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What Lies Beneath: Exploring the Soilborne

Microbial Complex of PrunusReplant Disease

David DollJanuary 22nd, 2008

A little bit about me…• From Southeastern

Indiana• Raised on an apple

and peach orchard• Undergraduate

studies at Purdue University in Plant Biology

• Completed my Master of Science in Plant Pathology at UC Davis under Greg Browne

Prunus Replant Problems• Heavily researched,

starting in the 1940’s• Symptoms appear to be

universal– Stunted shoot growth– Loss of fine feeder roots

• Identified a wide variety of causalities– Abiotic– Biotic

• Disease control through fumigation Healthy almond tree (L) vs. PRD-affected tree (R)

Prunus Replant Problems

0

1000

2000

3000

4000

0.5 1 1.5 2 2.5 3 3.5 4 4.5 5

Root diameter (mm)

Control

Pre-plant chloropcirin

0

1000

2000

3000

4000

0.5 1 1.5 2 2.5 3 3.5 4 4.5 5

Root diameter (mm)

Control

Pre-plant chloropcirin

Marianna 2624 rootstockLovell peach rootstock

Tota

l roo

t len

gth

per s

ampl

e (c

m)

>

(Root length densities, as determined by root excavation and digital imaging)Durham, CA, 2004

Biological Mediated Replant Problems of Prunus

• Plant parasitic nematodes(ring, lesion, root knot), approx. 35% of almond and fresh stone fruit acreage, 60% of cling peach acreage infested (McKenry)

• Aggressive pathogens (i.e., Phytophthora, Armillaria, Verticillium spp.)

• Prunus replant disease (PRD): incidence nearly universal in Prunus planted after Prunus; severity varies greatly (Browne)

Control of Prunus Replant Disease

•Soil from old peach orchard

•Pre-plant fumigation treatments applied to microplots before planting with Nemaguard

Control MBr 400 lb/A MBr 2700 lb/A

Chloropicrin 400 lb/A Chloropicrin 2700 lb/A

At end of first growing season, Nemaguard peach seedlings

The Soilborne Microbial Community: What is that?

• Soil provides a diverse habitat– Arthropods– Bacteria– Fungi– Nematodes– Protozoa– Viruses

• Each member serves a purpose

The Soilborne Microbial Community:Affecting the way Plants Grow

• Conditions determine community shifts– Environmental– Plant Hosts– Community members and

substrate present

• Communities have demonstrated the ability to cause beneficial and negative effects on plant growth.

Examining Etiology of PRD

• Microbial agents that may cause PRD:– Protozoa– Nematodes– Fungi– Bacteria– Arthropods– Viruses

Examining Etiology of PRD

Roots from healthy tree Roots from RD-affected tree

Roots and soil from healthy and RD-affected trees

Whole-organism assays• Direct quantification

(nematodes)• Isolation and quantification

in culture (fungi, bacteria)

Molecular assays• DNA fingerprinting (PCR

of rDNA; fungi, bacteria,• cloning of fragments,

DNA-sequence-based id)

Soil treatments• Semi-

selective chemicals or treatments in Microplot, GH tests

Pathogenicity tests

Nematode involvement in PRD• Butte County orchard RD trials (2001-2004):

– No significant counts of plant parasitic nematodes (3 orchards)– Sugar floatation and mist chamber extractions

• Parlier microplot trials (2002-2005): – Only pin nematode (Paratylenchus sp.) present in significant number (3

repeated trials); not correlated with RD incidence

• Madera County orchard trials (2003- ):– To date, minor or no nematode involvement; sampling continuing

• Conclusion: – RD and nematode parasitism not the same, they are distinct replant problems– Associated with biological agent(s) other than plant parasitic nematodes

Examining Etiology of PRD

• Microbial agents that may cause PRD:

– Nematodes– Fungi– Bacteria– Arthropods

Determining effects of semi-selective soil treatments on severity of PRD

Effect of pre-plant semi-selective treatments on the growth of replanted Nemaguard peach

0 10 20 30 40 50 60 70 80

Com. yeast extract (high)

Com. yeast extract (low)

Lorsban (chloropyriphos)

Ridomil (mefenoxam)

Folicur (tebuconazole)

Cannonball (fludioxonil)

Chloropicrin

Control da

bc

cdd

bcab

Top fresh wt. of plants per plot (g)

Preplant treatment

d

6 Replicates in CRD, One trial year

Examining Etiology of PRD

• Microbial agents that may cause PRD:

– Fungi– Bacteria– Arthropods

Examining Etiology of PRD

Roots from healthy tree Roots from RD-affected tree

Roots and soil from healthy and RD-affected trees

Whole-organism assays• Direct quantification

(nematodes)• Isolation and quantification

in culture (fungi, bacteria)

Molecular assays• DNA fingerprinting (PCR

of rDNA; fungi, bacteria,• cloning of fragments,

DNA-sequence-based id)

Soil treatments• Semi-

selective chemicals or treatments in Microplot, GH tests

Pathogenicity tests

Examination of fungal microbial community shifts involved in PRD.

