Graduate Theses and Dissertations Iowa State University Capstones, Theses andDissertations
2017
Soybean root rot caused by Fusarium oxysporumand Fusarium graminearum: interactions withbiotic and abiotic factorsDavid Ricardo Cruz JimenezIowa State University
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Recommended CitationCruz Jimenez, David Ricardo, "Soybean root rot caused by Fusarium oxysporum and Fusarium graminearum: interactions with bioticand abiotic factors" (2017). Graduate Theses and Dissertations. 15505.https://lib.dr.iastate.edu/etd/15505
Soybean root rot caused by Fusarium oxysporum and Fusarium graminearum:
interactions with biotic and abiotic factors
by
David R Cruz Jimenez
A dissertation submitted to the graduate faculty
in partial fulfillment of the requirements for the degree of
DOCTOR OF PHILOSOPHY
Major: Plant Pathology
Program of Study Committee:
Gary P. Munkvold, Co-Major Professor
Leonor F. S. Leandro, Co-Major Professor
Alison E. Robertson
Daren S. Mueller
Daniel J. Nordman
The student author and the program of study committee are solely responsible for the
content of this dissertation. The Graduate College will ensure this dissertation is globally
accessible and will not permit alterations after a degree is conferred.
Iowa State University
Ames, Iowa
2017
Copyright © David R Cruz Jimenez, 2017. All rights reserved.
ii
TABLE OF CONTENTS
Page
ABSTRACT………………………………. .............................................................. iv
CHAPTER 1 GENERAL INTRODUCTION ...................................................... 1
Dissertation organization ..................................................................................... 1
Literature review .................................................................................................. 1
Thesis justification ............................................................................................... 14
Literature cited ..................................................................................................... 15
CHAPTER 2 ISOLATE x CULTIVAR INTERACTIONS, IN VITRO
GROWTH AND FUNGICIDE SENSITIVITY OF FUSARIUM OXYSPORUM
ISOLATES CAUSING SEEDLING DISEASE ON SOYBEAN ............................. 32
Abstract ......................................................................................................... 32
Introduction ......................................................................................................... 33
Materials and methods ......................................................................................... 36
Results ......................................................................................................... 42
Discussion ......................................................................................................... 46
Literature cited ..................................................................................................... 52
Tables ......................................................................................................... 61
Figures ......................................................................................................... 66
CHAPTER 3 EFFECTS OF TEMPERATURE AND pH ON FUSASRIUM
OXYSPORUM AND SOYBEAN SEEDLING DISEASE ........................................ 73
Abstract ......................................................................................................... 73
Introduction ......................................................................................................... 74
Materials and methods ......................................................................................... 77
Results ......................................................................................................... 81
Discussion ......................................................................................................... 86
Literature cited ..................................................................................................... 94
Tables ......................................................................................................... 105
Figures ......................................................................................................... 107
iii
CHAPTER 4 EFFECTS OF SOIL CONDITIONS ON ROOT ROT OF SOYBEAN
CAUSED BY FUSARIUM GRAMINEARUM........................................................... 114
Abstract ......................................................................................................... 114
Introduction ......................................................................................................... 115
Materials and methods ......................................................................................... 118
Results ......................................................................................................... 122
Discussion ......................................................................................................... 125
Literature cited ..................................................................................................... 130
Tables ......................................................................................................... 139
Figures ......................................................................................................... 141
CHAPTER 5 CONCLUSIONS AND RECOMMENDATIONS ......................... 147
iv
ABSTRACT
Fusarium oxysporum (Fo) and Fusarium graminearum (Fg) are important
components of the Fusarium root rot complex in soybean. Fo is one of the species most
frequently associated with soybean root rot, and Fg isolates that colonize wheat and maize
have been found to be highly pathogenic on soybean, in the United States. Fo and Fg cause
seed decay, damping-off, crown and root rots and pod blight.
The goal of this research was to characterize the biology of Fo and Fg and determine
their role as soybean seedling pathogens in the Fusarium root rot complex. The objectives
were to: i) assess the phenotypic characteristics of Fo isolates from soybean, including the
interaction between Fo isolates and soybean cultivars, growth characteristics in culture, and
sensitivity to fungicides, ii) evaluate the effect of pH and temperature on the development of
soybean root rot caused by Fo, and iii) determine the impact of soil texture, soil pH and soil
water content on seedling disease caused by Fg.
For objective 1, pathogenicity of fourteen Fo isolates was evaluated on eleven
soybean cultivars in rolled-towel and petri-dish assays. Our study revealed that cultivars
differed in susceptibility to Fo, and there were significant isolate × cultivar interactions.
These results suggests that the pattern of resistance or susceptibility for each soybean cultivar
differs among isolates. In addition, soybean cultivars differed in susceptibility to Fo,
illustrating the variability among Fo isolates from soybean and the potential for their
management through cultivar selection.
v
Fo isolates also differed in radial growth on PDA. Pyraclostrobin and trifloxystrobin
effectively reduced conidial germination, and ipconazole effectively reduced fungal growth,
but fludioxonil was ineffective against Fo fungal growth. These results illustrate the
variability among Fo isolates from soybean and the potential for their management through
cultivar selection or seed treatment.
For objective 2, a growth chamber study was performed to assess the effects of pH
and temperature on Fo fungal growth and seedling disease. Fo isolates were grown on
artificial culture media at four pH levels (4, 5, 6, 7, 8), and incubated at four temperatures (15
20, 25, or 30ᵒC). In a rolled-towel assay, seeds were inoculated with a suspension of a
pathogenic or a non-pathogenic Fo isolate. We found that Fo isolates had the greatest radial
growth at pH 6 and 25ᵒC, and caused the most severe root rot at pH 6 and 25ᵒC. In addition, a
Gaussian model was performed to estimate optimal pH and temperature for fungal growth
and disease severity. Optimal conditions estimated using a Gaussian model were pH 6.4 at
27.4 ᵒC for maximal fungal growth, and pH 5.9 at 30ᵒC for maximal root rot severity. These
results indicate that optimal pH and temperature conditions for Fo growth are similar to
optimal conditions for infection and disease in soybean seedlings, and suggest that Fo may
be a more important seedling pathogen when soybeans are planted later, under warm
conditions.
For objective 3, we tested the effect of four artificial soil textures (sand, loamy sand,
sandy loam and loam), two levels of soil pH (6 and 8), and three levels of soil moisture
(permanent wilting point, field capacity and saturation) on root rot of soybean caused by Fg.
We found a significant interaction between soil moisture and soil texture for root rot. The
greatest severity (~70%) was observed at pH 6 and permanent wilting point in sandy loam
vi
soils. In contrast, pot saturation resulted in the lowest levels of disease in sandy loam and
loam soils (11.6 and 10.8%, respectively). Percentages of reduction on seedling growth
parameters relative to the non-inoculated control, such as root length, foliar area, shoot and
root dry weights and root tips were significantly higher in sandy loam soils. In contrast, there
were no relative growth reductions in sandy soils. Our results suggest root disease caused by
Fg increases in water-stressed plants, resulting in detrimental effects on plant development.
1
CHAPTER 1. GENERAL INTRODUCTION
Dissertation Organization
This dissertation is organized in five chapters. The first chapter contains the general
introduction, literature review, and research justification. Chapter two describes the
phenotypic characterization of Fusarium oxysporum isolates collected in Iowa, including the
interaction with soybean cultivars, fungal growth characteristics in culture, and sensitivity to
fungicides. Chapter three describes the effects of pH and temperature on Fusarium
oxysporum fungal growth and soybean seedling disease under growth chamber conditions.
Chapter four is a greenhouse study to determine the effects of soil pH, soil texture and soil
moisture on root rot of soybean caused by Fusarium graminearum. Finally, chapter five
includes general conclusions and recommendations from this research project.
Literature Review
Fusarium oxysporum
Fusarium oxysporum (Fo) is a highly ubiquitous, anamorphic species that infects a
wide range of hosts causing various diseases like vascular wilts, yellows, pre-emergence and
post-emergence damping-off, and root rot. Fo is also considered a common constituent of
fungal communities in the rhizosphere of plants (Fravel et al. 2003). All isolates of Fo are
considered saprophytic since they can survive in the soil organic matter. Moreover, some
2
isolates are pathogenic to plant species and many other isolates do not invade the root tissues
or cause disease (Alabouvette et al. 1993).
Fusarium wilt pathogens present a high level of host specificity, therefore intra-
specific subdivision has been done mainly on the concept of forma speciales (f. sp.) based on
the infected plant host (Laurence et al. 2014).
Fo is very diverse at the species level, with more than 120 f. sp. causing disease on a
wide range of plant families (Michielse and Rep 2009). Fo causes vascular wilts in a wide
variety of economically important crops including banana (Fo f. sp. cubense) (Groenewald et
al. 2006), watermelon (Fo f. sp. niveum) (Wu et al. 2009), asparagus (Fo f. sp. asparagi)
(Reid et al. 2002), beans (Fo f. sp. fabae) (Ivanovic et al. 1987), spinach (Fo f. sp. spinaceae)
(Naiki and Morita 1983), guava (Fo f. sp. psidii) (Gupta et al. 2010), sugar beet (Fo f. sp.
betae) (Webb et al. 2015), carnations (Fo f. sp. dianthi) (Harling et al. 1988), chickpea (Fo f.
sp. ciceris) (Landa et al. 2004), lettuce (Fo f. sp. latucae) (Scott et al. 2010), tomato (Fo f. sp.
lycopersici) (Borrego-Benjumea et al. 2014), cucumber (Fo f. sp. cucumerinum) (Chen et al.
2013), flax (Fo f. sp. lini) (Hoper et al. 1995), muskmelon (Fo f. sp. melonis) (Gordon and
Okamoto 1990), cotton (Fo f. sp. vasinfectu) (Smith and Snyder 1975).
The paucity and variability of morphological characters of the asexual reproductive
structures led to a short definition of Fo that did not reflect the inherent variability of this
species. Molecular tools have provided a new insight in the taxonomic framework and
species classification (Fravel et al. 2003). Even though, the use of molecular tools has
allowed successful identification of pathogenic strains and races, determination f. sp. still
widely relies on bioassays (Gordon and Martyn 1997).
3
Fo sexual stage has never been observed in nature or induced in the laboratory,
therefore is considered to reproduce clonally. Fo has been hypothesized to undergo
recombination besides sexual reproduction, such as parasexual recombination, although this
has not been proven to occur (Gordon and Martyn 1997). Horizontal gene transfer may play
an important role in generating new genetic diversity in Fo (Ma et al. 2010).
One interesting outcome of the genome sequence analysis of Fo f. sp. lycopersici is
that pathogenicity-related genes seem to be non-randomly distributed. Chromosome 14
contains more avirulence genes in a region that is conserved between clonal linages of Fo f.
sp. lycopersici than other genes on other chromosomes, indicating that chromosome 14 has
been subjected to horizontal gene transfer (Michielse and Rep 2009; van der Does et al.
2008).
Fusarium oxysporum species complex (FOSC)
Due to the predominant asexual reproduction of Fo, it is regarded as a species
complex; a collection of clonal lines or isolates within the genus Fusarium. Members of the
FOSC collectively represent pathogenic, saprophytic and non-pathogenic depending on the
interactions they have with host vegetation (Gordon and Martyn 1997). A number of these
Fusarium are also clinically important, since they cause threatening infections in humans and
other animals (O'Donnell et al. 2004b).
Distinguishing species boundaries within the FOSC is challenging due to the lack of
taxonomic characters, its broad geographic distribution, the diverse biology of isolates, and
finally the anthropogenic distribution of pathogens and its influence on fungal evolutionary
aspects (Laurence et al. 2014; O'Donnell et al. 2009).
4
Four clades in the FOSC were identified based on the gene sequence information
from two genes proven to be useful to distinguish among members of the FOSC, the
translation elongation factor (tef1α) and the mitochondrial small subunit (mtSSU). The five
clades were obtained including Fo isolates from a diverse number of plant species lettuce
(Lactuca sativa L.), maize (Zea maiz L), banana (Musa spp.), cotton (Gossypium hirsutum
L.), chickpea (Cicer arietinum L.), soybean (Glycine max (L.) Merr.) and other sources
(Baayen et al. 2000; Ellis et al. 2014; O'Donnell et al. 1998; O'Donnell et al. 2004b).
Fusarium oxysporum in soybeans
There are several Fusarium species occurring on debris and soils samples in soybean
fields, such as F. oxysporum F. acuminatum, F. equiseti, F. moniliforme, F. graminearum,
F. solani, F. semitectum, F. chamydosporium, F. compactum, F. merismoides, and F.
proliferatum (Leslie et al. 1990). Many of these species cause seed and seedling diseases, and
vascular wilts in soybean in vegetative and reproductive developmental stages (Armstrong
and Armstrong 1950; Broders et al. 2007; Díaz Arias et al. 2013a; Díaz Arias et al. 2013b;
Killebrew et al. 1993a). However, Fo is the most common species associated to soybean
roots in Iowa (Díaz Arias et al. 2013b).
One of the first reports of Fusarium root in Iowa was described by Dunleavy in 1953
(Niblack et al. 2002), identifying the causal agent as F. orthoceras. Later, Fo was reported as
the predominant species isolated form symptomatic roots (French and Kennedy 1963). The
causal agents of Fusarium root rot or wilt may act as primary pathogens, or secondary
pathogens that colonize root tissues along with other soilborne pathogens, making it difficult
to diagnose correctly. (Avanzato et al. 2008; Datnoff and Sinclair 1988; Farias and Griffin
1990; French and Kennedy 1963).
5
Soybean symptoms produced by Fo pathogenic isolates include pre and post
emergence damping-off, vascular discoloration, necrosis of cotyledons, water-soaked lesions
on the stems, wilting, and brown and black root rot (Backmand et al. 1993; Ellis et al. 2014;
Nelson 1999).
Formae speciales have not been reported for Fo isolates causing seedling disease and
root rot in soybean (Ellis et al. 2014). However, two other Fo forma speciales from cotton
(Fo. f. sp. vasinfectum, race 2) and cowpea (Fo. f. sp. tracheiphilium, race 1) have been
reported to cause wilt in soybean in the cultivar ‘Yelredo’ (Armstrong and Armstrong 1958).
A genotypic and phenotypic characterization of Fo isolates collected in the
Midwestern and southeastern United States from soybean roots, demonstrated a large genetic
diversity in all five FOSC clades; three of these clades previously described by O’Donell et
al. (2004b). Isolates form Iowa varied from highly aggressive to almost non-pathogenic to
soybean seedling, but there was not a clear association between clades and level of
pathogenicity; except for clade 2, in which many of the non-pathogenic isolates were
classified (Cruz et al. 2013; Ellis et al. 2014).
Fusarium graminearum
Fusarium graminearum (synonym Gibberella zeae (Schwein.) Petch) is an important
pathogen of plant cereal world-wide causing Fusarium head blight of wheat (Triticum
aestivum L.), barley (Hordeum vulgare L.), and oat (Avena sativa L.), and gibberella ear and
stalk rot of corn (McMullen et al. 1997; Xue et al. 2007). In addition to head blight and stalk
rot, F. graminearum is an important seedling pathogen of corn and wheat (Carter et al. 2002;
Jones 1999). F. graminearum has also been reported to be a pathogen in non-cereal crops
such as potato (Solanum tuberosum L.) (Ali et al. 2005), canola (Brassica rapa L.) (Chongo
6
et al. 2001), dry bean (Phaseolus vulgaris L.) (Bilgi et al. 2011), and sugar beet (Beta
vulgaris L.) (Hanson 2006).
Since the year 2000 members of the F. graminearum species complex started being
classified into different linages and are no longer considered as a single species (O'Donnell et
al. 2008; O'Donnell et al. 2004a; Sarver et al. 2011). High phenotypic diversity has been
reported within populations of F. graminearum in the USA and this has been associated with
sexual outcrossing (Walker et al. 2001). F. graminearum commonly produce type B
trichothecene mycotoxins, which are divided into three genotypes 3-acetyldeoxynivalenol (3-
ADON), 15-acetyldeoxynivalenol (15-ADON), or nivalenol (NIV) (Ellis and Munkvold
2014; Ward et al. 2008). Evidence of population subdivision within F. graminearum in the
USA correlates with the trichothecene chemotype, most North American F. graminearum
isolates predominantly produce 15-ADON, and a small number of isolates from Minnesota
and North Dakota produce 3-ADON suggesting a division of the larger population from 15-
ADON (Gale et al. 2003).
Fusarium graminearum in soybeans
F. graminearum is cosmopolitan and has been reported recently as a major
necrotrophic pathogen of soybean (Broders et al. 2007; Xue et al. 2007), causing seed decay,
pre- and post-emergence damping-off, crown and root rot, and pod blight in the United
States, Canada and other temperate regions (Martinelli et al. 2004; Sella et al. 2014; Xue et
al. 2007).
F. graminearum overwinters and colonizes corn residues, providing a source of
primary inoculum for subsequent crops (Sutton 1982; Windels et al. 1988). F. graminearum
has been isolated from various parts of the soybean plants including stems, seeds and roots
7
(Anderson et al. 1988). In the past F. graminearum was not considered to be pathogenic on
soybean (Fernandez and Fernandes 1990; Garcia-Romera et al. 1998). However, subsequent
studies have demostrated that F. graminearum produces soybean seedling diseases in the
USA (Broders et al. 2007; Xue et al. 2007), and soybean pod blight and root rot in south
America (Martinelli et al. 2004).
Most of the soybean production areas in the USA and Canada are used in rotation
with corn or wheat, in combination with reduced-tillage or no tillage systems (Miller et al.
1998). Reduced tillage practices substantially increase F. graminearum inoculum and keep
the soil surface cool and wet, therefore increasing the amount of time required for the seed to
germinate increasing the chances for soybean seedling diseases (Broders et al. 2007; Griffith
et al. 1997; Vandoren and Triplett 1973). Xue et al. (2007) concluded that the emergence of
F. graminearum as a soybean pathogen is may be due to selection pressure for highly
aggressive isolates trought crop rotation.
Even though, the emergence of F. graminearum as a soybean pathogen is linked to
the scarce development and delivery of new cultivars by the industry and the limited
diversity of soybean lines from some companies, disease resistance is promising for F.
graminerum management (Sneller 2003).
The type of disease resistance (innate or basal, qualitative and quantitative) often
depends on the biology of the pathogen (Glazebrook 2005; Hammond-Kosack and Parker
2003; Poland et al. 2009). Due to the recent emergence of F. graminearum as an important
pathogen of soybean there are a few studies characterizing the resistance in this pathosystem;
however, in other pathosystems resistance to F. graminearum is quantitative (Bai et al. 1999;
Gervais et al. 2003; Zhou et al. 2002).
