenhouse bioassays at different application rates.

Bioassays under controlled conditions using spore solutions as inoculum showed that the

spring pea variety Santana was considerably more susceptible to all four pathogens tested in

comparison to the winter pea variety EFB33 in both sterile sand and non-sterilized field soil.

In sterile sand F. avenaceum was the most aggressive pathogen, followed by P. medicaginis,

D. pinodes, and F. solani. Aggressiveness of all pathogens was greatly reduced in non-

sterile field soil. Reduction of growing temperatures from 19/16°C day/night to 16/12°C and

13/10°C reduced plant growth and slightly increased disease on EFB33 whereas the highest

disease severity on Santana was observed at the highest temperature, 19/16°C. The two

isolates of F. avenaceum caused severe symptoms on roots of all nine plant species tested.

Especially susceptible were White clover (Trifolium repens), Subterranean clover (Trifolium

SUMMARY

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subterraneum), Indian mustard (Brassica juncea) and White mustard (Sinapis alba) in

addition to peas.

Application of 20% v/v of YWC reduced disease on peas due to F. avenaceum, F. solani, P.

medicaginis and D. pinodes. The intensity of suppression of root and foot rot of peas

depended on pea variety, pathogen and growing media used. As disease severity on EFB33

was overall low due to its high resistance no statistically significant effects of compost

application were observed in sterile sand or non-sterile field soil. In contrast, on Santana

when sterilized sand was used as growing substrate compost application successfully

reduced disease caused by F. avenaceum, P. medicaginis, and D. pinodes. Furthermore,

fresh weight was significantly increased with compost application in all treatments. In non-

sterilized field soil as well Santana as EFB33 were moderately diseased in the uninoculated

controls due to the presence of native inoculum of F. solani. At the same time, overall

disease in the inoculated plants was considerably lower than in the sterile sand suggesting

that a certain degree of natural suppression was occurring in the field soil. In order to

determine if YWC applied at the rate allowed in the field was able to suppress disease in

greenhouse trials under controlled conditions, tests were performed with compost application

rate of 3.5% v/v. Suppression was also achieved with this lower application rate, although

there was a tendency that plants were healthier when substrate was amended with 20% v/v

compost.

To investigate if the cause for the suppressive effects was of biotic origin experiments were

conducted comparing the effects of non-sterile with γ sterilized compost. The suppressive

effect was lost in the pathosystems with Fusarium spp. and sterile compost while in the

systems with D. pinodes and P. medicaginis sterilized compost still decreased disease. This

suggests that different mechanisms of suppression may be involved and that the

suppression of disease caused by Fusarium spp. is biological in origin, whereas chemical

and physical properties of compost are playing an additional role in the suppression of

disease caused by D. pinodes and P. medicaginis. In contrast to sterilized sand when non-

sterilized field soil was used as substrate, compost application did not result in significant

reduction of disease, and in treatments with γ-irradiated compost disease symptoms were

even more severe than without compost application. This suggests that the sterilized

compost may have served as a food base for the native inoculum present in the soil.

In conclusion, the winter pea variety EFB33 can be considered as a good alternative to the

commonly grown spring variety Santana due to its broad resistance towards predominant

root rot pathogens of peas in German conditions. Most field experiments are conducted with

one-time applications of compost. Compost based agriculture will apply compost on a regular

basis, however. Although inconsistency in the compost induced disease suppression

SUMMARY

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between field and greenhouse tests was observed, YWC has the potential to improve pea

health. Longer term regular compost applications in the field should be tested for their

capacity to build resilient soils that are the basis of sustainable agricultural systems.

ACKNOWLEDGMENTS

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Acknowledgments

First and foremost I want to thank my supervisor Professor Dr. Maria R. Finckh for all of her

support, guidance, motivation and contribution of time and ideas, especially during the final

stages of this PhD. I am thankful for the excellent example she has provided as a scientist

and professor. My gratitude goes also to Professor Dr. Gunter Backes for his kind and

valuable advices.

I gratefully acknowledge the project “KLIFF - Climate Impact Research in Lower Saxony" and

the University of Kassel for founding my research. Also, Dr. Tivoli from the National Institute

of Agronomic Research (INRA) France and Prof. Dr. von Tiedemann from the Plant

Pathology Department of the Georg-August-University in Göttingen for kindly providing

isolates of P. medicaginis and D. pinodes used in this work.

I cannot find adequate words to thank enough all my colleagues from the Department of

Ecological Plant Protection for all of their help and assistance during the last five years, but

even more I am grateful for enriching my life with new friends.

I would like to thank Adnan for his friendship and patience through my ups and downs,

especially in the last several months. I am grateful for his company and support and most of

all for making me laugh.

To my dearest friends Marija Pavleska Wood, Marija Kalentić and Miroslava Spernjak I am

eternally grateful for being always there for me, accompanying me through good and bad

times. You are truly the best friends one can wish for.

Lastly, I would like to thank my parents Radmila and Nemanja for their unconditional love

and encouragement. They raised me with a love of science and encouraged me to explore

the world with eyes wide open always having in mind that it is not only the destination that

matters rather the road you take.

Without all of you this would be just a work, but you all made it an experience that I would

cherish forever.

With all of my gratitude,

Jelena

ERKLÄRUNG

121

Erklärung

Hiermit versichere ich, dass ich die vorliegende Dissertation selbstständig, ohne unerlaubte

Hilfe Dritter angefertigt und andere als die in der Dissertation angegebenen Hilfsmittel nicht

benutzt habe. Alle Stellen, die wörtlich oder sinn-gemäß aus veröffentlichten oder

unveröffentlichten Schriften entnommen sind, habe ich als solche kenntlich gemacht. Dritte

waren an der inhaltlich-materiellen Erstellung der Dissertation nicht beteiligt; insbesondere

habe ich hierfür nicht die Hilfe eines Promotionsberaters in Anspruch genommen. Kein Teil

dieser Arbeit ist in einem anderen Promotions- oder Habilitationsverfahren verwendet

worden.

Witzenhausen, 22. Januar 2015

________________________________

Jelena Baćanović

Pathogens occuring in the winter pea - maize

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