Transcript Document

Microbes and Mulches
Marianne Powell
Masters Student
Dr. Debra Inglis, Graduate Advisor
Dr. Marion Brodhagen, Committee Member; USDA Lab Host
Dr. Carol Miles, Committee Member
Dr. Karen Leonas, Materials Lab Host
Graduate Student Symposium
SCRI Team Meeting Lubbock, TX
June 2, 2011
1
Today’s Outline
I.
Isolation of potential biodegradable mulch utilizing microbes from TN,
TX, and WA mesh bag studies
A. Colony forming units per gram of mulch
B. Number of isolates that degraded BDMs
C. Conclusions from the Mesh Bag Study
II. 2010 WA disease assessments in the field over time
A. Tomato diseases and disorders in the BDM experiment
B. Koch’s Postulates of Verticillium wilt on lettuce
C. Conclusions from the 2010 growing season
2
2
Mesh Bag Study: Isolate Microbes Utilizing BDM
Material as C Source
Visit to Brodhagen laboratory at USDA
ARS Forage Seed and Cereal Research Unit, Corvallis, OR
3
3
BDM Samples Examined
4
4
Microbe Isolation and BDM Biodegradation Assay
1
3
2
5
4
Dilution plates
Multipipetter
Serial dilutions
0.5 g mulch in PBS
6
After incubation
Minimal media with BDM
sample
5
5
1: WA/IN/SB Fungi
1st
BDM Test
2: TN/IN/BT #9 Bacteria
1000X, ˽ = 3.0 µm
3: TX/OUT/BB #2 Fungi
4: TN/IN/BT #9 Bacteria
20X, growing on mulch
75X,growing on mulch
6
6
TN Mesh Bag Study:
Culturable Bacteria
TX Mesh Bag Study:
Culturable Bacteria
9.0E+08
9.0E+08
8.0E+08
7.0E+08
CFU/g mulch, +/- S.E.
CFU/g mulch, +/- S.E.
8.0E+08
6.0E+08
5.0E+08
4.0E+08
3.0E+08
TN/IN
2.0E+08
TN/OUT
1.0E+08
7.0E+08
6.0E+08
5.0E+08
4.0E+08
3.0E+08
TX/IN
2.0E+08
TX/OUT
1.0E+08
0.0E+00
Cellulose
BioTelo
Biobag
SB-PLA
0.0E+00
Soil only
Cellulose
BioTelo
Treatment
1.5E+07
1.5E+07
1.0E+07
TN/IN
TN/OUT
5.0E+06
0.0E+00
Treatment
SB-PLA
Soil only
CFU/g mulch, +/- S.E.
CFU/g mulch, +/- S.E.
2.0E+07
Biobag
Soil only
TX Mesh Bag Study: Culturable Fungi
2.0E+07
BioTelo
SB-PLA
Treatment
TN Mesh Bag Study: Culturable Fungi
Cellulose
Biobag
1.0E+07
TX/IN
TX/OUT
5.0E+06
0.0E+00
Cellulose
BioTelo
Biobag
Treatment
SB-PLA
Soil only
77
Fifty microbes capable of utilizing BDMs were
obtained from TN and TX.
Number of fungal isolates that degrade BDMs
Mulch type TN/IN TN/OUT TX/IN TX/OUT SUM
CC
5
1
1
1
8
SB
5
3
2
6 16
BB
3
1
2
3
9
BT
6
0
4
1 11
Number of bacterial isolates that degrade BDMs
Mulch type TN/IN TN/OUT TX/IN TX/OUT SUM
CC
3
0
2
0
5
SB
4
0
1
1
6
BB
2
2
0
1
5
BT
1
1
0
1
3
Bacteria and Fungi
Grand Total
13
22
14
14
36 fungal and 14 bacterial isolates have been
shown to degrade SB, BB, and BT.
WA samples are still in progress.
8
Biodegradation Assay Conclusions
• We have developed a novel assay for testing
BDM utilization by soil bacteria and fungi.
• We have isolated native bacteria and fungi
capable of degrading each of the BDMs from
TN and TX.
TN/IN/BT/ #15
Fungi
• The Brodhagen lab will confirm our initial
results in future tests and identify microbes
with universal bacteria and fungal primers
• Some of these isolates are likely to be
redundant (e.g. same species).
• In future studies, BDM degradation by these
microbes could potentially be tested via
composting studies - ASTM D5338 (2003)
9
9
Tomato Disease Ratings in WA BDM Study
• Weekly visual assessments of disease incidence and severity
Physiological leaf roll
Leaf mold suspect (HT)
Verticillium wilt suspect (HT)
10
10
Culturing Fungal Organisms
from Symptomatic Tissues
Ulocladium spp.
ITS-PCR sequencing
Botrytis spp.
Morphology ID
Fulvia fulva
Morphology ID
11
11
AUDPC for Physiological Leaf Roll
Mulch
Biobag
High Tunnel
Open Field
2529
1954 c
BioTelo
2381
2011 c
Cellulose
control
2409
1543 b
Non-BDM
2334
2146 c
BT
BB
SB
Non-mulch
2053
488 a
Spunbond
PLA
2170
637 a
P Value
NSD
NM
<.0001
12
12
Pathogens in Nearby Plots
Lettuce is susceptible to Verticillium wilt, caused by V. dahliae;
we isolated and confirmed (ITS) V. tricorpus.
13
13
Source of Verticillium sp. on Lettuce?
Seedborne?
Soilborne?
Soil dilution plates from
plots with diseased lettuce
Tested seeds from lettuce
seed lot for 2010 planting
V. Colonies
growing on NP-10
Counting V. spp.
14
14
Lettuce: Verticillium Assays
From the seed?
From the soil?
Cultivar
V. dahliae
V. tricorpus
Plot
V. dahliae
CFU/g soil
V. tricorpus
CFU/g soil
Coastal
Star
0
0
IN
7
1
Jericho
Star
0
0
OUT
4
0
15
15
Completing Koch’s Postulates on Lettuce
Grow inoculum
Inoculate seedling
Evaluate disease
Culture and characterize
16
16
Conclusions for 2010
• V. tricorpus isolated from lettuce in 2010 is pathogenic on lettuce
and this is a first report for WA state (Japan)
• The non-mulch and spundbond-PLA treatments in the WA open
field plots showed reduced physiological leaf roll severity and this
could be an important clue to as what causes the disorder, which is
currently unknown.
• No BDM treatment differences to leaf mold (F. fulva) noted on
tomato cultivar ‘Celebrity’, but Celebrity showed resistance in 2010
(Inglis tomato cultivar study finding).
17
17
Special Thanks
• Dr. Debra Ann Inglis
• Babette Gunderson
• Jeremy Cowen
• Dr. Marion Brodhagen
• Ashley Florence
• Carl Evans
18