8
Ellis et al. (2012) reported a wide range of reactions in soybean cultivars following
inoculation with F. graminearum, finding cultivar that exhibit high levels of resistance
suggesting that resistance to F. graminearum may be common in soybean and plant breeding
effort sould be focusing on screeninig advance breeding lines.
Fusarium life cycle
The life cycle starts with a saprophytic phase of the chlamydospores surviving in soil
(Gordon and Okamoto 1990). Pathogenic isolates haven been demonstrated to survive up to
25 years in the absence of a susceptible host (Smith and Snyder 1975). Nutrients released
form the growing roots stimulate the dormant chlamydospores or conidia to germinate,
producing hyphae from 6 to 8 hours if conditions are favorable. Fusarium then penetrates the
epidermal cells and gets into the root system, colonizing the root cortex (Beckman and
Roberts 1995). Penetration occurs directly through root epidermal cells or indirectly through
wounds that naturally occur from secondary root development (Nelson et al. 1997; Nelson et
al. 1981).
The fungus spreads intercellularly into the vascular bundle and proliferates into the
xylem. In wilt causing strains, microconidia are transported into the xylem, germinate and
blocks the vessels, producing a significant negative impact on the water economy of the host
plant due to vessel clogging. The combined effect of fungal growth and the production of
plant defenses accentuate the problem resulting in severe wilting and eventual death
(Beckman and Roberts 1995).
In advanced stages of disease, the pathogen keeps growing in the vascular system in
adjacent parenchyma cells, producing vast quantities of conidia. Some Fusarium species
causing root rot specialized in infecting and destroying the cortical tissue of the host (Nelson
9
et al. 1997). In resistant hosts, penetration into root tissues by Fusarium occurs but further
spread is blocked, limiting tissue colonization (Baayen et al. 1989; Harrison and Beckman
1982; Rodriguez-Molina et al. 2003).
Abiotic factors contributing to pathogenesis in Fusarium wilts
Temperature
Optimal growth temperature for Fo has been found between 25 to 28ᵒC, with growth
inhibition above 33ᵒC and below 17ᵒC (Cook and Baker 1983). Fusarium wilts are clearly
influenced by soil temperature; for example, cabbage yellows, flax and tomato wilt are
favored by high soil temperature, whereas and tobacco root rot are conditioned by low soil
temperature (Jones 1924). Most of Fusarium wilts describe a parabolic trend in disease
intensity, indicating that in low and high extremes of temperature symptoms do not develop.
For example, in Fusarium wilt of carnation caused by Fo f. sp. dianthi, stems were highly
colonized with severe symptoms in a range of temperatures between 23-26ᵒC, but remained
symptomless and with very little colonization in a range of temperatures between 14 to 20ᵒC
(Ben-Yephet and Shtienberg 1994).
Temperature and climate also determines the distribution and composition of
Fusarium spp. in soil (Burgess and Summerell 1992). For example, in observational studies
F. compactum occurs only in the warm areas of central and northern Australia, whereas F.
acuminatum is restricted to the southern Australia (Backhouse and Burgess 1995). Similarly,
in a 12-month field study, soils inoculated with five Fusarium species and subjected to
different temperature regimes, displayed significant differences in community and structure
in which F. solani and F. compactum presented a higher propagule density at high
temperatures (25-30ᵒC) (Saremi et al. 1999).
10
Soil moisture
Soil characteristics significantly influence most of the variation in disease intensity
for soilborne pathogens (Dixon and Tilston 2010). Soil temperature and soil moisture have
been recognized as important factors for disease development by Fusarium spp. (Brownell
and Schneider 1985; Cook and Papendick 1972). Literature contains dissimilar results on the
effects of soil moisture on Fusarium wilts, these discrepancies may depend on the Fusarium
species, f. sp., physiological or geographical adaptations of isolates (Burgess et al. 1988;
Jones 1924; Manshor et al. 2012; Walker and White 2005). However, there is strong
evidence suggesting that Fusarium soil populations can be greatly reduced by maintaining
high soil moisture conditions in the absence of a host (Stover 1953).
Soil flooding produces physical, biological and chemical changes in the soil,
anaerobic conditions and alterations in soil structure (Unger et al. 2010), increase the
ammonia concentration, and decrease the nitrate nitrogen levels (Shelton et al. 2000). One or
more of these physiochemical soil changes may play a role in the survival of soilborne
pathogens. Stover (1953), observed significant reductions of Fo, F. graminearum, and F.
moniliforme soil populations increasing soil moisture by 85% of saturation in a loam soil
with the maximum bacterial soil population at 75% of saturation. Therefore, low soil
moisture and light-textured soils with low bacterial populations favored the survival of
Fusarium propagules in the soil. In similar studies, flooding for 15 days with the
incorporation of maize or rice straw into the soil reduced propagules of Fo f. sp. cubense up
to 90% (Wen et al. 2015).
In soybean, one of the first reports of the combined effects of temperature and soil
moisture on Fo was made by French (1963), in which disease severity was dependent on
11
temperature when plants were subjected to saturated infested soils. Roots were healthy form
26 to 32ᵒC, but symptoms developed and were more severe from 14 to 23ᵒC. Soybeans are
sensitive to flooding conditions (Komatsu et al. 2010; Russell et al. 1990). Common
soilborne pathogens recovered from soybean diseased roots under flooding or anoxic
conditions are Pythium spp., Phytophthora sojae, and Rhizoctonia solani (Brown and
Kennedy 1966; Killebrew et al. 1993b; Rao et al. 1978). Conversely, pathogens such as
Fusarium spp. and Macrophomina phaseolina were less frequently associated to root rot
diseases under flood conditions (Kirkpatrick et al. 2006).
Fugal structures such as micro and macroconidia, chlamydospores have outer
protective walls that maintain the cellular water potential above of the external dry
surrounding environment, providing more tolerance to desiccation (Cook and Papendick
1972). Conversely, bacterial cells are less tolerant to low soil water potentials (Marshall
1975). As soil pores dry, water films become thinner and diffusion of substrate molecules
become difficult, reducing the nutrient flux to the bacterial cell surface (Stark and Firestone
1995). In addition, solutes used by the cell to balance internal and external water potentials
may interfere with specific essential biochemical processes (Csonka 1989).
pH
Soil pH influences plant disease development directly on the soilborne pathogen
population and indirectly throughout the availability of nutrients to the plant host (Ghorbani
et al. 2008). In in vitro conditions, fungi grow maximally over a narrow range of pH values,
describing a parabolic trend in which fungal growth is negligible at high and low extremes;
most plant pathogens grow best in media with pH from 5 to 6.5 (Cochrane 1958). In general,
12
acid soil conditions enhance spore germination, mycelial growth and production of
conidiophores (Burpee 1990).
Physiological studies on plant pathogenic isolates in the Fusarium genus indicated
that the most suitable pH for optimal vegetative growth is in the range of 6 and 6.5 (Cochrane
1958; Srobar 1978). However, some Fusarium spp. display optimal growth in acid pH media.
For example, F. graminearum, F. equiseti and F. solani showed optimal growth in pH range
from 3.5 to 4.5 (Agarwal and Sarbhoy 1978). Fusarium soil populations were observed to
survive and grow in soils at a pH of 4.2; however, soil pH close to neutrality negatively
affected this growth (Wilson 1946).
Raising soil pH appears to be a common cultural practice for the control of a number
of Fusarium wilts, which is a disease commonly associated to acidic sandy dry soils (Woltz
and Jones 1981). Comparisons between bacteria and fungi populations in a silty loam soil
with a natural gradient of pH from 4 to 8.3 indicated a fivefold increase in bacterial growth
and a fivefold decrease in fungal growth with high pH (Rousk et al. 2009). In vitro studies
suggest that adverse effects of high pH on fungal populations may be due to microbial
competition and bacterial antibiosis. In addition, bacteria and actinomycetes compete for
nutrients at high pH (Marshall 1960).
Manipulation of the nutrient status of the soil by changing pH has demonstrated the
role of microbial competition for nutrients and mechanisms of fungal soil suppression
(Alabouvette 1999). Iron is an essential element for growth and development of living
organisms, participating in a number of cellular processes including oxygen transport, ATP
generation and detoxification; particularly for fungal pathogens iron is a major virulence
factor (Symeonidis and Marangos 2012).
13
Iron bioavailability is reduced in alkaline soils (Expert 2009). The effect of soil
fungal suppression at a given soil pH depends on the ability of the fungus to acquire iron
compared to its antagonists under iron-limiting conditions (Hoper and Alabouvette 1996).
For example, fluorescent pseudomonas produce high affinity iron siderophores that enhance
the acquisition of iron (Scher and Baker 1980). High pH Fusarium suppressive soils contain
siderophore-producing bacteria that complex iron, making it less available to microflora not
capable of producing efficient iron-transport compounds, suggesting that management of iron
availability in the soil through competition may induce suppressiveness of Fusarium wilt
pathogens (Scher and Baker 1982).
Soil texture
Soil clay content appears to be an important abiotic factor that regulates microbial
communities (Hoper et al. 1995). It has been demonstrated that addition of clay minerals
such as kaolinite, illite or montmorillonite suppress the severity of Fusarium wilts (Hoper and
Alabouvette 1996). Montmorillonite clays increase bacterial metabolic activity against soil
fungi through a pH buffering effect related to a high cationic exchange capacity and rapid
utilization of nutrients by the bacteria (Burpee 1990).
The spread of Fusarium oxysporum f. sp. cubense across Central America is a
well- documented example of the effects of clay in Fusarium spp. Soils with high contents of
montmorillonite clays maintain the fungus excluded by effective bacterial competition.
Conversely, rapid disease spread was observed in soils lacking montmorillonite clays
(Marshall 1975). Combined effects of soil pH and type of clay has been found to be effective
in the suppression of Fusarium wilts, addition of illite in a sandy soil induced disease
suppression of Fusarium wilt of flax in soils with a pH 7 or higher (Hoper et al. 1995).
14
The main physiochemical soil characteristics are completely interrelated. For
example, there is a significant correlation between organic matter content and soil clay
content (Chaussod et al. 1986). Modification of one factor such as clay content may result in
the increase of the cationic exchange capacity, pore sizes, aggregation and level of organic
matte that impacts microbial populations (Amir and Alabouvette 1993). For example,
addition of organic amendments have positive effects in the suppression of Fusarium wilts
(Fang et al. 2012).
Thesis Justification
Fusarium root rot complex of soybean is comprised by numerous Fusarium species
capable of causing different soybean diseases, such as pre and post-emergence damping-off,
root rot, seed rot and wilting of adult plants. Some of the most common Fusarium spp. in the
root rot complex include Fusarium oxysporum and F. graminearum. However, the effects of
abiotic factors such as soil texture, soil pH, soil moisture and temperature associated with
disease development are unknown. The main goal of this research project was to make
efforts to better understand which abiotic factors contribute to root rot development and are
more deserving for further exploration. The results from these studies will contribute to
increase understanding in the biology of F. oxysporum and F. graminearum and their role in
the Fusarium root rot complex, which can help to effectively design management strategies
in the future. The objectives of this research were to:
1. Phenotypic characterize F. oxysporum isolates associated with soybean root from
Iowa.
15
2. Determine the effects of pH ad temperature in the development of soybean root rot
caused by F. oxysporum.
3. Measure the effects of important soil variables in the development of root rot of
soybean caused by F. graminearum.
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toxigenic Fusarium graminearum in North America. Fungal Genet. Biol. 45:473-484.
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32
CHAPTER 2. ISOLATE x CULTIVAR INTERACTIONS, IN-VITRO GROWTH AND
FUNGICIDE SENSITIVITY OF FUSARIUM OXYSPORUM ISOLATES CAUSING
SEEDLING DISEASE IN SOYBEAN
A paper accepted for publication in the journal Plant Disease
D. R. Cruz, M. L. Ellis, G. P. Munkvold and L. F. S. Leandro
Department of Plant Pathology and Microbiology, Iowa State University, Ames. 50011.
Abstract
Fusarium oxysporum Schlechtend:Fr (Fo) is one of the species most frequently
associated with soybean root rot in the United States. Information about pathogenicity and
phenotypic characteristics of Fo populations is limited. The goal of this study was to assess
phenotypic characteristics of Fo isolates from soybean, including the interaction between Fo
isolates and soybean cultivars, growth characteristics in culture, and sensitivity to fungicides.
Pathogenicity of 14 isolates was evaluated in rolled-towel and petri-dish assays. In the rolled-
towel assay, seeds were inoculated with a conidial suspension and rated for disease severity
after 7 days. In the petri-dish assay, Fo isolates were grown on 2% water agar, seeds were
placed on the Fo colony, and seedling disease symptoms were rated. Soybean cultivars
differed in susceptibility to Fo, and there were significant isolate × cultivar interactions
(P≤0.05). Fo isolates differed in their radial growth on PDA at 25ᵒC. Pyraclostrobin and
trifloxystrobin effectively reduced conidial germination with average EC50 of 0.15 and 0.20
33
(µg a.i./ml), respectively. Ipconazole effectively reduced fungal growth with average EC50 of
0.23 (µg a.i./ml), whereas fludioxonil was ineffective against Fo fungal growth. These results
illustrate the variability among Fo isolates from soybean and the potential for their
management through cultivar selection or seed treatment.
Introduction
Fusarium root rot is an important soybean disease in the United States and Canada.
Common symptoms of Fusarium root rot vary from wilting, damping off, slow emergence,
root rot, and vascular discoloration (Nelson 1999). Yield reduction caused by this disease has
been particularly significant when cool and wet conditions are predominant during the
planting season (Farias and Griffin 1990; Wrather and Koenning 2009; Wrather et al. 2001).
In field conditions, Fusarium spp. associated with soybean wilts and root rot show maximum
disease at temperatures between 14 to 23ᵒC (Carson et al. 1991), whereas several Fo isolates
have been reported to grow at a temperature range of 22 to 28ᵒC in vitro (Agarwal and
Sarbhoy 1978; Balasu et al. 2015; Groenewald et al. 2006; Nelson et al. 1990b).
Diverse Fusarium species have been associated with soybean root rot. Although
fungal populations may vary due to geographic and climatic conditions, the most prevalent
and frequent Fusarium species found in roots, soil, or crop residues in a number of surveys
performed across soybean fields in the Unites States were Fo, F. solani (Mart.) Sacc., and F.
graminearum Schwabe (Díaz Arias et al. 2013b; Leslie et al. 1990; Marburger et al. 2015;
Nyvall 1976; Zhang et al. 2010). Fo is composed of diverse cryptic species known as the
Fusarium oxysporum species complex (FOSC), members within this group are responsible to
cause vascular wilts, damping-off and root rots in a wide range of economically important
34
crops (Michielse and Rep 2009; O'Donnell et al. 2009). In previous studies new Fusarium
species have been identified within the FOSC, including F. commune Skovgaard, O’Donnell
et Nierenberg (Skovgaard et al. 2003). In Iowa, Fo isolates associated to soybean seedling
and root rot diseases correspond to five clades within the FOSC.
Soybean isolates within this species complex are genotypically and phenotypically
diverse. Pathogenic capabilities of these isolates in soybeans range from highly pathogenic to
non-pathogenic and saprophytic (Ellis et al. 2014; Laurence et al. 2014). Even though Fo is
the species most commonly associated with soybean root rot, there have been inconsistent
findings about its role and importance in the Fusarium root rot complex. For example, in
greenhouse studies using artificially-infested soils, Fo caused less severe root rot compared
to F. graminearum, F. avenaceum and F. tricinctum (REF). However, Fo produced the
highest seed mortality (Zhang et al. 2010). In artificially-infested microplots, Fo did not
significantly reduce yield but regression analysis performed on individual Fo isolates
indicated a negative correlation between root rot severity and yield (Díaz Arias et al. 2013a).
Similarly other reports estimated reductions up to 59% on yield under very similar field
conditions (Leath and Carroll 1981).
Little information is available about the range of pathogenicity of Fo isolates toward
different soybean cultivars. In one of the first reports about Fo and its interactions with
soybeans cultivars, a differential response in susceptibility was evident across different
soybean cultivars (Leath and Carroll 1981). However, this study was performed only with
one Fo isolate, not addressing the vast diversity of the FOSC. For this reason, it is
imperative to study the biology of FOSC associated with soybeans roots to elucidate its
35
unknown role as a part of the Fusarium root rot complex, and to reveal better management
strategies.
Currently, one the most efficient disease management strategies to control seedling
diseases in soybean is the use of fungicide seed treatments. These are typically used in
circumstances where environmental conditions delay germination and prevent infection by
soilborne pathogens that cause pre- and post-emergence damping-off, seedling blight and
root rot. Although the use of seed treatments on soybeans is a relatively recent practice, it is
becoming more common as many commercial seed treatments include a combination of
nematicides and fungicides (Hartman et al. 1999; Mueller et al. 2013).
Among the most widely used active ingredients, are the quinone outside inhibitors
(QoI), such as trifloxystrobin and pyraclostrobin, which are extensively used on soybeans
and maize in combination with fludioxonil to improve efficacy against a wide range of
soilborne pathogens including Fusarium spp. (Broders et al. 2007a; Munkvold 2009).
However, QoIs have site-specific activity and represent a high risk for development of
resistance (Mueller et al. 2013). Increased insensitivity to QoIs have been reported for
Fusarium spp. in tomato (Chapin et al. 2006), Alternaria alternata in citrus and apple
(Mondal et al. 2005; Reuveni and Sheglov 2002), and Pythium spp. in soybeans (Broders et
al. 2007b).
The objectives of this research were to clarify the importance of Fo as a soybean
seedling pathogen by: (i) studying the reactions of soybean cultivars to different Fo isolates
collected from soybean production fields in Iowa, (ii) examining the impact of commonly
36
used fungicidal active ingredients on conidial survival and radial growth of Fo isolates, and
(iii) comparing radial growth of Fo isolates under optimum temperature conditions.
Materials and Methods
Inoculum and plant source
Fourteen Fo isolates collected from symptomatic soybean roots across Iowa in 2007
(Díaz Arias et al. 2013a) were used in all experiments (Table 1). Isolates originated from
single conidia and were maintained on silica gel beads at 4°C until use. The fourteen isolates
had been tested previously in greenhouse studies and information regarding their
aggressiveness in causing root rot and seedling mortality on the soybean cultivar Asgrow
2403 (SDS susceptible) (Monsanto Co., St. Louis, MO) was available (Díaz Arias et al.
2013a). In addition, the same isolates had also been previously characterized phenotypically
in a rolled towel assay with the susceptible cultivar MN1805 (Ellis et al. 2014).
