What Lies Beneath: Exploring the Soilborne ... - Stanislaus...
Transcript of What Lies Beneath: Exploring the Soilborne ... - Stanislaus...
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.