What Lies Beneath: Exploring the Soilborne Microbial Complex of Prunus Replant Disease. David Doll January 22nd, 2008

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1 What Lies Beneath: Exploring the Soilborne Microbial Complex of Prunus Replant Disease David Doll January 22nd, 2008

2 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

3 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)

4 Prunus Replant Problems Total root length per sample (cm) Lovell peach rootstock Marianna 2624 rootstock Control Pre-plant chloropcirin Root diameter (mm) > Control Pre-plant chloropcirin Root diameter (mm) (Root length densities, as determined by root excavation and digital imaging) Durham, CA, 2004

5 Biological Mediated Replant 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) Problems of Prunus

6 Control of Prunus Replant Disease Control MBr 400 lb/a MBr 2700 lb/a Soil from old peach orchard Pre-plant fumigation treatments applied to microplots before planting with Nemaguard Chloropicrin 400 lb/a Chloropicrin 2700 lb/a At end of first growing season, Nemaguard peach seedlings

7 The Soilborne Microbial Community: What is that? Soil provides a diverse habitat Arthropods Bacteria Fungi Nematodes Protozoa Viruses Each member serves a purpose

8 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.

9 Examining Etiology of PRD Microbial agents that may cause PRD: Protozoa Nematodes Fungi Bacteria Arthropods Viruses

10 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) Pathogenicity tests Molecular assays DNA fingerprinting (PCR of rdna; fungi, bacteria, cloning of fragments, DNA-sequence-based id) Soil treatments Semiselective chemicals or treatments in Microplot, GH tests

11 Nematode involvement in PRD Butte County orchard RD trials ( ): No significant counts of plant parasitic nematodes (3 orchards) Sugar floatation and mist chamber extractions Parlier microplot trials ( ): 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

12 Examining Etiology of PRD Microbial agents that may cause PRD: Nematodes Fungi Bacteria Arthropods

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

14 Effect of pre-plant semi-selective treatments on the growth of replanted Nemaguard peach Preplant treatment Control Chloropicrin Cannonball (fludioxonil) Folicur (tebuconazole) Ridomil (mefenoxam) Lorsban (chloropyriphos) Com. yeast extract (low) Com. yeast extract (high) d d d cd bc bc ab a Top fresh wt. of plants per plot (g) 6 Replicates in CRD, One trial year

15 Examining Etiology of PRD Microbial agents that may cause PRD: Fungi Bacteria Arthropods

16 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) Pathogenicity tests Molecular assays DNA fingerprinting (PCR of rdna; fungi, bacteria, cloning of fragments, DNA-sequence-based id) Soil treatments Semiselective chemicals or treatments in Microplot, GH tests

17 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 2007 Firebaugh trial. t Tricho-2 Chloropicrin*Bleach Chloropicrin Fus4 Unk-C Acremon Fus3 Control*Rinse Chloropicrin*Rinse N=175 Asper Fusoxy1 Control Control*Bleach Cylind Fus

18 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 Chloropicrin*Bleach Chloropicrin Chloropicrin*Rinse UnkA Glio FusI FusUkn FusH Mort Pyth Rhizoc Alt FusE FusD Unkgen FusA FusC Cyl N=4155 Control*Rinse Control Control*Bleach

19 Examination of bacterial microbial community shifts involved in PRD Methyl Bromide Bacill-5 Bacill-6 Methyl Bromide*Rinse Microb-1 Bacill-4 Pseud-1 Phyllo Arthro-2 Nocard-2 Vario-1 Pseud-2 Chloropicrin Chloropicrin*Rinse n=351 Pseud-3 Lyso-2 Chloropicrin*Bleach Control*Rinse Methyl Bromide*Bleach Rhiz-1 Control RhizA-1 RhizA-2 Control*Bleach Control*Bleach Rhiz-1 RhizA-1 Control RhizA-2 Pseud-2 Pseud-1 Entero Methyl Bromide Flav-4 Chloropicrin*Bleach Microb-1 Bacill-6 Vario-1 Control*Rinse Microb-3 Chloropicrin Pseud-3 Arthro-2 Bacill-4 Methyl Bromide*Rinse n=259 Chloropicrin*Rinse Methyl Bromide*Bleach Trial Year 2004 Trial Year 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.

20 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.

21 Greenhouse Experiments: Disease Expression in the Greenhouse PRD-affected Soil Sterilized Soil Height of Plants (cm)

22 Greenhouse Experiments: Application of Associated Bacteria RhizA-c RhizA-1 Rhiz-c Rhiz-1 Vario-c Vario-1 Pseud-c Pseud-1 Flav Control PRD-Affected Plant Height (cm) Sterile

23 Examination of Culture Independent Bacterial community shifts involved in PRD. 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 Pseud-21 Cellv-2 Vario-2 Microb-2 Brevun-2 Acidov-0 Actpl-1 Hydrog-3 Xanth-1 Methph-5 Gamma-4 Caulo-1 Xanth-3 Rhiz-6 Microb-1 Lyso-2 Methb Chloropicrin Methyl Bromide N=4155 N=497 Rhiza-4 Comam-2 Gamma-2 TM7-3 Anaer0-1 Control

24 Examining Etiology of PRD Microbial agents that may cause PRD: Fungi Bacteria

25 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

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