Each Fo isolate was grown on potato dextrose agar (PDA) for 7-14 days, in an
incubator at 25˚C with a 12 h photoperiod, in order to promote conidial formation. Sterile
distilled water (SDW) was added to the culture which was gently scrubbed with a sterile stick
to collect the macro and/or microconidia. The conidia suspension was passed through sterile
cheesecloth to remove mycelial fragments. Inoculum concentration was calculated by
counting conidia in a hemacytometer (Bright-line Hemacytometer, American Optical,
Buffalo, NY) under the microscope, and conidial suspensions were adjusted to a
concentration of 1×104 conidia/ml.
37
Eleven soybean cultivars were selected for these studies, including the susceptible
cultivar MN1805. These cultivars had been tested previously for resistance to soybean death
syndrome (SDS) and soybean cyst nematode (SCN) under greenhouse and field conditions,
and represented a wide range of resistance and susceptibility to those pathogens across
different soybean maturity groups (Table 2).
Rolled towel pathogenicity assay
Fifteen soybean seeds of the same cultivar were placed equidistantly in a row on a
moist paper towel. Each seed was inoculated with 100 µl of the conidia 1×104 conidia/ml
suspension. After inoculation, seeds were covered with another moist towel to keep them in
place and to provide enough moisture for germination. There were three inoculated and one
mock-inoculated rolls per cultivar. Inoculations were performed with one Fo isolate on
eleven soybean cultivars at a time. The 44 rolls were placed in a 25 L bucket containing 300
ml of distilled water, and incubated for 7 days at room temperature (25˚C). Mock-inoculated
rolls were confined within the bucket with a plastic tray to avoid cross contamination. One
roll was considered as an experimental unit with fifteen seeds or subsamples on each roll.
Each of the fourteen Fo isolates were tested one at a time. The experimental design was a
randomized complete block, in which each bucket was considered as a random factor for the
statistical analysis. The experiment was conducted two times.
Disease intensity was evaluated by two methods as described by Ellis et al. (2011).
Disease severity index (DSI) was calculated as the ratio between the length of discolored
hypocotyl lesion and the total plant length, multiplied by 100. When seeds did not germinate
and were completely colonized, they were assigned a DSI value of 100%. Seedlings were
38
also evaluated using a 1-5 severity scale, in which 1 = germination, no symptoms of disease
or colonization on seedling; 2 = germination, little colonization of the seedling, 1 to 19% of
the root with lesions; 3 = germination, some colonization of seedling, and 20 to 74% of the
root with lesions; 4 = germination, complete colonization of the seedling, and 75% or more
of the root with lesions; 5 = no germination, complete colonization of the seed (Fig. 1). Root
length, root weight and plant length were also measured.
Petri dish pathogenicity assay
A petri dish assay was performed to test pathogenicity of fourteen Fo isolates and
eleven soybean cultivars following a methodology similar to what has been previously
published (Broders et al. 2007b; Muyolo et al. 1993; Zhang and Yang 2000). A 3-mm plug
was transferred from a 7 day-old PDA culture of an isolate to the center of a petri dish
containing 2% water agar, and incubated for 4 days under a 12 h photoperiod at 25˚C. Eight
soybean seeds were surface disinfested in a 0.5% NaOCl solution for 2 min and rinsed twice
with SDW for 2 minutes. Seeds were then placed and spaced evenly around the dish,
approximately 1 cm from the edge of the fungal colony. Plates were incubated at 25˚C for 7
days with a 12 h photoperiod. Disease severity was rated using a 0 to 3 scale proposed by
Broders et al. (2007): 0 = 100% germination with no symptoms of root infection; 1 = 70 to
99% germination with lesion formation on the roots; 2= 30 to 69% germination with
coalesced lesions; and 3 = 0 to 29% germination and all seed tissues were colonized (Fig. 2).
The experimental design was arranged in incomplete blocks with 3 soybean cultivars each,
and cultivars Jack and MN1805 as a control checks on each of the blocks. Therefore, there
were 3 incomplete blocks that included eleven soybean cultivars. For each treatment
39
combination of cultivar and isolate there were three petri dishes and the experiment was
conducted twice. Blocks were considered as random effect for the statistical analysis.
Radial growth on PDA
To compare the growth rate of the fourteen Fo isolates, 5 mm diameter mycelial
plugs of each isolate were aseptically transferred from a 7 day-old PDA culture to the center
of 90-mm diameter PDA petri dishes, and incubated for 4 days at 25˚C, under a 12 h
photoperiod (Balasu et al. 2015; Groenewald et al. 2006). Colony diameter was measured in
two perpendicular directions on each plate, and an average was calculated, after subtracting
5-mm of the colonized plug from the final values. The experiment followed a completely
randomized design with 4 replicate plates per isolate, and the experiment was conducted
twice.
Fungicide sensitivity assays
The fourteen isolates were tested for sensitivity to growth inhibition by the fungicides
ipconazole and fludioxonil. Active ingredients (a.i.) ipconazole (Bayer CropScience) and
fludioxonil (Syngenta Crop Protection) were dissolved in acetone and added to PDA to
obtain final concentrations of 0.01, 0.1, 1, 10, and 100 µg a.i./ml. The non-amended control
contained acetone at 0.1% v/v only. A colonized 5 mm plug was transferred from the original
PDA cultures to 90-mm diam petri dishes with the amended PDA.
Colony diameter was measured after 4 days of incubation at 25˚C with a 12 h
photoperiod, as described above. Percent growth inhibition was obtained by dividing the
diameter of the colonies in the fungicide treatments by the mean colony diameter of the non-
amended control. The experiment followed a completely randomized factorial design with
40
two replicate petri dishes per fungicide x concentration x isolate combination. The
experiment was repeated three times.
Isolates were also tested for sensitivity to conidial germination inhibition by the
fungicides pyraclostrobin and trifloxystrobin. Active ingredients pyraclostrobin (BASF
Corp.) and trifloxystrobin (Bayer CropScience) were dissolved in acetone to prepare stock
solutions of 1 x 104 µg a.i./ml. PDA was amended with each a.i, to obtain final
concentrations of 0.01, 0.1, 1, 10, and 100 µg a.i./ml. Salicylhydroxamic acid (SHAM, Sigma
Aldrich, St. Louis, MO) was dissolved in methanol at a rate of 100 µg a.i./ml to inhibit the
alternative oxidase respiratory pathway. All dilutions, including the non-amended control,
contained both acetone and methanol at 0.1% v/v and SHAM at 100 µg a.i./ml.
Conidia (macro and microconidia) were collected from the pure cultures by adding 10
ml of sterile distilled water per plate and dislodging conidia from the surface of the culture
with a sterile glass rod. Culture suspensions were passed through three layers of cheesecloth
in order to remove mycelial fragments, and conidia concentrations were adjusted to 2.0 × 105
conidia/ml using a hemacytometer (Bright-line Hemacytometer, American Optical, Buffalo,
NY). One hundred µl of the adjusted conidia suspension were added to a single 60mm x
15mm petri dish with PDA. Plates were incubated at 25˚C under continuous light for 18 h,
then placed at 5˚C and immediately scored for germination rate by direct observation under a
light microscope at 100X magnification. One hundred arbitrarily selected conidia per plate
were examined and considered germinated if the germ tube was twice the length of the
conidium. The experiment followed a completely randomized factorial design with three
replicate petri dishes per fungicide x concentration x isolate combination.
41
Data analysis
For the rolled towel assay, the percentage of DSI was arcsin transformed, and root
length, plant length, and root weight were log transformed to meet the assumptions of the
ANOVA. Levene’s test of homogeneity of variance was performed on the transformed data
to compare the two experimental runs for each isolate separately using PROC GLM of SAS
version 9.3 (SAS Institute Inc., Cary, NC). Since there were no significant differences in the
response variables between the two experimental runs, all data were combined.
Subsequently, ANOVA was performed on the transformed data using PROC GLIMMIX. In
addition, averages of DSI and severity for the two experimental runs were used to calculate
linear correlation coefficients using the PROC CORR statement. For the petri dish assay, the
nonparametric disease data from the response variable severity were analyzed using the
PROC GLIMMIX statement of SAS. For the radial growth on PDA, Levene’s tests of
homogeneity and analysis of variance were performed using PROC GLM. Data for the two
experimental runs were combined since there were no significant differences between the
variances. For all experiments, mean separation analyses were performed using a Fisher’s
protected least significant difference (LSD) test at P = 0.05.
EC50s for the growth inhibitors ipconazole and fludioxonil were calculated in a probit
regression analysis. For each Fo isolate, the averaged percentage of growth inhibition on
each dose was converted to a numerical equivalent of the probit function by using the normal
inverse distribution function “NORMINV” in Excel (Microsoft). A linear equation was
obtained from the regression between the logarithm of the doses and their respective probit
values, and the EC50 was calculated to the corresponding dose value where probit was equal
to 0.5. For the conidial germination inhibitors pyraclostrobin and trifloxystrobin, EC50
42
calculations were performed using the PROC PROBIT statement for each Fo isolate. Data
for this analysis were based on the average of the percent inhibition as compared to the non-
amended control.
Results
Rolled-towel assay
The analysis of variance indicated a significant effect for the main factors soybean
cultivar and Fo isolate, as well as a significant cultivar × isolate interaction for DSI (P =
0.0140) and the visual severity scale (P = 0.0083). These significant effects indicate that the
level of disease varied among all cultivars and also the level of aggressiveness differed
among the Fo isolates (Table 3). Significant interactions between cultivar and isolate indicate
that relative susceptibility of soybean cultivars was not uniform among Fo isolates.
Mean separation analysis for the main factors indicated a wide range of
aggressiveness among isolates and susceptibility among cultivars. Not all the isolates were
able to cause disease; in particular, FO36, FO42, and FO46 colonized the seed coat, but no
lesions or fungal colonization were observed in the radicle or hypocotyls. In contrast, poor
germination, fungal colonization of cotyledons and or large lesions on the hypocotyl and
radicle were observed when soybean seeds were inoculated with isolates FO38, FO41, and
FO40, which resulted in higher levels of DSI (Fig. 3A)
Soybean cultivars used in this experiment showed different degrees of susceptibility
when inoculated with Fo. Cultivars Ripley and 299N had little or no symptoms of root rot
43
across all Fo isolates. Conversely, cultivars MN1805 and Jack displayed the highest mean
levels of disease across Fo isolates (Fig. 3B). The DSI ranged from 2.6 % on the soybean
cultivar Ripley when inoculated with isolate FO46 to 60.8% on cultivar MN1805 when
inoculated with isolate FO40.
The visual scale of severity presented results similar to those of the DSI. Visual
severity ratings in the rolled-towel assay also indicated a significant interaction (P = 0.00083)
between cultivars and isolates (Table 3). Furthermore, the two methods for root rot
assessment (DSI and visual rating scale) were linearly related (P < 0.0001) (Fig 5).
Pearson’s correlation coefficients for the two disease evaluation methods were highly
significant for all cultivars (Table 4).
Pathogenic isolates negatively impacted plant development at different magnitudes.
Plant length and root weight were significantly reduced for the plants inoculated with the
highly aggressive isolates (FO38, FO41, FO40) when compared to the mock-inoculated
controls. Conversely, non-pathogenic isolates (FO42, FO46, FO36, FO48), which showed the
lowest levels of DSI, did not significantly affect the total plant length or root weight (Fig 4A,
4C).
Plant length was negatively affected for almost all cultivars when they were
inoculated with pathogenic isolates of Fo, with the exceptions of CM396 and MN1805 (Fig
4B). On the other hand, only a few cultivars showed reductions in root weight or root length
compared to non-inoculated controls; only highly susceptible cultivars H2494 and Jack
exhibited significant reductions in root length (Fig 4D, 4F).
44
Pearson coefficients indicated a significant strong and negative linear correlation
between DSI and plant length. In contrast, plant variables such as root length and root weight
were not strongly correlated with DSI for most of the cultivars (Table 4).
Petri-dish assay
Based on the visual severity scale for the petri dish assay (Fig 2), cultivars varied in
their degree of susceptibility to seed rot, and there was a significant interaction between the
main factors cultivars and isolates (P = 0.0455) (Table 3). Overall, cultivars MN1805 and
MAC02 were the most susceptible, showing poor germination and completely colonized
seeds, with disease severity mean scores of 2.35 and 1.96, respectively. In contrast, cultivars
299N and MYC5171 presented the lowest disease severity mean scores of 1.35 and 1.40,
respectively, with no symptoms or very small dark brown discoloration on seedling roots
(Fig 3D).
Disease levels and type of symptoms varied widely among the isolates. Isolates such
as FO36 and FO42 were almost non-pathogenic, producing small brown lesions on roots or
no symptoms, such as in the cultivars MYC5171 and 299N. In contrast, isolates FO40, FO41
and FO43 were highly aggressive; they rapidly colonized the seed, causing pre-emergence
damping-off. Cultivar MN1805 scored the highest severity average of 2.8 when inoculated
with isolate FO40, and 2.7 with FO41. In addition, MN1805 was given the highest score for
disease severity in 12 out of the 14 Fo isolates.
Radial growth on PDA
Radial growth at 25˚C differed significantly (P < 0.001) among the fourteen Fo
isolates. Radial growth after 4 days varied from 2.3 cm (FO46) to 4.3 cm (FO47). Isolates
45
FO47, FO42, and FO36 had significantly faster growth than the rest of the isolates. Average
diameter among all isolates was 3.34 cm (Fig 6).
Fungicide sensitivity assays
Inhibition in radial growth Fo isolates due to fludioxonil was variable and did not
exhibit a dose response; therefore, EC50 values could not be calculated. Growth inhibition did
not exceed 40% compared to the control (Fig 7). In contrast, ipconazole effectively reduced
the growth of all isolates; at the highest two concentrations (10 and 100 µg a.i./ml),
ipconazole inhibited growth by 97.3% and 99.3%, respectively (Fig 7). Ipconazole EC50
values varied among isolates; however, isolates FO38 and FO41 showed the highest EC50
values (0.747 and 1.667 µg a.i./ml, respectively) (Table 5).
QoIs effectively inhibited conidial germination for all Fo isolates. At the highest
concentrations (10 and 100 µg a.i./ml), trifloxystrobin inhibited germination by 90% and
99%, respectively. Pyraclostrobin performed better than trifloxystrobin for the 10 µg/ml
concentration (94.6% inhibition) (Fig 7). Even though QoIs displayed similar results,
pyraclostrobin was more effective and had a mean EC50 value lower than that of
trifloxystrobin (Table 5). Interestingly, pyraclostrobin EC50 values were high for individual
isolates FO38 and FO41 (0.481, and 0.358 mg a.i./L, respectively), compared to the rest of
the isolates. In addition, FO38 also showed a high EC50 for trifloxystrobin (1.479 µg a.i./ml)
(Table 5).
46
Discussion
This study provides information about the interaction of Fo with soybean cultivars
and describes phenotypic diversity of Fo that may affect its role in the Fusarium root rot
complex. The fourteen Fo isolates collected in Iowa and evaluated in this study displayed a
high degree of variability in aggressiveness, ranging from almost non-pathogenic to very
aggressive. Similarly, soybean cultivars displayed a wide range of susceptibility, with some
cultivars showing only small lesions on seedling tissues and a few other cultivars
experiencing extensive colonization leading to low germination.
Fo is an important component of the Fusarium root rot complex in soybean, and is
usually found in significantly higher frequency than other Fusarium species in soybean roots
(Díaz Arias et al. 2013b; Leslie et al. 1990; Nelson and Windels 1992). However, the role of
Fo as a pathogen has been elusive due to the high degree of variability in disease severity
caused by different strains. Although previous studies have reported significant differences
on susceptibility among cultivars or aggressiveness among Fo isolates, they have included
only one isolate across several cultivars at a time or vice versa. Therefore, conclusions from
these studies were contradictory, proposing Fo as a primary pathogen of soybeans or as a
secondary weak pathogen (Armstrong and Armstrong 1950, 1965; Ferrant and Carroll 1981;
French and Kennedy 1963; Jester 1973; Killebrew et al. 1993; Zhang et al. 2010). Although
we report a great variation in disease intensity as well, these data provide a more complete
overview from the FOSC including interactions among several isolates and soybean
cultivars.
47
In this study, we evaluated different aspects of Fo pathogenicity (root rot and
damping-off), using two methodologies. The rolled towel and petri dish assays provide
information on the ability of the isolates to cause disease at a controlled level of inoculum
and when the seed is in contact with mycelium, respectively. Despite the difference in
methodologies and the high degree of variation in disease levels, many isolates and cultivars
showed consistent levels of susceptibility or aggressiveness in causing both root rot and
damping-off in both assays. However, some isolates were aggressive causing either damping-
off or root rot. This finding emphasizes the value of using assays that measure both root rot
and damping-off, in order to fully understand the impact of specific isolate-cultivar
interactions. More studies are necessary to determine how the interaction between different
isolates may affect the expression of symptoms.
The significant cultivar x isolate interactions suggest that the pattern of resistance or
susceptibility for each soybean cultivar differs among isolates. However, significant
interactions were more attributable to differing magnitudes of disease severity ranges among
cultivars, rather than changes in rank. Additionally, we report here two cultivars (Ripley and
299N) that consistently presented resistance to the isolates used in this study, and may serve
as a future source for plant breeders.
The DSI and the visual scale of severity used in the rolled towel assay to measure
disease intensity had a significant linear relationship suggesting they are similar indicators of
disease across different soybean cultivars. The visual scale of severity could be a quicker
technique to evaluate several cultivars in a short period of time as compared to the DSI.
Although the visual scale is not as precise as the index based on lesion measurement, it was
48
accurate enough to present similar results in the ranking of the less susceptible cultivars and
the most aggressive isolates compared to the DSI.
Correlation analysis between DSI and plant growth characteristics indicated the
negative effects of Fo on seedling health. Plant length was negatively correlated with DSI
and significant reductions in plant length occurred due to Fo inoculation. However, it is
important to note that despite this high correlation, plant length was not indicative of
susceptibility. Some soybean cultivars that exhibited resistance according to the DSI or
visual severity still experienced a significant reduction in plant length. In addition, root
weight and root length were highly variable within and among cultivars; therefore, it was
difficult to observe significant differences between inoculated and control treatments.
Analysis of root weight reductions was, however, more useful when it was performed
by isolate, since it pinpointed the 3 most aggressive isolates (FO38, FO41 and FO40)
compared to the mock-inoculated controls. These results are consistent with Zhang et al.,
(2010) who reported no significant correlations between plant growth variables and root rot
severity and reported that resistant soybean cultivars displayed reductions in fresh root
weight and plant height.
Fo isolates differed in colony morphology and radial growth when growing on PDA
media at 25˚C. In similar previous studies, the optimum growth temperature has been
reported at 25˚C for a vast majority of members of the Fusarium genera and some formae
speciales of Fo from beans, spinach and banana (Groenewald et al. 2006; Ivanovic 1987;
Naiki and Morita 1983; Nelson et al. 1990a). Our data did not suggest any significant
correlation between levels of disease intensity and radial growth. For example, at 25˚C
49
isolates FO41 and FO46 presented the highest and the lowest radial growth respectively and
they are almost non-pathogenic on soybeans; in the same way, aggressive isolates FO41 and
FO40 differed significantly in their relative radial growth. Similarly, temperature in our
pathogenicity assays was close to 25˚C and we obtained a wide range of variability in disease
intensity; thus vegetative radial growth at 25˚C was not an indicator of aggressiveness.