-0.8 0.8

-0.6

1.0

Cylind

Fusoxy1

Fus1

Fus3Fus4Unk-C

Tricho-2

Acremon

Asper

Control

ChloropicrinControl*Rinse

Control*Bleach

Chloropicrin*Rinse

Chloropicrin*Bleach

t N=175

•Redundancy analysis of fungal communities associated with PRD using culture based technique, at right are shown shifts in fungal community in the 2007 Firebaugh trial.

Examination of fungal microbial community shifts involved in PRD.

•Redundancy analysis of fungal communities associated with PRD using culture based technique, at right are shown shifts in fungal community in the Durham 2004 trial.

-1.0 1.0

-1.0

1.0

Alt

Cyl

FusUkn

FusAFusCFusD

FusE

FusH

FusIGlio

MortPythRhizoc

UnkgenUnkA

Control

Chloropicrin

Control*Bleach

Control*Rinse

Chloropicrin*Bleach

Chloropicrin*Rinse

N=4155

Examination of bacterial microbial community shifts involved in PRD.

Redundancy analysis of bacterial communities associated with PRD using culture based technique, at right are shown shifts in fungal community in the Parlier 2003 and 2004 trial.

-1.0 1.0

-1.0

1.0

Arthro-2Bacill-4

Bacill-6

EnteroFlav-4 Microb-1

Microb-3

Pseud-1RhizA-2

Pseud-2

Pseud-3

Rhiz-1RhizA-1

Vario-1

Control

Chloropicrin

Methyl Bromide

Control*Bleach

Control*Rinse

Chloropicrin*Rinse

Chloropicrin*Bleach

Methyl Bromide*Bleach

Methyl Bromide*Rinse

n=259

-1.0 1.0

-1.0

1.0

Arthro-2

Bacill-4

Bacill-5Bacill-6

Lyso-2

Microb-1

Nocard-2

PhylloPseud-1

Pseud-2

Pseud-3

Rhiz-1

RhizA-1RhizA-2Vario-1

Control

Chloropicrin

Methyl Bromide

Control*Rinse

Control*Bleach

Chloropicrin*BleachChloropicrin*Rinse

Methyl Bromide*Rinse

Methyl Bromide*Bleach

n=351

2003 Trial Year 2004 Trial Year

Greenhouse Experiments: Application of Associated Bacteria

Bacterial isolates were applied to sterilize and PRD-affected soil.

Nemaguard peach seedlings were planted immediately after application of bacteria.

Greenhouse Experiments:Disease Expression in the Greenhouse

0 5 10 15 20 25 30 35

SterilizedSoil

PRD-affectedSoil

Height of Plants (cm)

Greenhouse Experiments: Application of Associated Bacteria

0 5 10 15 20 25 30 35 40

Control

Flav

Pseud-1

Pseud-c

Vario-1

Vario-c

Rhiz-1

Rhiz-c

RhizA-1

RhizA-c

Plant Height (cm)PRD-AffectedSterile

•Redundancy analysis of culture independent bacterial communities associated with PRD using culture based technique, at right are shown shifts in bacterial community in the Parlier 2003 trial.

Examination of Culture Independent Bacterial community shifts involved in PRD.

N=4155

-1.0 1.0

-1.0

1.0

Anaer0-1TM7-3Lyso-2

Xanth-1

Gamma-2

Methb

Hydrog-3

Vario-2

Acidov-0

Gamma-4Caulo-1

Rhiza-4Microb-2

Actpl-1

Microb-1

Cellv-2

Pseud-21

Methph-5

Comam-2

Xanth-3Rhiz-6

Brevun-2

Control

Chloropicrin

Methyl Bromide

N=497

Examining Etiology of PRD

• Microbial agents that may cause PRD:

– Fungi– Bacteria

Conclusions• Replant disease (RD) of almond is a biologically

induced disease showing poor growth or survival in almond planted after Prunus. – Prevented by pre-plant fumigation with fumigants

containing Chloropicrin.

• Some fungi and bacteria are being implicated, multiple approaches are being used to determine causal agents and developing greenhouse assays.

• An unfavorable microbial community may be responsible for the disease:– Shifting the microbial community in favor of the newly

planted Prunus sp. tree may provide some level of disease control.

– Pre-plant Application of treatments or cover cropping may provide alternative control reducing the use of fumigants

Thank You!

Project involved multiple collaborations with UC, UCCE, USDA-ARS scientists, UC Faculty, Growers, Private Industry, and Funding sources.