What Lies Beneath: Exploring the Soilborne Microbial Complex of Prunus Replant Disease David Doll January 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 Total root length per sample (cm) Lovell peach rootstock Marianna 2624 rootstock 4000 3000 Control Pre-plant chloropcirin 2000 1000 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 Root diameter (mm) > 4000 3000 Control Pre-plant chloropcirin 2000 1000 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 Root diameter (mm) (Root length densities, as determined by root excavation and digital imaging) Durham, CA, 2004
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
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
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) 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
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 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 0 10 20 30 40 50 60 70 80 Top fresh wt. of plants per plot (g) 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) 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
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 -0.6 1.0 Tricho-2 Chloropicrin*Bleach Chloropicrin Fus4 Unk-C Acremon Fus3 Control*Rinse Chloropicrin*Rinse N=175 Asper Fusoxy1 Control Control*Bleach Cylind Fus1-0.8 0.8
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 Chloropicrin*Bleach Chloropicrin Chloropicrin*Rinse UnkA Glio FusI FusUkn FusH Mort Pyth Rhizoc Alt FusE FusD Unkgen FusA FusC -1.0 1.0 Cyl N=4155 Control*Rinse Control Control*Bleach
Examination of bacterial microbial community shifts involved in PRD. -1.0 1.0 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 -1.0 1.0-1.0 1.0 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 -1.0 1.0 2003 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.
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 PRD-affected Soil Sterilized Soil 0 5 10 15 20 25 30 35 Height of Plants (cm)
Greenhouse Experiments: Application of Associated Bacteria RhizA-c RhizA-1 Rhiz-c Rhiz-1 Vario-c Vario-1 Pseud-c Pseud-1 Flav Control 0 5 10 15 20 25 30 35 40 PRD-Affected Plant Height (cm) Sterile
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. -1.0 1.0 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 -1.0 1.0
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.