However, it would be interesting to observe radial growth of these isolates at different
temperatures and correlate it to pathogenicity data.
This study reports fungicide sensitivity analyses for Fo isolates obtained from
soybean roots. Inhibition of spore germination by trifloxystrobin and pyraclostrobin indicated
little variability in sensitivity across Fo isolates with EC50 values ranging from 0.005 to 1.49
µg a.i./ml. These findings contrast with previous research showing that F. graminearum
isolates collected from corn and soybean in Ohio have reduced sensitivity to trifloxystrobin;
this fungicide inhibited growth of some isolates only up to 35% of the control at 100 µg
a.i./ml (Broders et al. 2007a).
According to our sensitivity studies ipconazole significantly reduced the growth of all
Fo isolates, these findings agree with previous reports in which ipconazole significantly
reduced Fusarium wilt caused by Fo f. sp. niveum in watermelon field trials (Everts et al.
2014). It has also been shown that ipconazole used in combination with QoIs have positive
effects on emergence in chickpea plants infected with Aschochyta rabiei (Wise et al. 2009).
Fo isolates tested in our studies were insensitive to fludioxonil. These results indicate
that fludioxonil activity varies widely among Fusarium spp. For example, fludioxonil has
been reported to be effective against F. graminearum from soybeans and maize providing
50
effective inhibition of mycelial growth when used as a seed treatment (Broders et al. 2007a;
Ellis et al. 2011; Munkvold and O'Mara 2002). Fludioxonil significantly reduced plant death
in asparagus plants inoculated with Fo f. sp. asparagi and F. proliferatum (Reid et al. 2002),
and improved stands of wheat under field conditions (Mueller et al. 1997). However,
fludioxonil-insensitive isolates in the genus Fusarium have been reported in Canada and
United States (Gachango et al. 2012; Peters et al. 2008; Vignutelli et al. 2002).
Although this is unknown, it is likely that the Fo isolates tested in our studies had
been previously in contact with the active ingredients used in the fungicide sensitivity
analyses. These are widely used active ingredients, and have been used for a considerable
period of time for the control of seedling disease in soybean and maize. Fludioxonil was
introduced as a seed treatment for several crops including soybeans and maize in 1994, and
trifloxistrobin and pyraclostrobin were approved for the same use in 1999 and 2008,
respectively (Munkvold 2009). To the best of our knowledge, there are no previous reports of
sensitivity profiles on Fo isolates associated with soybean roots, and there is a lack of true
baseline sensitivity data. These results serve as a reference for future studies and emphasize
the need to include Fo isolates from other locations and to test these active ingredients on in
vivo trials.
The sensitivity profiles for Fo isolates provided here, in conjunction with previous
research on fungicide sensitivity on other root rot pathogens associated with soybean root rot,
such as Pythium spp. and F. graminearum, will provide insight into effective disease
management strategies. For example, QoIs may provide excellent control for Fo but not for
Pythium spp. or F. graminearum. On the other hand, fludioxonil effectively reduced the
growth of F. graminearum, whereas Fo is insensitive to it. All these differences in fungicide
51
sensitivity, and the diverse number of species that constitute the disease root rot complexes
make it challenging to determine the best combinations of active ingredients to be included
as seed treatments.
In this study we report the effect of fungicides on individual strains on amended
plates, but more information should be added on the direct effect of these molecules on seed
treatments under greenhouse and field conditions at different temperatures. Temperature may
play an important role in the level of efficacy and relative control for this type of assay, as it
has been demonstrated in the case of Pythium spp. (Matthiesen et al. 2016). This knowledge
represents a strong motivation to further investigate the efficacy of these active ingredients at
different temperatures.
FOSC is a genetically diverse group with polyphyletic origins (Ellis et al. 2014). Not
surprisingly, a great deal of variability was observed regarding colony characteristics and
pathogenicity in this study. It is important to remember that soybean seedling diseases and
root rot always appear as disease complexes and, as a consequence, the interactions of the
FOSC with other soil pathogens outside the Fusarium genus may influence the outcomes of
seed treatments. In the future, it would be of great importance to generate information about
the interaction between Fo isolates other genera and spp., not only for pathogenicity or
disease intensity, but also for the implications it may have on fungicide seed treatments.
The most effective strategies to manage Fusarium root rot are crop rotation, high
quality seed, delayed planting and fungicide seed treatments (Nelson 1999). However,
cultivar resistance and pathogenicity information are a key point to explore possibilities to
develop long-term strategies in plant breeding programs (Zhang et al. 2013). For example,
52
host transcriptome profiles have been done for soybean plants inoculated with Fo isolates
FO40 and FO36, identified to be pathogenic and non-pathogenic respectively. Substantial
differences were observed in the gene expression profiles in which FO40 activated stronger
expression of defense-related genes compared to the non-pathogenic FO36, in response to
which there was a very low number of differentially expressed genes (Lanubile et al. 2015).
Pathogenicity information generated in this study along with transcriptome profiles could be
used as a strategy for a more accurate selection of strains that may serve as biocontrol for Fo
such as the soybean non-pathogenic isolate FO36. Although more studies on the effects of
pathogenicity due to the interaction between pathogenic and non-pathogenic isolates must be
done and the mode of actions of biocontrol must be elucidated (Fravel et al. 2003), there is a
good chance that these studies will serve as a first step to develop biological seed treatments
based on the accurate selection of Fo isolates in the near future.
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61
Table 1. Description of F. oxysporum isolates collected from soybeans roots across Iowa in
2007 (Díaz Arias et al. 2013a), and genotypic characterization including mating type loci and
clades within the F. oxysporum species complex (FOSC) (Ellis et al. 2014).
Isolate
ID County
Growth
stagea
Mating
type
FOSC
clade
Type of conidiab
Microconidia Macroconidia
FO36 Lyon V3 MAT1-2 5 P A
FO37 Lyon V3 MAT1-2 5 A P
FO38 Hamilton R1 MAT1-2 5 P P
FO39 Lee V3 MAT1-2 5 P A
FO40 Butler V3 MAT1-1 3 P P
FO41 Jefferson V3 MAT1-1 6 P A
FO42 Lee V3 MAT1-2 2 P P
FO43 Crawford V2 MAT1-2 5 P P
FO44 Winneshiek V2 MAT1-2 5 P A
FO45 Lyon V3 MAT1-2 5 P A
FO46 Crawford V2 MAT1-2 5 A P
FO47 Dickinson V3 MAT1-2 3 P A
FO48 Crawford V2 MAT1-1 3 A P
FO49 Allamakee V3 MAT1-2 6 P A
a Soybean developmental stage at the time of root sampling b Type of conidia produced on PDA after 7 days of incubation in a 12h light period at 25˚C
P present
A absent
62
Table 2. Description of eleven soybean cultivars used to study its interaction to Fusarium oxysporum
*SDS Soybean death syndrome
†SCN Soybean cyst nematode
S susceptible
MS moderately susceptible
MR moderately resistant
PR partially resistant
R resistant
Cultivar Maturity
Group Pedigree
Year
released Developer reference
Disease reactions References
SDS * SCN †
Ripley IV
Hodgson x
V68-1034 1985
Ohio ARDC and
USDA-ARS PR S
(Cianzio et al. 2016; Cooper et al. 1990;
Luckew et al. 2012; Tatalović 2014)
299N II Mycogen Seeds S MS
(Cianzio et al. 2016; Luckew et al. 2012;
Tatalović 2014)
Forrest V
Dyer x
Bragg 1972
Mississippi,Tennessee
AES, and USDA R R (Hartwig and Epps 1973; Tatalović 2014)
MAC02 IV
University of
Missouri S MR (Luckew et al. 2012; Tatalović 2014)
MN1606 I R S (Luckew et al. 2012; Tatalović 2014)
CM396 III R S (Schmidt et al. 2014; Tatalović 2014)
MYC5171 I Mycogen Seeds MS S/MS (Luckew et al. 2012; Tatalović 2014)
Williams
82 III
Williams(7)
x Kingwa 1981
Illinois AES and
USDA S S
(Bernard and Cremeens 1988; Schmidt et
al. 2009; Tatalović 2014)
H2494 II S S (Cianzio et al. 2016; Tatalović 2014)
MN1805 I MR S (Luckew et al. 2012; Tatalović 2014)
Jack II
Fayette x
Hardin 1989 Illinois AES MR R
(Cianzio et al. 2016; Nickell et al. 1990;
Schmidt et al. 2009; Tatalović 2014)
62
63
Table 3. Analysis of variance indicating the effects of soybean cultivars and F. oxysporum isolates on disease severity index
(DSI), visual severity rating, plant length (PL), root length (RL), and root weight (RW).
*DSI was arcsin transformed. Visual scale of severity, plant length, root weight, and root length were log transformed.
Experiments consisted of 3 replications (towels) for each treatment combination and two experiments for the rolled towel assay.
Petri dish assay experiments consisted of 3 petri dishes per treatment combination in an incomplete block design, and using
soybean cultivars Ripley and Jack as a control check for each block. Data are from 11 soybean cultivars (C) and fourteen F.
oxysporum isolates (I).
Rolled towel Petri dish
DSI* Severity PL RL RW Severity
Source df F P > F F P > F F P > F F P > F F P > F F P > F
Cultivar (C) 10 30.98 <0.0001 23.05 <0.0001 15.27 <0.0001 46.95 <0.0001 26.73 <0.0001 30.70 <0.0001
Isolates (I) 13 65.27 <0.0001 126.84 <0.0001 35.67 <0.0001 13.14 <0.0001 9.72 <0.0001 7.27 0.0001
(C×I) 130 1.32 0.0140 1.36 0.0083 1.21 0.0717 1.64 <0.0001 1.14 0.1587 1.24 0.0455
63
64
Table 4. Pearson correlation coefficients performed by soybean cultivar on disease severity
index (DSI), Plant length (PL), root length (RL), and root weight (RW), and visual scale for
severity.
Pearson correlations between DSI (%) and plant variables / Prob
> |r|
Cultivar PL RL RW SEVERITY
Ripley -0.71 -0.44 -0.49 0.88
<0.0001 0.016 0.007 <0.0001
299N -0.61 -0.39 -0.25 0.86
0.0005 0.038 0.189 <0.0001
Forrest -0.72 -0.42 -0.32 0.87
<0.0001 0.023 0.092 <0.0001
MAC02 -0.69 -0.50 -0.36 0.94
<0.0001 0.006 0.056 <0.0001
MN1606 -0.72 -0.63 -0.08 0.95
<0.0001 0.0003 0.654 <0.0001
CM396 -0.73 -0.31 -0.21 0.94
<0.0001 0.098 0.272 <0.0001
MYC5171 -0.41 -0.22 -0.13 0.93
0.027 0.258 0.484 <0.0001
Williams82 -0.68 0.02 -0.08 0.97
<0.0001 0.881 0.659 <0.0001
H2494 -0.79 -0.59 -0.64 0.97
<0.0001 0.0008 0.0002 <0.0001
MN1805 -0.56 -0.48 -0.24 0.87
0.001 0.008 0.218 <0.0001
Jack -0.59 -0.26 -0.08 0.93
0.0008 0.178 0.669 <0.0001
65
Table 5. Fungicide sensitivity analysis (EC50) for fourteen Fusarium oxysporum isolates
recovered from soybeans. EC50 for the a.i. fludioxonil were not calculated since there was no
apparent inhibition of mycelial radial growth with respect to the non-amended control.
Pyraclostrobin Trifloxystrobin Ipconazole
Isolates EC50
Log10 (dose)
EC50
µg a.i./ml
EC50
Log10 (dose)
EC50
µg a.i./ml
EC50
Log10 (dose)
EC50
µg a.i./ml
FO36 -0.777 0.166 -1.193 0.063 -1.125 0.056
FO37 -0.991 0.102 -0.717 0.191 -1.120 0.075
FO38 -0.317 0.481 0.170 1.479 -0.747 0.747
FO39 -1.025 0.094 -1.040 0.091 -1.095 0.080
FO40 -1.392 0.040 -1.331 0.046 -2.109 0.007
FO41 -0.445 0.358 -1.560 0.027 0.222 1.667
FO42 -1.222 0.059 -1.661 0.021 -0.972 0.106
FO43 -1.164 0.068 -1.238 0.057 -1.107 0.078
FO44 -0.758 0.174 -0.519 0.302 -1.099 0.079
FO45 -1.156 0.069 -2.236 0.005 -1.271 0.053
FO46 -0.641 0.228 -1.126 0.074 -1.165 0.068
FO47 -0.990 0.102 -0.521 0.300 -1.248 0.056
FO48 -0.949 0.112 -1.469 0.033 -1.018 0.095
FO49 -0.905 0.124 -0.761 0.173 -1.109 0.077
Average -0.909 0.155 -1.085 0.204 -1.068 0.231
66
Fig 1. Fusarium root rot disease intensity evaluated by an ordinal severity scale 1 =
germination, no symptoms of disease or colonization on seedling; 2 = germination, little
colonization of the seedling, 1 to 19% of the root with lesions; 3 = germination, some
colonization of seedling, and 20 to 74% of the root with lesions; 4 = germination, complete
colonization of the seedling, and 75% or more of the seedling root with lesions; 5 = no
germination, complete colonization of the seed.
67
Fig 2. Fusarium root rot disease intensity evaluated by a visual severity scale 0 = 100%
germination with no symptoms of root infection; 1 = 70 to 99% germination with lesion
formation on the roots; 2= 30 to 69% germination with coalesced lesions; and 3 = 0 to 29%
germination and all seed tissues were colonized.
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Fig 3. Mean Disease severity index (DSI%) (A and B rolled towel assay) and visual severity
rating (C and D petri dish assay) for the main effects of isolate and soybean cultivar for
Fusarium oxysporum isolates from Iowa. Means with same letter are not significantly
different using Fisher’s protected least significant difference (P = 0.05), on arcsin
transformed data for the rolled towel assay. Data from the mock-inoculated seeds were not
included in the analysis.
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Fig 4. Differences in plant characteristics for the main effects of isolate and soybean cultivar.
A and B Plant length (cm). C and D Root weight (g). E and F Root length (cm). * indicates
significant differences from the non-inoculated control (P = 0.05) with a T-test for mean
separation. Comparisons were made individually by isolate and soybean cultivar.
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Fig 5. Relationship between the visual scale for severity and the disease severity index (DSI)
for the rolled towel assay. Each point represents an average of 3 towels containing 15
soybean seeds each.
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Fig 6. Radial growth of fourteen Fusarium oxysporum isolates on PDA after 4 d at 24˚C.
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Fig 7. Inhibitory effects of fungicides on radial growth (fludioxonil, ipconazole) and
conidial germination (trifloxystrobin and pyraclostrobin) with respect to dose concentration
on fourteen Fusarium oxysporum isolates.
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CHAPTER 3. EFFECTS OF TEMPERATURE AND pH ON FUSASRIUM
OXYSPORUM AND SOYBEAN SEEDLING DISEASE
A paper to be submitted to the journal Plant Disease
D. R. Cruz, L. F. S. Leandro and G. P. Munkvold
Department of Plant Pathology and Microbiology, Iowa State University, Ames. 50011.
Abstract
Fusarium oxysporum (Fo) is an important pathogen that reduces soybean yield by
causing seedling disease and root rot. This study the effects of pH and temperature on Fo
fungal growth and seedling disease were assessed. In an in vitro assay, 14 Fo isolates were
grown on artificial culture media at five pH levels (4, 5, 6, 7, 8), and incubated at four
temperatures (15 20, 25, or 30ᵒC). In a rolled-towel assay, seeds were inoculated with a
suspension of a pathogenic or a non-pathogenic Fo isolate. The seeds were placed in rolled
germination paper and the rolls were incubated in all combinations of buffer solutions at four
pH levels (4, 5, 6, 7), and four temperatures (15, 20, 25, or 30ᵒC). There was a significant
interaction between temperature and pH (P < 0.05) for in vitro radial growth and root rot
severity. Isolates showed the most in vitro radial growth after 5 days of incubation at pH 6
and 25ᵒC. For the rolled-towel assay, the pathogenic isolate caused the most severe root rot at
pH 6 and 25ᵒC. Gaussian regression analyses were performed to estimate optimal pH and
temperature for fungal growth and disease severity. Optimal estimated conditions were pH
6.4 at 27.4 ᵒC for maximal fungal growth, and pH 5.9 at 30ᵒC for maximal root rot severity.
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These results indicate that optimal pH and temperature conditions are similar for Fo growth
and disease in soybean seedlings, and suggest that Fo may be a more important seedling
pathogen when soybeans are planted later in the planting season, when soil conditions tend to
be warmer.
Introduction
Fusarium oxysporum Schltdl. (Fo) is one of the fungal species most commonly
isolated from soybean roots in the soybean producing regions of North America (Díaz Arias
et al. 2013a; Ellis et al. 2014; Zhang et al. 2010). Fo has been associated with soybean
damping-off, seed and seedling rot, root rot, and vascular wilt (Armstrong and Armstrong
1965; Datnoff and Sinclair 1988; Díaz Arias et al. 2013a; Díaz Arias et al. 2013b; Leath and
Carroll 1981). Significant differences in root rot severity have been observed among Fo
isolates varying from weakly pathogenic to highly aggressive (Cruz et al. 2013; Díaz Arias et
al. 2013a; Ellis et al. 2014). These variations in disease severity may be influenced by abiotic
factors such as temperature and pH that alter host-pathogen dynamics, making it more
difficult to understand the disease processes involved in soybean root rot development.
However, the effects of abiotic factors on vegetative fungal growth and root rot severity of
Fo on soybeans are unknown.
In the case of plant pathogenic isolates in the Fusarium genus, the most suitable pH
for growth has been reported in the 6 to 6.5 range (Cochrane 1958; Srobar 1978). However,
there are instances in which the greatest pH in vitro growth in Fusarium spp. has been found
in more acidic ranges (Srivastava et al. 2011). For example, F. graminearum and F. equiseti
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grew best at pH 3.5, and F. solani at pH 4.5 (Agarwal and Sarbhoy 1978). In addition, a
number of Fo forma speciales (f. sp.) display a wide range of optimal pH for growth. For
example, isolates from Fo f. sp. cubense grew optimally at pH 6.0 but no growth was
observed at pH 8.0 (Groenewald et al., 2006). Gupta et al., (2010), on the other hand,
observed a maximum growth of Fo f. sp. psidii at pH 5.5.
Maximal in vitro radial growth in Fo plant pathogenic isolates have been found at
temperatures between 25 to 28ᵒC (Brownell and Schneider 1985; Gupta et al. 2010; Marin et
al. 1995; Nelson et al. 1990). However, there are isolates of many other Fusarium spp. and
Fo f. sp. isolates, such as F. beomiforme, F. proliferatum, F. verticillioides, and Fo f. sp.
betae, which display higher temperatures for maximal radial growth (≥30ᵒC) (Miller and
Burke 1985; Nelson et al. 1990; Webb et al. 2015). Although there is evidence of significant
interactions between pH and temperature on fungal radial growth in Fusarium spp. (Chen et
al. 2013; Marin et al. 1995; Schuerger and Mitchell 1992), interactions between these two
factors are not often considered during Fusarium inoculum preparation for research
experiments.
Temperature alters the expression of host defenses mechanisms favoring resistance or
susceptibility reactions (Harling et al. 1988; Stuthman et al. 2007). For example, moderately
resistant or highly resistant cultivars of banana, carnation, chickpea or lettuce, become
susceptible under high temperatures (>25ᵒC) after inoculation with their respective Fo f. sp.
(Brake et al. 1995; Harling et al. 1988; Landa et al. 2006; Scott et al. 2010). The effects of
temperature and pH in the soybean-Fo interaction are unknown, although there is some
understanding of Fo genetic and phenotypic diversity, including the existence of pathogenic
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and non-pathogenic isolates, and variable levels of resistance among soybean cultivars (Cruz
et al. 2013; Ellis et al. 2014; Lanubile et al. 2015).
One approach to understand the interactions of abiotic factors on pathogens is to
model. Gaussian and quasi-Gaussian models are used to describe spatial dispersion of spores
over long distances (Skelsey et al. 2008; Soubeyrand et al. 2008). Gaussian models have
been used in plant pathology to simulate spore wind dispersal of Phytophthora infestans in
potatoes (Spijkerboer et al. 2002), and describe spatial movement of wind-dispersed spores
of Gibberella zeae foci on wheat plots (Paulitz et al. 1999; Prussin et al. 2015). With FO, a
few studies have modeled the effects of the interaction between pH and temperature on
Fusarium wilts caused by Fusarium spp. Polynomial models have been used to describe
temperature and pH effects on Fusarium wilts under greenhouse and hydroponic conditions
in cucumber and mung bean (Chen et al. 2013; Schuerger and Mitchell 1992). Development
of a Gaussian model may be useful to describe how these factors impact disease
development.
The objectives of this research were to evaluate the importance of pH and
temperature in the development of soybean root rot caused by Fo by studying (i) the effects
of the pH and temperature interaction on in vitro radial growth, (ii) the effects of the pH and
temperature interaction on soybean root rot under growth chamber conditions, and (iii) the
differences between a pathogenic and non-pathogenic Fo isolate in causing disease under
different pH and temperature conditions.
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Materials and Methods
Effects of pH and temperature on in vitro radial growth
Fourteen single- spore Fo isolates collected from symptomatic soybean roots in Iowa
in 2007 (Díaz Arias et al. 2013a) were maintained on dry silica beads at 4˚C until use. To
prepare inoculum for the experiments, the Fo isolates were grown on potato dextrose agar
(PDA) at 39g/L (Difco, Becton, Dickinson and Co, Spark, MD, USA) at 25˚C for seven days,
with 12 h photoperiod. A 5-mm plug was punched from the PDA culture and transferred to
the center of a 90 mm diameter petri dish containing differential media with one of five pH
levels (4, 5, 6, 7, 8), and additionally full strength PDA media (pH ~ 6.5).
The media differing in pH levels were prepared as described by Groenewald et al.
(2006). Citrate – phosphate buffer was prepared by titrating 0.1 M citric acid (Armesco,
Ohio, USA) with 0.2 M Na2HPO4 12H2O (Acros, New Jersey, USA) to reach a pH of 4 or 5.
Phosphate buffer was prepared by titrating 0.2 M Na2HPO4 12H2O (Acros, New Jersey,
USA) with 0.2 M NaH2PO4 2H2O (Acros, New Jersey, USA) to reach a pH of 6 or 7. Boric
acid buffer was prepared by titrating 0.2 M of boric acid (Fisher, Illinois, USA) with 0.05M
of sodium tetraborate Na2B4O7 10H2O (Fisher, Illinois, USA) to reach a pH of 8. Once the
buffers for pH 4, 5, 6, 7, and 8 were prepared, a basal medium was added to each liter of
buffer. The basal medium consisted of 45g sucrose (C&H, Crocket, CA, USA), 3g of NaNO3
(Sigma-Aldrich), 1.5g K2HPO4 (Fisher, Illinois, USA), 0.75g MgSO4 7H2O (Sigma-Aldrich),
0.75 KCl (Fisher) and 0.015g FeSO4 7H2O (Fisher, Illinois, USA). Agar (Difco, Becton,
Dickinson and Co, Spark, MD, USA) was added at a rate of 18 g/L to the pH 4 buffer and 15
g/L of agar for pH buffers 5, 6, 7, 8. In addition to the pH differential media a full strength
PDA media at 39g/L (pH ~6.5) was included in the experiments.
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Fo cultures on the pH differential media and full strength PDA media (pH ~6.5), were
incubated at four different temperatures (15, 20, 25, 30˚C) for five days, in an incubator
(Hoffman manufacturing Inc. Oregon, USA) at 65% relative humidity and in the dark.
Diameter of each colony was then measured in two perpendicular directions, and an average
of the two measurements was calculated after subtracting the 5-mm diameter of the colonized
plug. The experiment followed a split-plot design, with temperature as the main plot and
media and Fo isolates as sub-plot factors. Each petri plate was considered as an experimental
unit, and there were four replicate petri dishes per isolate, pH level, and temperature
combination (14 isolates x 6 media x 4 temps x 4 reps = 1,344 experimental units). The
experiment was conducted twice.
Effects of pH and temperature on seedling disease
Fo isolates FO38 and FO42, previously identified as pathogenic and non-pathogenic
to soybeans, respectively, (Cruz et al. 2013; Díaz Arias et al. 2013a; Ellis et al. 2014), were
grown on PDA for 7 days at 25ᵒC, with a 12 h photoperiod, to promote conidial formation.
Macro and microconidia were collected by rinsing the plates with sterile distilled water and
scraping the surface of the cultures with a sterile stick. The suspension was filtered through
sterile cheesecloth to remove mycelial fragments. Inoculum concentration was calculated by
counting conidia in a hemocytometer (Bright-line Hemocytometer, American Optical,
Buffalo, NY) under the microscope, and conidial suspensions were adjusted to a
concentration of 1×106 conidia/ml.
Fifteen seeds of Fo-susceptible cultivar Jack were placed on moist towels and
individually inoculated with 200 µl of the spore solution of FO38 or FO42. Paper towels
were moistened with a buffer solution adjusted to pH levels 4, 5, 6, or 7 using citric acid
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(Amresco, Ohio, USA) and sodium hydroxide (NaOH) (Fisher, Illinois, USA). Towels were
rolled up and placed in 1500 ml glass jars containing 250 ml of the respective buffer and
incubated during 8 days in growth chambers (Hoffman manufacturing Inc. Oregon, USA) at
four different temperatures (15, 20, 25, and 30ᵒC) at 65% relative humidity, and with a 12 h
photoperiod.
Seedlings were rated for root rot using two methods: a disease severity index (DSI)
described by Ellis et al. (2011) and a visual disease severity scale (1-5). Disease severity
index (DSI) was calculated as the ratio between the lesion length (discolored or colonized
hypocotyl and radicle) and the total plant length, multiplied by 100. When seeds did not
germinate and were completely colonized, they were assigned a DSI value of 100%. The
visual 1-5 disease severity scale was assessed according to the following ratings: 1 =
germination, no symptoms of disease or colonization on seedling; 2 = germination, little
colonization of the seedling, 1 to 19% of the root with lesions; 3 = germination, some
colonization of seedling, and 20 to 74% of the root with lesions; 4 = germination, complete
colonization of the seedling, and 75% or more of the root with lesions; 5 = no germination,
complete colonization of the seed. Seedling growth variables (root weight, root length and
shoot length) were also evaluated.
Each towel roll was considered as an experimental unit with fifteen seeds or
subsamples on each roll. There were three inoculated rolls for each of the Fo isolates and
three mock-inoculated rolls for each pH and temperature treatment combination (4 temps x 4
pH x 3 inoculation trts x 3 reps = 144 experimental units). The experiment followed a split
plot design with temperature as the main-plot factor, and pH buffers and Fo isolates as sub-
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plot factors. Treatment combinations of pH levels and isolates were assigned randomly on
each growth chamber. The experiment was conducted twice.
Data analysis
Variances of the raw data for the two experiments of radial growth were compared by
using Lavene’s test for homogeneity under the general linear model procedure (PROC GLM)
of SAS version 9.3 (SAS Institute Inc., Cary, NC). Variances of the two experiments were
not statistically different according to the test (P = 0.95), therefore the raw data for the two
experiments were combined for subsequent analysis. In addition, normal distribution of the
residuals was tested under the PROC UNIVARIATE procedure. Analysis of variance was
performed under PROC GLIMMIX procedure, in which the two experimental replications
and its interaction with temperature were considered as random effects. pH differential media
and Fo isolates and full strength PDA media were considered as fixed factors for the
statistical analysis. Mean separation analyses were performed using a Fisher’s protected least
significant difference (LSD) test at P = 0.05.
The disease severity index (DSI) for the pathogenicity assay was arcsin transformed
and the disease severity scale was square-root transformed. In addition, root weight, root
length and plant length were log transformed. Analysis of variance was performed under the
PROC GLIMMIX procedure in which the two experimental replications and its interaction
with temperature were considered as random effects. pH buffers and Fo isolates were
considered as fixed factors for the statistical analysis. Mean separation analyses on the
transformed data were performed using a Fisher’s protected least significant difference
(LSD) test at P = 0.05.
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Linear correlation coefficients were calculated using the PROC CORR statement on
the average raw data for DSI and the visual scale for severity.
Exponential, polynomial, logistic, Gompertz and Gaussian models were tested to
describe the radial growth and root rot of Fo under the pH and temperature levels selected for
the experiments. Model selection was based on the following criteria described by
Byamukama et al. (2011), low standard error of the estimate (SEE), significant F statistics (P
≤ 0.05) and high coefficients of determination (R2). The model that best described and fit to
the data was selected to obtain parameter estimates.
Gaussian regression analyses for fungal radial growth and disease severity index,
were performed for each individual Fo isolate using SigmaPlot version 13 (Systat Software,
Inc., San Jose, CA) under the following function: .
Estimated values of temperature and pH for maximal fungal radial growth and disease
severity were obtained by using a non-linear optimization procedure (solver add-in) from
Excel (Microsoft) under the following constraints: pH range between 4 and 7 and a
temperature range between 15ᵒC and 30ᵒC.
Results
Effects of pH and temperature on in vitro radial growth
Analysis of variance indicated that main effects of temperature, pH and Fo isolate and
their interactions were highly significant (P < 0.0001) for fungal radial growth. The greatest
mean growth averaged among isolates (5.18 cm) was observed in the combination of 25ᵒC
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and pH 6, and the lowest radial growth (0.96 cm) was observed in the combination of 15ᵒC at
pH 5. No growth was observed at pH 8 for any of the Fo isolates evaluated in this study.
Maximal radial growth was observed at pH 6 at 25ᵒC and 30ᵒC, for isolates FO41 and
FO49 (6.39 cm and 6.30 cm, respectively). Minimal radial growth was observed at 15ᵒC at
pH 4 for isolate FO38 (0.3 cm) (Fig. 1). When mean separation analyses were performed for
radial growth across temperatures for each pH level, growth was significantly higher at 25ᵒC,
followed by 30ᵒC, 20ᵒC and 15ᵒC. This trend was observed across all pH buffer media and on
full strength PDA, which had pH of around 6.5.
There was a strong effect of temperature and isolate on radial growth on full strength
PDA and a significant interaction between these two factors (P < 0.0001). Although
individual isolates had significant differences in their final radial growth, maximum growth
across all isolates occurred at 25ᵒC, except for FO42, which had greater growth at 30ᵒC.
The Gaussian model best described the changes in radial growth across levels of pH
and temperature for all Fo isolates, with significant F statistics ranging from 6.8012 to
37.3336, indicating there was a strong relationship between temperature, pH and radial
growth (Table 1). Coefficients of determination (R2) from the Gaussian regression analyses
varied among isolates, explaining up to 93.1% of the variation in fungal radial growth. In
addition, the Gaussian model had lower standard error of the estimates with values ranging
from 0.3907 to 0.9157 (Table 1), compared to exponential, logistic and Gompertz models
(data not shown).
Estimated values of optimal pH and temperature for maximal radial growth obtained
from the Gaussian regression analysis indicated variation among isolates. pH estimated
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values varied between 5.6 and 6.8, and temperatures varied between 25.4ᵒC and 30ᵒC. A
separate regression was performed on the average data of fungal radial growth across Fo
isolates, the average estimate of pH and temperature for maximal radial growth over all Fo
isolates was 6.3 and 27.1ᵒC, respectively (Table 1) (Fig 2).
Effects of pH and temperature on seedling disease
According to the analysis of variance, pH, temperature and their interaction had
significant effects (P = 0.0005) on DSI. In addition, there was a significant effect of isolate
(P < 0.0001), indicating that disease severity differed between the pathogenic isolate FO38
and the nonpathogenic isolate FO42 (Table 2).
Disease severity index varied across levels of pH and temperature. For the pathogenic
isolate, the highest level of root rot was observed at pH 6 and 30ᵒC (67%), and the lowest
levels of disease severity were observed at 15ᵒC at pH 4 and 5 (4.2%) (Fig. 3A).
Interestingly, when the mean separation analyses were performed across temperatures
separately for each pH buffer media, 30ᵒC and 25ᵒC had significantly higher levels of disease
in all pH levels. Root rot severity at 15 and 20ᵒC displayed the lowest levels of disease for all
pH levels (Fig. 3A).
Disease levels for the non-pathogenic FO42 isolate were significantly lower than the
pathogenic isolate, but showed similar trends. The highest disease severity (17.2%) was
observed at the same level of pH and temperature as the pathogenic isolate; pH 6 at 30ᵒC
(Fig. 3B). Mean separation analysis indicated no differences in disease severity between 15,
20, and 25ᵒC for all pH levels (Fig. 3B).
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Root rot symptoms differed between the pathogenic and non-pathogenic Fo isolates.
Soybean seed inoculations performed with the pathogenic isolate FO38 caused extended
colonization of radicles, hypocotyls, cotyledons and tissue maceration. Subsequently,
germination was lower, and soybean seedlings had poor root and shoot development (Fig.
4A-D). Conversely, inoculations performed with the non-pathogenic isolate FO42 caused a
few small spots of tissue discoloration on the hypocotyls and radicles but tissue structure and
consistency were intact with no maceration or softening (Fig. 4E-H).
Analysis of variance also revealed highly significant effects of isolate for the plant
variables root weight, root length, and plant length (Table 2) (Fig. 5). Mean separation
analysis indicated that the pathogenic isolate FO38 produced significant detrimental effects
on plant length and root weight compared to the mock-inoculated control. Conversely,
overall means for root length and plant length were significantly higher when seeds were
inoculated with the non-pathogenic FO42 isolate when compared to the pathogenic isolate
and to the non-inoculated control.
The highest root weight reductions were observed at 30ᵒC ranging from 35 to 47%,
and the lowest reductions at 15ᵒC, ranging from 8.2 to 22% when seeds were inoculated with
the pathogenic isolate FO38 (Fig. 5A). Conversely, seeds inoculated with the non-pathogenic
FO42 isolate presented significantly lower reductions in root weight (<20%) (Fig. 5B).
The highest root length reductions were observed when seeds were inoculated with
the pathogenic isolate FO38 and incubated at 25ᵒC and pH 5, 6, and 7; ranging from 16 to
21%. Similarly, seeds incubated at 25ᵒC and pH 4 displayed a root length reduction of 20%.
The lowest root length reductions were observed at 15ᵒC, fluctuating from 6 to 8% (Fig. 5B).
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In contrasts, seeds inoculated with the non-pathogenic FO42 isolate showed significantly
lower reductions in root length (<13%) (Fig. 5D).
Plant length reductions were the highest when seeds were inoculated with the
pathogenic isolate FO38 and incubated at 30ᵒC and pH 4 and 5 (25%). The lowest plant
length reductions were observed at 15ᵒC ranging from 4.1 to 9.3% (Fig. 5C). Reductions in
plant length were <9% when seeds were inoculated with the non-pathogenic FO42 isolate,
with a few observations presenting no reductions (Fig. 5F).
The visual scale of severity presented similar results to DSI. The main effects of pH,
isolate, and the interaction between temperature and isolates were highly significant (P <
0.0001). In addition, pH and its interaction with temperature were significant (P = 0.0021)
(Table 2). Disease severity index and the visual scale for severity were linearly related.
Pearson’s correlation coefficients performed separately by Fo isolates were highly significant
(P < 0.0001) indicating a strong positive linear relationship (Fig. 6).
Although there were highly significant differences in the disease intensity caused by
the Fo isolates, the Gaussian regression analysis described with high precision the variation
in disease across pH levels and temperatures. Gaussian regressions performed separately on
each Fo isolate had low standard errors of the estimates, indicating higher precision for
predicted values of disease, compared to other standard models (data not shown). In addition,
regression analysis showed significant F statistics (P < 0.0001), and high coefficients of
determination (R2) explaining 98% and 96% of the variation in disease for the pathogenic
and non-pathogenic isolate, respectively (Table 1).
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Estimated optimal values of pH and temperature for maximal root rot obtained from
the Gaussian regression analysis were equivalent for the pathogenic and non-pathogenic Fo
isolates. Interestingly, estimated values of pH at 5.9 and 30ᵒC produced the highest level of
root rot for both isolates. Under these optimal pH and temperature conditions, the pathogenic
Fo isolate FO38 had a maximal estimated root rot of 64% and the non-pathogenic FO42 had
a maximal estimated root rot of 17%. (Table 1) (Figure 7).
Discussion
This study provides evidence that pH and temperature have significantly effects on
Fo fungal radial growth and severity of Fusarium root rot of soybean seedlings, increasing
our understanding of the epidemiology of the disease. The combination of pH 6 and 25ᵒC
resulted in the fastest in vitro radial growth and most severe root rot development. Using
Gaussian models, the optimal estimated conditions were pH 6.4 at 27.4 ᵒC for maximal
fungal growth, and pH 5.9 at 30ᵒC for maximal root rot severity, showing that Fo growth and
seedling disease are favored by similar conditions.
The results from this study are similar to the findings of earlier reports for in vitro
radial growth of other Fusarium species and formae speciales. For example, Fo f. sp.
cubense displayed a maximum radial growth at pH 6 at 25ᵒC, with a minimum growth at pH
4 (Groenewald et al. 2006). Similar results for optimal fungal growth at 25ᵒC were reported
for F. moniliforme (Shahadat et al. 2015), Fo f. sp. lactucae (Scott et al. 2010), Fo f. sp.
fabae (Ivanovic et al. 1987), and Fo f. sp. spinaciae (Naiki and Morita 1983).
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Our findings were similar to those on Fo f. sp. cubense (Groenewald et al. 2006),
using the same citrate – phosphate buffer media in which the most radial growth was
observed at pH 6 at 25ᵒC. However, in our studies, radial growth was significantly lower on
PDA at pH 6.5 at 25ᵒC compared to the buffered media at pH 6 and 7 at the same
temperature. These results suggest that studies to determine optimal temperature for fungal
growth must be viewed with some caution when PDA is used. Growth of fungi affects the pH
of the media due to the production of secondary metabolites, pigments, absorption of anions
and production of ammonia (Cochrane 1958). These metabolic activities tend to acidify the
medium as has been demonstrated in Fo (Srivastava et al. 2011), making it complex to draw
conclusions when poorly buffered growth media such as PDA is used. If the fungus is able to
change the pH media in order to favor its growth, then conclusions about the optimal pH for
growth could be misleading and a buffered media must be used.
pH is not a unitary factor influencing the mechanisms of growth; other physical
parameters such as temperature, water potential and relative humidity may interact to a
certain degree (Cochrane 1958; Pehrson 1948). Optimal pH for fungal growth can change
along with increasing or decreasing temperatures. For example, optimal pH for growth of
Phacidium infestans on liquid buffered medium decreased with temperature (Pehrson 1948).
This contrasts with our findings on Fo in which the optimal pH remained at 6.0 for growth
across all temperatures.
Fungal growth also depends on genetic background and physiological adaptations to
predominant temperatures of geographical areas (Burgess et al. 1988; Manshor et al. 2012;
Walker and White 2005). Isolates from tropical regions may have higher optimal growth
temperature than isolates from temperate and subtropical regions (Cochrane 1958; Nelson et
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al. 1990). For example, greatest fungal radial growth tends to occur at 25ᵒC for Fo f. sp.
betae isolates collected from temperate regions in the United States (Webb et al. 2015), as
well for Fo f. sp. psiidi and F. solani isolates collected from subtropical regions in India
(Gupta et al. 2010). In contrast, isolates of different Fusarium species occurring in arid and
tropical regions of Australia grow the best at 30ᵒC (Nelson et al. 1990; Sangalang et al.
1995). It would be interesting to include Fo isolates from tropical regions and compare them
to those from Iowa in future pH and temperature studies.
Optimal pH and temperature conditions for soilborne plant diseases may or may not
mimic optimal conditions for in vitro radial growth (Cochrane 1958). Our results showed the
highest root rot severity occurred at pH 6 at 30ᵒC, followed by pH 5 at 25ᵒC. These values
correspond to the highest in vitro radial growth, suggesting that pH and temperature have
similar effects on Fo growth and soybean root rot severity caused by Fo.
Our results on root rot severity are similar to those reported for Fusarium root rot in
other Fusarium species, such as Fo f. sp. dianthi, cucumerinum, cubense, ciceris, spinaceae,
phaseoli, and lactucae, and F. graminearum, (Ben-Yephet and Shtienberg 1994; Chen et al.
2013; Ellis et al. 2011; Groenewald et al. 2006; Landa et al. 2006; Naiki and Morita 1983;
Schuerger and Mitchell 1992; Scott et al. 2010), in which the highest disease severity was
observed at temperatures between 25 to 30ᵒC. Interestingly, the majority of these species
were predominantly from humid subtropical climatic zones.
Our results indicate that pathogen fungal growth and root rot severity temperature
curves have similar trends and follow each other closely. Similarities in these curves might
indicate that disease development and severity are mainly influenced by the effect of
temperature on the pathogen (Dickson 1923; Jones 1924).
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In addition to the slower growth of Fo at lower temperatures, soybean seedling
resistance to Fo may be increased. Low temperatures have been reported to favor plant
defenses mechanisms making soybean seedlings more resistant to Fo. There is evidence of
overexpression of resistance related proteins (esterase and PR-2) after soybean cultivars were
inoculated with pathogenic Fo isolates at low temperatures. Fo-inoculated seedlings
incubated at 8ᵒC had higher 1,3-β-glucanases activity compared to the seedlings incubated at
24ᵒC (Koretsky 2001). Therefore, low temperatures may favor resistance to Fo on soybeans,
agreeing with our observations of low root rot severity and low percentage of reduction in
seedling growth variables, such as root weight, root length and plant length, at 15 and 20ᵒC
under growth chamber conditions. Root rot at temperatures between 25 to 30ᵒC may be
influenced by a lack of soybean resistance, which, combined with faster growth and
colonization of Fo at its optimal growth temperature, resulted in more severe root rot.
However, these temperature dependent defense mechanisms need further study.
Our findings regarding optimal temperature for disease development conflicts with
some greenhouse and field observations in which Fusarium root rot of soybean occurs at low
temperatures (<18ᵒC) and disease severity was reduced when temperature increases from 26
to 32ᵒC in Fo infested saturated soils (Carson et al. 1991; Farias and Griffin 1990; French
1963). A possible reason for these conflicting results is the variability in soil temperature and
soil moisture under field conditions. For example, in Iowa, soybean is planted in late April or
early May and emergence usually takes between 7 to 14 days when average soil temperatures
are 10ᵒC (De Bruin and Pedersen 2008); seedling disease risk increases before emergence if
temperature drops and soils are wet. In our experiments, seeds were maintained at a constant
temperature and incubated in soilless pH media for only 8 days making it difficult to
90
compare with the variable temperatures that occur in the field. Another factor that may
explain these conflicting results is the fact that lower soil temperatures and high soil moisture
are associated with no-till soybean production, which may result in greater inoculum survival
that favors seedling root rot (Bockus and Shroyer 1998). Furthermore, seedling disease under
cool, moist conditions is most often due to other pathogens, such as Pythium spp. (Rizvi and
Yang 1996).
The ideal soybean germination and emergence temperatures are between 25 to 30ᵒC
(Hatfield and Egli 1974). However, these temperatures only occur during late-May or early-
June (4 or 5 weeks after planting) when soybean plants are already in active vegetative
growth (V3-V5) (Licht et al. 2013). In the field in Iowa, soybean planting would rarely take
place when soils are at 25 or 30ᵒC. However, in other regions, especially where soybean is
double-cropped with winter cereals, temperatures in this range may be common at planting
(Dillon 2014). It is important to observe the effect of various temperatures on disease
development at early soybean stages. Likewise, these observations could lead to a new
research to describe root rot disease development in relation to soybean growth stages.
Fo is an important component of the Fusarium rot root complex in soybeans, which
includes other Fusarium spp. such as F. graminearum, F. pseudograminearum, F. commune,
F. proliferatum, F. redolens, and F. solani (Broders et al. 2007; Díaz Arias et al. 2013a; Ellis
et al. 2013; Ellis et al. 2014; Killebrew et al. 1993; Skovgaard et al. 2003). In addition,
Pythium and Rhizoctonia spp. are primary soybean pathogens and may interact with the
members of the Fusarium root rot complex to cause disease (Datnoff and Sinclair 1988;
Pieczarka and Abawi 1978). The effect of interaction among these species and soil microbial
populations on Fusarium root rot is still not well understood.
91
Our findings of the effect of pH on fungal growth and disease severity agreed with a
number of studies on Fusarium spp. in which slightly acidic growth media at around pH 6
promotes fungal growth and sporulation (Agarwal and Sarbhoy 1978; Cochrane 1958;
Groenewald et al. 2006; Gupta et al. 2010; Wu et al. 2009). According to our results, it
appears that there were inhibitory effects on disease severity at pH 4 and 7. Given the results
of this and other pH studies performed under similar conditions (Schuerger and Mitchell
1992), we propose that under growth chamber conditions, root rot severity is mainly
influenced by effects of pH and temperature on fungal growth, combined with the effect of
temperature in host resistance.
Interestingly, alkalizing soil amendments to rise soil pH may result in a transitory or
partial suppression of Fusarium wilts by reducing fungal biomass and availability of iron to
the pathogen (Gatch and du Toit 2017; Hoper et al. 1995; Peng et al. 1999). On the other
hand; in the case of soybeans, high soil pH levels could lead to iron-deficiency chlorosis, high
soybean cyst nematode infections, and more severe root rot symptoms, resulting in yield
losses (Charlson et al. 2004; Chen et al. 2007; Hansen et al. 2004; Naeve 2006; Rogovska et
al. 2007). Soil pH as a management strategy for root rot of soybeans is still difficult to
elucidate, since optimal soil pH from 6.2 to 7 is important for root nodulation (Hartman et al.
1999). However, the evidence that none of the Fo isolates grew at pH 8 could provide a
motivation to explore the effects of alkaline soils on root rot of soybeans under greenhouse
and field conditions.
Disease severity and root rot symptoms varied significantly between the pathogenic
and non-pathogenic Fo isolates. Lanubile et al. (2015) demonstrated significant differences
in soybean defense gene expression profiles in response to pathogenic and non-pathogenic
92
Fo isolates. Pathogenic Fo isolates induced a strong defense reaction compared to non-
pathogenic, suggesting that the non-pathogenic isolates did not induce a response in the host
to infection. These descriptions agreed with our observations in which the non-pathogenic
FO42 isolate produced small patches of root discoloration but not evident damage to root
tissues or evident signs of root colonization.
The non-pathogenic isolate FO42 showed a notable ability to promote plant growth;
seedlings inoculated with this isolate showed significantly higher root weight and total plant
length compared to the mock-inoculated control. Plant growth stimulation by non-pathogenic
Fo isolates has also been observed in other hosts (Fracchia et al. 2000; Thongkamngam and
Jaenaksorn 2017). This effect has been associated with the interaction of certain fungal
metabolites and volatiles with the host hormone transport and signaling of auxins (Bitas et al.
2015), and the overexpression of PR genes along with the activation of jasmonic and
salicylic acid pathways upon inoculation (Saldajeno and Hyakumachi 2011; Shcherbakova et
al. 2016; Veloso et al. 2016). Additional research on the interaction between pathogenic and
non-pathogenic Fo isolates must be done in order to identify the potential for biocontrol and
how biocontrol efficacy might be influenced by abiotic factors such as pH and temperature in
soybeans.
Although, disease severity between FO38 and FO42 isolates had similar trends in
response to environmental factors, severity for the non-pathogenic FO42 isolate remained
low in all combinations of pH and temperature. In addition, estimates of percent reduction in
seedling growth variables for the non-pathogenic FO42 isolate were significantly lower in all
the pH and temperature combinations in which the pathogenic isolate FO38 displayed the
highest levels of reductions. These results suggest it is unlikely that the non-pathogenic Fo
93
isolates become pathogenic to soybean at least under the environmental conditions set in our
studies. Our results suggest that abiotic factors influence the level of disease caused by Fo
pathogenic isolates; however, pathogenicity may be conferred by other mechanisms, such as
horizontal gene transfer of genomic regions rich in transposons or pathogenicity-related
genes that contributes to the origin of pathogenic genotypes (Ma et al. 2010).
This is the first report quantifying and modeling the effects of abiotic factors on Fo in
soybeans. We found that the Gaussian model best explained the effects of temperature and
pH on fungal radial growth and disease severity. Previous studies on Fusarium wilts in other
pathosystems have used polynomial models to describe the interaction between temperature
and pH for fungal growth in soil (Chen et al. 2013), and spore attachment under hydroponic
conditions (Schuerger and Mitchell 1992). In addition, Navas-Cortes et al. (2007) developed
exponential models to describe effects of soil temperature and inoculum density on Fusarium
wilt of chickpea. Interestingly, these polynomial and exponential models describe trends and
optimal temperatures similar to our studies of Fo on soybeans. The models developed for Fo
growth and root rot will give insights on high and low risk scenarios to growers and farmers.
This simple quantitative model captures crucial features of host - pathogen interactions on
key abiotic variables such as pH and temperature. Additional variables could be added to
create more complex and representative models.
The information generated in this research will increase knowledge about the
epidemiology of Fo and may help to predict the risk of Fusarium root rot. This knowledge
may assist breeders to better identify soybean resistant cultivars, taking into account that
temperature significantly influences disease development and host susceptibility. Quantifying
the impact on environmental factors on root rot disease development by the use of
94
quantitative models may help predict scenarios in which root rot may be more severe.
However, we acknowledge that the predictability of our model will decrease if the effects of
soil moisture, soil temperature or soil pH and their interaction are considered. For these
reasons these soil variables must be considered in future studies.
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Table 1. Estimates of pH and temperature obtained from the non-linear optimization
procedure from the Gaussian model for in vitro fungal radial growth and root rot on
Fusarium oxysporum isolates.
xMaximal radial growth and maximal disease severity index estimates were obtained by
using a non-linear optimization procedure including pH range from 4 to 7 and temperature
range from 15 to 30ᵒC for each isolate. ySEE Standard error of estimate zR2 Coefficients of determination
Gaussian model parameters and statistics for in vitro radial growth
Isolates pH Temperature (ᵒC) Radial
growth (cm)x SEEy F statistics P > F
Gaussian
regression R2 z
FO36 6.2 27.8 5.2 0.6540 15.8475 0.0002 0.8521
FO37 6.7 28.1 5.5 0.6252 16.3469 0.0001 0.8560
FO38 5.8 28.1 3.0 0.4427 9.9208 0.0001 0.7830
FO39 6.7 27.6 4.5 0.7199 9.1845 0.0016 0.7696
FO40 6.1 27.3 5.3 0.3907 37.3336 <0.0001 0.9314
FO41 6.0 25.4 5.8 0.8204 11.8469 0.0006 0.8116
FO42 6.4 27.1 4.9 0.5791 17.1491 0.0001 0.8618
FO43 6.8 27.0 5.0 0.9150 6.8012 0.0052 0.7121
FO44 6.4 26.6 5.1 0.7169 13.9697 0.0003 0.8355
FO45 6.4 28.9 5.2 0.6895 16.4253 0.0001 0.8566
FO46 6.7 30.0 4.5 0.8193 7.2480 0.0041 0.7249
FO47 6.5 26.7 5.4 0.5334 23.8329 <0.0001 0.8966
FO48 6.5 27.4 4.9 0.6307 15.3397 0.0002 0.8480
FO49 6.4 26.0 6.0 0.8846 11.1288 0.0007 0.8019
Average 6.3 27.1 4.9 0.5792 17.1491 0.0001 0.8618
Gaussian model parameters and statistics for root rot seedling disease
Isolates pH Temperature (ᵒC) DSI (%)x SEEy F statistics P > F Gaussian
regression R2 z
FO38 5.9 30 64.0 2.9389 168.7979 <0.0001 0.9840
FO42 5.9 30 17.1 1.0859 70.2363 <0.0001 0.9623
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Table 2. Analysis of variance indicating the effects of pH and temperature on seedling disease after inoculation of soybean seeds with
pathogenic (FO38), and non-pathogenic (FO42) Fusarium oxysporum isolates in a rolled-towel assay.
xDSI (disease severity index) was arcsin transformed. Visual scale of severity was square root transformed. Seedling growth variables
root weight, root length and plant length were log transformed. ANOVA do not include the mock-inoculated controls data.
Experiments consisted of 3 replications (towels) for each treatment combination and two experiments for the rolled towel assay.
Analyses of variance for all response variables included the mock-inoculated controls.
DSIx Severity Root Weight Root length Plant length
Source df F value P > F F value P > F F value P > F F value P > F F value P > F
pH 3 24.14 <0.0001 24.85 <0.0001 9.09 <0.0001 148.19 <0.0001 74.71 <0.0001
Temperature (T) 3 7.19 0.0434 11.45 0.0197 2.24 0.2255 4.72 0.0084 8.80 0.0310
pH×T 9 3.52 0.0005 3.07 0.0021 2.39 0.0148 6.18 <0.0001 2.42 0.0135
Isolate (I) 1 600.83 <0.0001 532.04 <0.0001 12.22 0.0006 14.04 0.0003 24.76 <0.0001
pH×I 3 0.20 0.8947 1.24 0.2961 1.39 0.2480 1.40 0.2443 0.32 0.8113
T×I 3 39.88 <0.0001 66.82 <0.0001 6.17 0.0005 0.76 0.5982 2.97 0.0337
pH×T×I 9 0.51 0.8646 1.60 0.1206 1.24 0.2753 0.98 0.4610 0.72 0.6920
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Figure 1. Radial growth of 14 Fusarium oxysporum isolates with respect to temperature and
pH of the growth medium. A, B, C, D isolates growing in Citrate – phosphate buffer set at
pH 4, 5, 6, and 7, respectively. Data represent the mean values of two experiments with four
replicate petri dishes for each treatment combination.
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Figure 2. Estimates of fungal radial growth (cm) from the Gaussian regression analysis
across the main factors pH at 4, 5, 6, and 7 and temperature at 15, 20, 25, 30ᵒC for the
average growth of 14 Fusarium oxysporum isolates. Data represent the mean values of two
experiments with four replicate petri dishes for each treatment combination.
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Figure 3. Bar graph for disease severity index (DSI) with respect to pH buffer and incubation
temperature for A, FO38 pathogenic Fo isolate and B, FO42 non-pathogenic Fo isolate.
Comparisons were made separately by pH buffer. Means with the same letter are not
significantly different according to Fisher’s protected least significant difference (P < 0.05).
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Figure 4. Soybean seedlings incubated at 30ᵒC for 8 days after seed inoculation with
Fusarium oxysporum isolates with a spore suspension of 1×106 conidia/ml. A-D, seedlings
inoculated with pathogenic isolate FO38. A, B, C, D seedlings growing in citric acid and
NaOH buffer set at pH 4, 5, 6, and 7, respectively. E-H, seedlings inoculated with the non-
pathogenic isolate FO42. E, F, G, H seedlings growing in citric acid and NaOH buffer set at
pH 4, 5, 6, and 7, respectively.
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Figure 5. Percentage of reduction on seedling growth variables relative to the mock-
inoculated control with respect to pH buffer and incubation temperature for A, C, E plants
inoculated with FO38 pathogenic Fo isolate and B, D, F plants inoculated with FO42 non-
pathogenic Fo isolate. Means with the same letter are not significantly different according to
Fisher’s protected least significant difference (P < 0.05).
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Figure 6. Linear relationship between visual scale for severity disease severity index (DSI).
Each data point represents the average disease of one towel containing 15 seeds inoculated
with A, FO38 pathogenic Fo isolate and B, FO42 non-pathogenic Fo isolate. r = Pearson’s
linear correlation coefficients.
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Figure 7. Estimates of disease severity index (DSI) from the Gaussian regression analysis
across the main factors pH at 4, 5, 6, and 7 and temperature at 15, 20, 25, 30ᵒC for A, FO38
pathogenic Fo isolate and B, FO42 non-pathogenic Fo isolate. Data represents the disease
mean values of two experimental replicates for each Fo isolate.
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CHAPTER 4. EFFECTS OF SOIL CONDITIONS ON ROOT ROT OF SOYBEAN
CAUSED BY FUSARIUM GRAMINEARUM
A paper to be submitted to the journal Plant Disease
D. R. Cruz, L. F. S. Leandro and G. P. Munkvold
Department of Plant Pathology and Microbiology, Iowa State University, Ames. 50011.
Abstract
Fusarium graminearum (Fg) is an important soybean pathogen that causes seedling
disease, root rot, pre- and post-emergence damping-off. However, effects of soil conditions
on the disease are not well understood. The objective of this greenhouse study was to observe
the impacts of soil texture, pH, and soil moisture on seedling root rot symptoms and
detrimental effects on seedling development caused by Fg. Fg-infested millet was added
(10% v/v) to soil with four different textures (sand, loamy sand, sandy loam, and loam). Soil
moisture was maintained at saturation, field capacity or permanent wilting point at soil pH
levels of 6 or 8. Seedlings were evaluated 4 weeks after planting for root rot, root length, root
and shoot dry weights, and leaf area. There was a significant interaction between soil
moisture and soil texture for root rot assessed visually (P<0.0001). Highest severity (~70%)
was observed at pH 6 and permanent wilting point in sandy loam soils. In contrast, pot
saturation resulted in the lowest levels of disease in sandy loam and loam soils (11.6 and
10.8%, respectively). Percent reductions in seedling growth parameters, including root
length, foliar area, shoot and root dry weights and root tips, relative to the non-infested
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control, were significantly greater in sandy loam soils. In contrast, there were no relative
growth reductions in sandy soils. This study showed that levels of root rot increased under
moisture-limiting conditions, producing detrimental effects on plant development.
Introduction
Fusarium graminearum (Fg) (synonym Gibberella zeae (Schwein.) Petch) is a major
necrotrophic pathogen of soybean, causing seed decay, damping-off, crown and root rots, and
pod blight in the United States, Canada and other temperate regions (Martinelli et al. 2004;
Sella et al. 2014; Xue et al. 2007). Soybean is often included in rotation with wheat, maize
and other cereal crops that are hosts of Fg. Fg colonizes and overwinters on host crop
residues, thus the residues provide a source of inoculum for subsequent seasons (Barros et al.
2014; Leslie et al. 1990; Windels et al. 1988). Fg isolates pathogenic on wheat and maize
have been also found to be highly pathogenic on soybean (Broders et al. 2007). Xue et al.
(2007) suggested that soybean in rotation with cereal crops provide strong selection pressure
for the development of highly aggressive Fg isolates on all crops.
Fg is highly prevalent in Iowa and has been associated with soybean seedlings with
severe root rot (Díaz Arias et al. 2013a; Díaz Arias et al. 2013b). Seedling diseases are
caused by diverse Fusarium and Pythium spp. and are often more prevalent when soils are
cool and wet at planting. Delayed germination and emergence in such conditions increase the
chances for seed and seedling infection (Broders et al. 2007; Licht et al. 2013). Disease
severity for some pathogens may also be linked to soil physiochemical conditions, and
biodiversity in soil microbial populations (Ghorbani et al. 2008). There is a lack of detailed
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knowledge about soil environment and soil conditions such as pH, soil texture and soil
moisture that influence disease development, incidence and severity of Fusarium root rot of
soybean caused by Fg.
Soil pH influences root rot disease severity by directly affecting soil microbial
communities and the balance between pathogens and their specific antagonists (Hoper and
Alabouvette 1996). Evidence suggests that lowering soil pH from 8 to 6 in Fusarium
suppressive soils drastically reduces the viability of Pseudomonas spp. and eliminates the
suppressive effects on Fusarium wilt diseases (Scher and Baker 1980). Similarly, changes in
soil pH indirectly affect nutrient availability to the pathogen and the plant host (Ghorbani et
al. 2008).
Soybeans are sensitive to changes in soil pH; greatest soybean yields in Iowa and
Minnesota have been reported in soil pH levels between 6.2 and 7.0 (McLean and Brown
1984; Rogovska et al. 2007). Altered nutrition induced by soil acidity can increase or
decrease the susceptibility of the plant host, affecting disease severity and incidence (Fang et
al. 2012; Ghorbani et al. 2008). Under high soil pH, iron availability is reduced and pathogen
establishment will depend on the ability to acquire and compete for iron. Fg has two types of
iron acquisition systems, secretion and uptake of iron-chelating molecules and production of
cell wall iron reductases to reduce ferric iron to ferrous iron (Greenshields et al. 2007;
Kosman 2003; Philpott 2006). Iron availability also affects soybean plants and can lead to
iron deficiency chlorosis (IDC) in high pH soils (Rogovska et al. 2007). Soybean plants with
IDC symptoms often display root rot symptoms, but the basis of this association is not clear.
Soil texture can also affect pathogen distribution in soil and plant resistance to
disease. For example, Fusarium wilts have been commonly associated with sandy soils
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(Dominguez et al. 1996; Toussoun 1975). Field observations revealed Fusarium wilts caused
by F. oxysporum are more severe in coarse sandy soils or loamy-textures soils, while clayey
soils seem to be root rot suppressive (Hoper and Alabouvette 1996; Scher and Baker 1980;
Yuen et al. 1983). An association between Fusarium root rot of soybean caused by Fg and
soil texture components have not been documented. Soil texture and type of clay have been
correlated to disease incidence and severity in a diverse number of Fusarium wilts on
muskmelon, banana, flax, barley, and tobacco (Alabouvette et al. 1979; Dominguez et al.
1996; Hoper et al. 1995; Strunnikova et al. 2015; Stutz et al. 1989; Toussoun 1975). Soil
clay minerals actively interact with bacteria due to the high cationic exchange capacity
(CEC), surface area and variable negative charge (Hoper and Alabouvette 1996); these
interactions consist mainly of buffering soil pH, providing available nutrients that can be
rapidly used. Similar to alkaline soil pH, the addition of clay minerals into the soil reduces
the availability of iron to fungal pathogens, promoting bacterial growth (Hoper et al. 1995). It
has been demonstrated that high clay CEC stimulates the metabolism of bacteria (Stotzky
and Rem 1966). In contrast silt-sand minerals are considered more inactive (Robert and
Chenu 1992).
Depending on the pathosystem, low and high soil water potential may affect on root
rot differently. For example, in Fusarium oxysporum infested soils near saturation, soybean
root rot severity increased, but only at low soil temperatures (French 1963). A three or seven
day duration of flooding at soybean emergence increased the incidence and severity of
Pythium spp. while Fusarium spp. were less frequently associated with seedling infection
under flooded conditions (Kirkpatrick et al. 2006). On the other hand, low water potentials
may predispose the host to infection and favor diseases such as dry rot of beans caused by F.
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solani, and seedling blight of cereals caused by F. roseum (Ghorbani et al. 2008; Hoper and
Alabouvette 1996). Fusarium root rot of sweet potatoes, peas and beans are also more severe
in dry soil conditions (Cook and Papendick 1972). Several Fusarium wilt diseases are also
favored by dry soil conditions, including Fusarium wilt of chickpea caused by F. oxysporum
f. sp. ciceris (Landa et al. 2006; Navas-Cortes et al. 2007), seedling blight of clover caused
by F. roseum (Graham et al. 1957), root and stem rot of peas and chickpeas caused by F.
solani f. sp. pisi (Bhatti and Kraft 1992).
The objectives of this research were to measure the effects of important soil variables
on the development of root rot of soybean caused by Fg by studying (i) the effects of soil pH
and soil water content interaction on root rot, (ii) the effects of artificial soil textures and its
interaction with soil pH and soil water content on root rot, and (iii) possible detrimental
effects on soybean seedling growth parameters under various edaphic conditions on Fg
infested soils.
Materials and Methods
Inoculum production
Fg isolate FG5 was collected from symptomatic soybean roots in Iowa in 2007 (Díaz
Arias et al. 2013b). FG5 was single spore and maintained on dry silica gel beds at 4˚C until
use. Aggressiveness and pathogenicity of the FG5 isolate was tested on soybean cultivar
Asgrow 2403 (SDS susceptible) (Monsanto Co., St. Louis, MO) under greenhouse conditions
(Díaz Arias et al. 2013a).
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Millet seeds were soaked for 24 h in water, drained and autoclaved at 121˚C for 1 h in
autoclave bags with a micro-porous filter patch on two consecutive days. Isolate FG5 was
grown on potato dextrose agar (PDA) 39g/L (Difco, Becton, Dickinson and Co, Spark, MD,
USA) for 14 days in an incubator at 25˚C with a 12 h photoperiod, in order to promote
conidial formation. Plugs (~1 cm2) from cultures of FG5 were added to the sterile millet in a
biosafety cabinet, and then sealed with a rubber band. Bags were then placed in an incubator
(Hoffman manufacturing Inc. Oregon, USA) for 8 days, at 65% relative humidity with a 12 h
photoperiod, and mixed by hand every day. FG5-infested millet was mixed with soil at 10%
concentration by volume. Non-infested control treatments contained autoclaved sterile millet
mixed with soil.
Artificial soil textures
Four artificial soil textures (sand, loamy sand, sandy loam and loam) were created by
mixing sand and silt loam in different proportions by volume (Table 1). Resulting textural
classes on the artificial soils were analyzed for particle size distribution following the
protocol by the USDA (1996). Soils were pasteurized in a soil steamer for 1.5 h, dried on a
greenhouse bench for 4 days and kept in plastic containers until use.
Soil pH
Initial soil pH for each soil textural class was measured by the electrometric standard
method, using a 1:1 soil/water ratio (Watson and Brown 1997). Ten ml of distilled water
were added to 10 g of soil in a 50 ml falcon tube, which was shaken for 10 min and settled
for 5 min, placing the pH meter electrode in the slurry. After the initial pH measurements,
soil pH was adjusted to 6 or 8 by gradually adding Al2(SO4)3 (Bonide Products, Inc. New
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York, USA) and CaCO3 (Fisher, Illinois, USA) to the soil, respectively. Soils were then wet,
dried and mixed twice a day during two weeks to allow the soil react with the chemical
compounds. Soil pH measurements were performed periodically until the soil remained at
constant pH equilibrium (Islam et al. 2004).
Soil moisture content
Three levels of soil moisture including pot saturation (PS), field capacity (FC) and
permanent wilting point (PWP) were set for each soil texture, derived from the volumetric
water content (θv) points for different textural classes described by Rowell (1994), and
Bradly and Weil (2004) (Table 2). For each soil textural class, 230 ml of dried soil were
weighed in a Styrofoam cup and a calculation the of volume of water needed for each type of
soil texture was performed by using the following formula
Where Vw is the volume of water contained in a soil, Vs is the total volume of soil,
and θv is the soil volumetric water content point. The weight of 230 ml of soil for each soil
textural class and the weight of the water needed to reach the desired water content point
were added to have a final weight per cup that was subsequently maintained by adding water
once a day.
Soil infestation and experimental design
After two weeks of incubation, inoculum was mixed with soil at 10% concentration
by volume. Non-infested control treatments contained 10% autoclaved sterile millet. Seeds of
soybean cultivar Jack were surface disinfested in 0.5% NaOCl for 2 min, rinsed twice for 2
min in sterile distilled water, and dried for 20 min in a laminar flow hood before planting.
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Three seeds per cup were planted into the infested or non-infested soil mix and watered daily
for 2 weeks. Soil moisture levels PS, FC, and PWP were imposed 2 weeks after planting and
maintained for 2 weeks. Four weeks after planting, seedlings were visually rated for root rot
severity (%), roots were scanned using a flatbed scanner (EPSON Expression, 10000XL,
Epson America, Inc.), and root images were analyzed using WinRhizo software 2008
(Regent Instruments Inc., Quebec, QC, Canada) to obtain estimates of root length, surface
area, root volume, root tips, and forks on each individual plant. In addition, foliar area was
estimated on individual plants using Assess 2.0 (The American Phytopathological Society
(APS), Saint Paul, Minnesota). Shoot and root dry weights were measured on each individual
plant after oven drying at 80˚C for 24 h. The experiment was organized in a split-plot design
with soil moisture (PS, FC, PWP) as the main-plot factor, and soil texture (sand, loamy sand,
sandy loam and loam), soil pH (6 or 8) and Fg infested or non-infested soils as sub-plot
factors. One cup was considered as an experimental unit with three seeds or subsamples in
each cup. The experiment had 8 replications and was conducted twice.
Data analysis
Visual root rot severity data (%) was arcsin transformed. Root length, surface area,
root volume, root tips, forks, foliar area shoot and dry weight were square-root transformed
to meet the assumptions of the ANOVA. In addition, normal distribution of the residuals was
tested for all the transformed data under the PROC UNIVARIATE procedure of SAS version
9.3 (SAS Institute Inc., Cary, NC). Separate analysis of the two experimental replications
showed similar trends, so data were combined for subsequent analyses. Analysis of variance
was performed on the transformed data using PROC GLIMMIX in which the main-plot
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effect; soil moisture, was tested against the whole-plot variation error, and sub-plot effects
were tested against the residual variability. Mean separation analyses on the transformed data
were performed using a Fisher’s protected least significant difference (LSD) test at P = 0.05.
Results
Effects on root rot severity
The analysis of variance indicated a significant effect for the main factors soil
moisture content (θv), soil pH, soil texture, as well as the interaction between θv and soil
texture (P < 0.0001) for the visual evaluation of root rot (Table 3). These significant
interactions indicate that the level of disease varied across the soil textures and the effect of
soil moisture content also varied among soil textures, primarily because soil moisture content
did not have an effect on root rot in sandy soil.
Soil pH significantly influence disease severity; soil at pH 6 showed more disease in
all treatment combinations compared to pH 8 (Fig. 1). Overall, across soil textures, the
highest level of root rot was observed at pH 6 and PWP (55.3%); and the lowest level of
disease was observed at pH 8 and PS (15.4%) (Fig. 1A).
The effects of soil moisture and soil texture on root rot severity displayed similar
trends for pH 6 and 8. Root rot severity increased from sandy to clayey soils at PWP;
conversely, disease severity decreased from sandy to clayey soils at PS; therefore, PWP and
PS revealed inverse trends on root rot severity. Disease severity at FC remained between the
highest and lowest disease levels in each soil texture. In addition, the sandy soil showed no
significant differences in the levels of disease across the soil moisture treatments (Fig. 2).
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The highest levels of disease corresponded to loamy sand and sandy loam soils at
PWP at both pH levels (Fig. 2). At soil pH 6, the highest levels of disease were 52.6% and
67%, in loamy sand and sandy loam soils, respectively, while at pH 8 they were 51.7% and
49.8%. In contrast, the lowest levels of root rot were found in sandy loam and loam soils
(10.2% and 9.7%, respectively) at PS and soil pH 8 (Fig. 2B), followed by sandy loam and
loam soils (21.5% and 19.9%, respectively) at PS and soil pH 6 (Fig. 2A).
Effects on seedling growth variables
Comparisons between Fg infested and non-infested treatments revealed detrimental
effects on shoot and root morphological characteristics, especially in loamy sand and sandy
loam soils. Plants growing in Fg infested soils had significantly shorter roots compared to the
non-infested control in loamy sand and sandy loam soil textures at all soil moisture levels. In
contrast, root length was not affected by Fg when plants were grown in sandy soil (Fig. 3A).
There were significant detrimental effects on foliar area for Fg infested sandy loam soil at all
soil moisture content levels, and loam soil at PS. Conversely, foliar area was significantly
higher in the infested treatments compared to non-infested treatments in sandy soils at FC
and PWP (Fig 3B).
Root and shoot dry weight were negatively impacted by Fg in sandy loam soil at PS
and FC. However, there were no detrimental effects on root and shoot dry weight for the
plants growing in infested sandy soil (Fig 3C-D).
Root volume was significantly affected by Fg; there were significant differences in
root volume between infested and non-infested plants for all soil textures and soil moisture
treatments except for sand (Fig. 3E). Number of root tips was significantly affected by Fg
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infection in sandy loam and loam soils but no adverse effects were observed in sand or loamy
sand soils (Fig. 3F).
Sandy loam soils generate the greatest percentages of reduction in the root and shoot
morphological characteristics (Fig. 4). The highest percentages of reduction in root length
were observed in sandy loam soils in all levels of soil moisture ranging from 26 to 21%
followed by loam soil at FC (18.6%) (Fig. 4A). Foliar area was significantly reduced in
sandy loam soil at PWP (53.2%) followed PS (23.7%). Loamy sand and sandy soils had no
reductions in foliar area (Fig. 4B).
Greatest percentages of reduction in root dry weight were observed in sandy loam and
loam soils at PS and FC ranging from 27 to 2%. In addition, there was no reduction in root
dry weight at PWP across all soil textures (Fig. 4C). Similarly, there was a significant
reduction in shoot dry weight in sandy loam and loam soils ranging from 19 to 3%. Seedlings
growing in loamy sand and sand soils did not displayed reductions in shoot dry weight (Fig
4D).
Greatest reductions in root volume were observed in plants growing in sandy loam
soils in all soil moisture treatments, followed by loamy and sandy loam soils at FC and PS
(Fig. 4E). Root tips reductions were significantly higher in plants growing in sandy loam
soils ranging from 16.2 to 6.5% (Fig. 4F). Sandy soils did not show reductions in the shoot
and root morphological characteristics measured in this study.
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Discussion
In this study, we provided the first analysis concerning the interactions among several
abiotic soil factors (soil moisture, soil pH, and soil texture) in the development of root rot of
soybean seedlings caused by Fg. The results suggest that edaphic characteristics have a
considerable influence on disease severity and seedling development. Acidic soils (pH 6)
were more conductive for root rot than alkaline soils (pH 8). Root rot severity was
significantly different across the soil moisture treatments, in which PWP displayed the
highest levels of root rot and PS the lowest.
Our findings that disease was most severe in acidic soils (pH 6) and least severe in
alkaline soils (pH 8) agree with several similar studies. For example, sorghum rot infection
by Fg was significantly higher at soil pH 5.1 than 6.1 on seedlings 36 days after planting
under field conditions (Davis et al. 1994). Strawberries infected by Fo f. sp. fragariae
displayed more severe root rot and reduced plant size at pH 5.2 than at pH 7.5 (Fang et al.
2012). Increasing soil pH has also been suggested as an alternative for disease management
in other Fusarium wilt diseases such as banana, spinach, tomato, cotton and melons
(Dominguez et al. 1996; Gatch and du Toit 2017; Groenewald et al. 2006; Jones et al. 1989;
Woltz and Jones 1973). Agarwal and Sarbhoy (1978) reported Fg grew best at pH 3.5 in in
vitro studies. Therefore, it seems plausible that increasing soil pH with CaCO3 might reduce
growth of Fg, decreasing the chances to effectively infect soybean roots.
Previous reports suggest that increasing soil pH by liming reduces soil fungal
populations and at the same time improves bacterial growth (Alabouvette 1999;
Muhlbachova and Tlustos 2006; Scher and Baker 1980). According to our observations,
liming with CaCO3 had a significant reduction effect on root rot of soybean caused by Fg
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during a four-week period. In addition, the adverse effects of liming on fungi are more
significant under limited iron in soil (Expert 2009; Ghini et al. 2000). These observations
may explain the low root rot severity at soil pH 8 in our studies, and suggest that disease
suppression is proportional to the reduction of available iron in the soil, as it has been
reported in other Fusarium wilts (Hoper et al. 1995).
Iron availability is affected in calcareous soils with high pH; the solubility of iron
Fe3+ decreases 1,000-fold with each unit increase in pH (Expert 2009). In addition, CaCO3
reduces the bioavailability of iron (Loeppert 1988). Iron is a limiting nutrient for fungi
metabolism and it is involved in mechanisms of disease virulence (Scher and Baker 1982;
Symeonidis and Marangos 2012; Weinberg 2009). From our results, low root rot severity at
soil pH 8 suggests that pathogenicity can be significantly influenced by the relative ability of
Fg to obtain essential iron under the limiting conditions imposed by high soil pH. High soil
pH can lead to IDC in soybean, which is associated with root rot symptoms often attributed
to Fusarium spp. However, our results do not support a role for Fg in enhanced root rot
symptoms under high pH conditions. Root rots associated with IDC are likely caused by
other fungal species.
Although several studies have demonstrated that clays favor bacterial activity and are
less favorable to fungal growth (Marshall 1975; Stotzky 1966; Stotzky and Rem 1967), in
our studies the effect of soil texture was highly dependent on soil moisture content. At PS,
root rot decreased with increasing clay content; at FC, sand had the most severe root rot but it
was similar among loamy sand, sandy loam, and loam; and at PWP, root rot was most severe
in sandy loam or loamy sand. Together, these results suggest that root rot was enhanced
under moisture-stress conditions; in sand, plant moisture stress may have been similar in all
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the moisture content treatments, due to rapid drainage of water added to the cups. The
interaction of soil texture with soil pH was not significant, but a trend toward lower root rot
severity with higher clay content was more evident at pH 8 than at pH 6 (Fig. 1B). This
observation is consistent with previous reports made by Hoper et al. (1995) and Amir and
Alabouvette (1993) on Fusarium wilts, which demonstrated that addition of clay
(montmorillonite or illite) provides the most disease suppression at high soil pH.
Soil texture affects plant disease due to the correlation with water holding capacity,
nutrient availability, porosity and root growth (Ghorbani et al. 2008). In our studies, we
observed that the combined effect of soil texture and soil moisture displayed a high variation
in disease severity. For example, sandy soils did not present significant differences in root rot
across soil moisture contents. However, effects of soil moisture treatment on root rot severity
became more accentuated with incremental clay content across soil textures, suggesting that
the water holding capacity effect of clay plays an important role on seedling root rot
development.
The lowest root rot severity and the greatest seedling growth were observed at PS in
the sandy loam and loam soils. The results suggest that high soil moisture given by the higher
water holding capacity of high clay content soils may have been detrimental for the Fg
inoculum viability. Previous studies on inoculum survival in loam soils indicated that soil
moisture content higher than 85% of saturation reduced soil populations of Fg, F.
moniliforme and F. oxysporum, and stimulated soil bacterial populations (Stover 1953).
Our observations of reduced root rot on high moisture soils agrees with reports in
other pathosystems. For example, Fusarium root rot of wheat caused by F. roseum f. sp.
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cerealis was less severe in high moisture fine-textured soils under field conditions
(Papendick and Cook 1974).
Root rot was significantly higher at PWP soil moisture treatments in all soil textures
except sand. Similar results regarding drought stress predisposing wheat seedlings to higher
Fg infection have been described by Beddis and Burgess (1992). In cereals, Fg tends to be
the most predominant Fusarium species in the warmer regions of the USA, and warm and
dry weather are considered to be the optimal conditions for foot rot development (Doohan et
al. 2003; Vigier et al. 1997). It is likely that there is an association between dry climatic
conditions optimal for Fg to produce disease and our observation on root rot of soybean.
Soybean seedlings growing in soils at PWP displayed reduced root and shoot growth.
The effects of low water stress in soybean include inhibition of cell division, reduction in
foliar area, suppression of shoot, root growth and root volume, and decrease in
photosynthetic rate (Hossain et al. 2014; Krizek et al. 1985). Although studying the adverse
effects of water stress on soybean seedling growth parameters was not our main objective, it
is important to note that the highest levels of root rot were observed on water-stressed
seedlings with reduced growth in root and shoot. For these reasons, it was more informative
to draw conclusions on the effects of plant growth variables by making comparisons between
the infested and non-infested seedlings in each treatment combination.
Soybean seedlings growing in Fg infested soils displayed significant reduction in
plant growth parameters. These findings are in general agreement with (Zhang et al. 2010)
and (Xue et al. 2007), in which seed or seedling Fg inoculation reduced root dry weight and
plant height up to 24 and 28%, respectively. Reductions in root length, root volume, number
of tips and forks by Fg soil inoculation have also been reported (Díaz Arias et al. 2013a).
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Differences in all plant growth parameters between infested and non-infested plants in sandy
soils were negligible. This effect may be due in part to the reduction of inoculum potential
associated with low amount of nutrients provided to Fg inoculum from the soil and the low
CEC of sand minerals (Robert and Chenu 1992). Moreover, the low impedance for root
elongation offered by the high sand content allows the root to scavenge, reducing detrimental
effects of Fg, also evident in the shoot weight and foliar area (Baligar et al. 1980).
Highly significant differences were observed between the infested and non-infested
treatments for all the root morphological variables in the PS and FC sandy loam soils, even
though these treatment combinations had the lowest root rot severity. A possible explanation
for the correlation between low root rot severity and high percentage of growth reduction is
that the visual evaluation of disease does not take into account root internal colonization and
the possible damage in the vascular system that might affect root growth and development.
Root morphological characteristics analyzed on WinRhizo are useful indicators of
root health. Estimates of root length, and root volume, root forks and root tips helped to
interpret the detrimental effects of Fg and its interactions with soil moisture, soil texture and
soil pH. Root volume however, seems to be the most sensitive variable in estimating
detrimental effects of root rot. For example, there were significant differences in root volume
comparing infested and non-infested treatments in all soil textures and soil moisture
treatment combinations except for sand.
The results from this study have provided information on abiotic factors of
importance for root rot of soybean caused by Fg. We demonstrated that changes in edaphic
factors and their interactions influence disease severity. The soil parameters studied the most
in relation to soilborne pathogens are soil pH, clay content and nitrogen content (Janvier et
130
al. 2007). There are not many reports on Fusarium wilts that included several soil factors in
the same study and a combined approach is needed due to the possible number of biotic and
abiotic factors related to soilborne diseases.
Additional variables such as temperature fluctuation and interactions between and
within fungal species should be included in future studies. For example, Tu (1994) performed
a study of the effects of soil moisture and temperature on the Fusarium root rot complex of
pea, including F. solani f. sp. pisi and F. oxysporum f. sp. pisi and found that both lack of
moisture and saturation increased disease severity. It is important to study these types of
interactions in the future since the soybean root rot complex is not only associated with Fg
but include a diverse number of Fusarium species (Díaz Arias et al. 2013a; Leslie et al.
1990).
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Table 1. Artificial soil textural classes generated by sand and silt loam mix.
Soil mix by volume Particle size distribution analysisx
Sand (%) Silt loam (%) Textural class Sand (%) Silt (%) Fine silt (%) Clay (%)
90 10 Sand 95.90 1.49 1.94 0.67
75 25 Loamy sand 87.14 4.62 3.96 4.28
25 75 Sandy loam 61.94 13.67 11.84 12.55
10 90 Loam 42.10 19.05 19.88 18.97 xParticle size distribution analyses were performed by the pipette method by the USDA 1996
Table 2. Values of volumetric water content of soils of varying soil textures
Soil volumetric water content (θv)
Soil textures PS FC PWP
Sand 0.13 0.09 0.07
Loamy sand 0.16 0.10 0.08
Sandy loam 0.25 0.17 0.09
Loam 0.28 0.21 0.11
Soil moisture levels PS = pot saturation, FC = field capacity, PWP = permanent wilting point
Table 3. Analysis of variance indicating the effects of soil moisture content, soil pH and soil
texture on root rot visual evaluation (%), following soil infestation with Fusarium
graminearum.
Source df F value P > F
Soil moisture content (θv) 2 73.09 <0.0001
Soil pH (pH) 1 50.42 <0.0001
θv×pH 2 0.57 0.5675
Soil texture (ST) 3 14.51 <0.0001
θv×ST 6 16.66 <0.0001
pH×ST 3 1.40 0.2406
θv×pH×ST 6 0.77 0.5956
Analysis of variance was performed only on plants growing on soils infester with Fusarium
graminearum. Visual root rot disease data (%) were arcsine transformed. The experiment
consisted of eight reps in a split-plot design, with soil moisture content as the main-plot
factor, and soil pH and soil texture as the sub-plot factors.
140
Table 4. F values for comparisons of seedling growth parameters between F. graminearum
infested and non-infested treatments on the main effects of soil texture and soil moisture
(PS= pot saturation; FC= field capacity; PWP= permanent wilting point) at different soil pH.
* indicates significant differences from the non-infested control (P ≤ 0.05) with a T-test for
mean separation.
ST = soil texture, S = sand, LS = loamy sand, SL = sandy loam, L = loam, RL = root length
(cm), FA = Foliar area (cm2), RDW = root dry weight (g), SDW = shoot dry weight (g), RV
= root volume (cm3), RT = root tips, SA = surface area. Data represent values for two
experiments with a total of 16 observations per treatment combination. Analyses were
performed on square-root transformed data.
pH 6
ST θv RL FA RDW SDW RV RT SA F
S PS 1.71 0.04 0.83 0.85 0.05 1.18 0.62 2.65
FC 2.24 2.65 2.48 3.06 0.37 0.95 1.08 3.05
PWP 2.06 0.50 0.85 0.32 1.94 1.77 2.05 5.06*
LS PS 3.27 0.01 3.30 0.41 2.86 0.14 3.56 0.10
FC 1.76 0.49 0.83 0.17 3.88 0.07 2.95 0.60
PWP 7.06* 0.66 2.61 0.06 15.75* 0.01 11.37* 0.31
SL PS 33.75* 22.67* 26.38* 23.25* 50.74* 9.40* 45.05* 18.17*
FC 14.98* 1.66 2.06 5.90* 12.76* 7.38* 14.37* 13.33*
PWP 9.27* 15.20* 3.53 3.92 22.67* 0.66 16.01* 2.85
L PS 10.45* 8.95* 13.51* 5.93* 15.97* 3.25* 13.98* 1.63
FC 5.38* 0.36 5.08* 0.65 12.98* 1.24 9.13* 2.19
PWP 2.32 6.52* 0.71 1.78 4.77 0.38 3.84 0.01
pH 8
ST θv RL FA RDW SDW RV RT SA F
S PS 1.25 1.96 0.17 6.13* 0.04 1.29 0.41 1.03
FC 0.51 3.81 0.78 11.74* 0.38 0.10 0.49 0.28
PWP 0.04 8.87* 1.81 2.94 0.05 0.67 0.25 0.70
LS PS 1.33 0.36 0.36 0.02 5.48* 1.07 3.26 0.08
FC 9.90 0.74 0.52 0.13 5.06* 1.13 7.65* 7.29*
PWP 0.37 0.18 0.04 0.10 1.47 0.84 0.90 0.13
SL PS 2.74 3.19 4.68* 2.34 3.89 1.65 3.44 1.39
FC 8.86* 7.86* 10.93* 13.06* 17.07* 2.46 13.91* 11.32*
PWP 7.01* 4.48* 0.01 2.04 16.66* 2.84 12.30* 2.84
L PS 0.27 0.01 0.71 0.02 1.45 2.14 0.17 5.15*
FC 8.78* 3.16 4.93* 0.12 8.60* 5.29* 8.81* 5.46*
PWP 0.54 0.29 0.39 1.56 2.49 5.71* 0.15 2.40
141
Figure 1. Effects of soil pH on root rot of soybean caused by F. graminearum. Average
values of visual disease evaluations over A, soil moisture content. B, soil texture. Means with
the same letter are not significantly different according to Fisher’s protected least significant
difference (P < 0.05).
PS = pot saturation, FC = field capacity, PWP = permanent wilting point
Comparisons were made separately by pH level on the arcsin transformed data. Data
represents values of two experimental replications. Analysis did not included data from non-
infested plants.
142
Figure 2. Combined effects of soil texture and soil moisture content on root rot of soybean
caused by F. graminearum. Average values of visual disease evaluations on A, soil at pH 6.
B, soil at pH 8. Means with the same letter are not significantly different according to
Fisher’s protected least significant difference (P < 0.05).
PS= pot saturation, FC= field capacity, PWP= permanent wilting point
Comparisons were made separately by pH level on the arcsin transformed data. Data
represents values for two experimental replications and do not include data from non-infested
plants.
143
Figure 3. Comparisons between infested and non-infested treatments on plant characteristics
on the main effects of soil texture, and soil moisture. A, root length (cm). B, foliar area
(cm2). C, root dry weight (g). D, shoot dry weight (g). E, root volume (cm3). F, root tips. *
indicates significant differences from the non-infested control (P = 0.05) with a T-test for
mean separation.
144
PS= pot saturation, FC= field capacity, PWP= permanent wilting point
Non-infested control contained sterile non-infested millet (10% v/v)
Mean comparisons were performed individually by soil moisture on each soil texture.
Analyses were performed on the square-root transformed data. Data represents values for two
experimental replications.
145
Figure 4. Percentage of growth reduction relative to the non-infested control on plant
variables. Differences in plant characteristics for the interaction between the main effects of
soil texture and soil moisture. A, root length. B, foliar area. C, root dry weight. D, shoot dry
weight. E, root volume. F, root tips. Means with the same letter are not significantly different
according to Fisher’s protected least significant difference (P < 0.05).
146
PS= pot saturation, FC= field capacity, PWP= permanent wilting point
Non-infested control = soil containing sterile non-infested millet (10% v/v)
Analyses were performed on the square-root transformed data. Data represents values for two
experimental replications.
147
CHAPTER 5. CONCLUSIONS AND RECOMMENDATIONS
The objectives of this dissertation were to characterize the biology of Fusarium
oxysporum and determine its role as a soybean seedling pathogen in the Fusarium root rot
complex. Additionally, these studies had the objective to determine the impact of
environmental factors, including pH, temperature, soil texture, and soil water content, on the
development of soybean root rot caused by F. oxysporum and F. graminearum under
laboratory and greenhouse conditions.
Pathogenicity data collected from a rolled towel and a petri dish assay, including 11
soybean cultivars and 14 F. oxysporum isolates provided valuable information on the
diversity of F. oxysporum collected from soybean roots in Iowa, demonstrating that isolates
varied phenotypically. Significant variation in cultivar susceptibility and isolate
aggressiveness was observed. Our research is novel in that it tested the interaction of
different cultivars and isolates. Previous reports of root rot of soybean caused by F.
oxysporum included only one cultivar or one isolate on different cultivars, which in most
cases bring incomplete, conflicting or wrong conclusions.
Although results of the petri dish and the rolled towel assays were similar, the
symptoms produced by each isolate sometimes differed between assays. For example, some
isolates caused only root rot symptoms in the rolled towel assay but caused both root rot and
damping-off symptoms in the petri dish assay. This observation raises the questions about
how the presence and interaction of various isolates in each field affects the predominance of
damping-off or root rot symptoms. In addition, there is no information about other Fusarium
spp. and other pathogens such as Pythium or Rhizoctonia interacting with F. oxysporum
148
causing soybean seedling disease. Future research may need to include within and among
species interactions, and determine how abiotic factors influence disease expression.
In growth chamber experiments investigating the effect of temperature and pH on F.
oxysporum fungal growth and soybean root rot, a significant interaction of temperature and
pH was found, with the combination of pH 6 and 25˚C resulting in the greatest fungal growth
and the most severe root. Interestingly, our results for temperature agreed with other studies
on Fusarium wilts performed under growth chamber conditions, but conflicted with previous
studies conducted in field conditions. These findings suggest that temperature fluctuations
and natural edaphic factors, such as soil texture, soil moisture, organic matter, nutrient
availability, and soil microbial populations, may also play an important role in the
development and severity of Fusarium wilts.
A Gaussian model helped to further describe the effects of pH and temperature on
fungal radial growth and root rot severity under controlled conditions. According to the
Gaussian regression analysis, the estimated pH and temperature optimal values for maximal
root rot were equivalent for the pathogenic and non-pathogenic F. oxysporum isolates.
However, inferences can only be made on the F. oxysporum isolates included in our studies.
Therefore, more isolates should be included in this type of pathogenicity studies to validate
these results. In addition, the level of predictability of the model could increase as more
significant variables are added, helping to explain or predict phenomena that occur under
more complex conditions.
Finally, greenhouse experiments testing the effects of soil pH, soil texture and soil
moisture on root rot caused by F. graminearum suggested that disease may be inhibited in
alkaline soils with pH levels around 8. In addition, soil moisture had a significant effect on
149
disease development. Under moisture-limiting conditions, levels of root rot increased
significantly. Conversely, seedlings subjected to soil saturation displayed significantly low
levels of disease. No correlation between disease severity and reduction in plant growth
parameters were observed in our studies. This may be due to the inability of visual disease
evaluations to take into account non-symptomatic root colonization or damage to internal
root vascular tissues. In future studies, it may be useful to measure or quantify root
colonization to estimate endophytic fungal biomass that can be correlated to other plant
growth variables and help to understand detrimental effects of Fusarium wilts on plant hosts.
Overall, the evidence collected in the different experiments contained in this thesis
demonstrated clear effects of temperature, soil pH, soil texture, and soil moisture on
Fusarium root rot. However, more biotic and abiotic variables and their interactions must be
studied in the future to generate useful information for designing management strategies that
can realistically fit in soybean production systems.