Microbes and MulchesMarianne Powell

Masters Student

Dr. Debra Inglis, Graduate AdvisorDr. Marion Brodhagen, Committee Member; USDA Lab Host

Dr. Carol Miles, Committee MemberDr. Karen Leonas, Materials Lab Host

Graduate Student SymposiumSCRI Team Meeting Lubbock, TX

June 2, 2011

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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

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Mesh Bag Study: Isolate Microbes Utilizing BDM Material as C Source

Visit to Brodhagen laboratory at USDAARS Forage Seed and Cereal Research Unit, Corvallis, OR

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BDM Samples Examined

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Microbe Isolation and BDM Biodegradation Assay

1 2 3

4 5 6

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0.5 g mulch in PBS Serial dilutions Multipipetter

Dilution plates After incubationMinimal media with BDM sample

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4: TN/IN/BT #9 Bacteria75X,growing on mulch

2: TN/IN/BT #9 Bacteria 1000X, ˽ = 3.0 µm

3: TX/OUT/BB #2 Fungi20X, growing on mulch

1: WA/IN/SB Fungi 1st BDM Test

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Cellulose BioTelo Biobag SB-PLA Soil only0.0E+00

1.0E+08

2.0E+08

3.0E+08

4.0E+08

5.0E+08

6.0E+08

7.0E+08

8.0E+08

9.0E+08

TN Mesh Bag Study: Culturable Bacteria

TN/INTN/OUT

Treatment

CFU/

g m

ulch

, +/-

S.E

.

Cel-lu-

lose

BioTelo

Biobag

SB-PLA

Soil only

0.0E+00

5.0E+06

1.0E+07

1.5E+07

2.0E+07TN Mesh Bag Study: Culturable Fungi

TN/IN

TN/OUT

Treatment

CFU/

g m

ulch

, +/-

S.E

.

Cellulose BioTelo Biobag SB-PLA Soil only0.0E+00

1.0E+08

2.0E+08

3.0E+08

4.0E+08

5.0E+08

6.0E+08

7.0E+08

8.0E+08

9.0E+08

TX Mesh Bag Study:Culturable Bacteria

TX/INTX/OUT

Treatment

CFU/

g m

ulch

, +/-

S.E

.

Cellulose BioTelo Biobag SB-PLA Soil only0.0E+00

5.0E+06

1.0E+07

1.5E+07

2.0E+07

TX Mesh Bag Study: Culturable Fungi

TX/INTX/OUT

Treatment

CFU/

g m

ulch

, +/-

S.E

.

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Number of fungal isolates that degrade BDMs Number of bacterial isolates that degrade BDMs Bacteria and FungiMulch type TN/IN TN/OUT TX/IN TX/OUT SUM Mulch type TN/IN TN/OUT TX/IN TX/OUT SUM Grand Total

CC 5 1 1 1 8 CC 3 0 2 0 5 13SB 5 3 2 6 16 SB 4 0 1 1 6 22BB 3 1 2 3 9 BB 2 2 0 1 5 14BT 6 0 4 1 11 BT 1 1 0 1 3 14

Fifty microbes capable of utilizing BDMs were obtained from TN and TX.

36 fungal and 14 bacterial isolates have been shown to degrade SB, BB, and BT.

WA samples are still in progress.8

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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.

• 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)

TN/IN/BT/ #15 Fungi

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Tomato Disease Ratings in WA BDM Study• Weekly visual assessments of disease incidence and severity

Physiological leaf roll

Verticillium wilt suspect (HT)Leaf mold suspect (HT)

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Culturing Fungal Organisms from Symptomatic Tissues

ITS-PCR sequencing Morphology ID Morphology ID

Ulocladium spp. Botrytis spp. Fulvia fulva

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AUDPC for Physiological Leaf RollMulch High Tunnel Open Field

Biobag 2529 1954 c

BioTelo 2381 2011 c

Cellulose control

2409 1543 b

Non-BDM 2334 2146 c

Non-mulch 2053 488 a

Spunbond PLA

2170 637 a

P Value NSD <.0001

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BT

BB

SB

NM

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Pathogens in Nearby Plots

Lettuce is susceptible to Verticillium wilt, caused by V. dahliae; we isolated and confirmed (ITS) V. tricorpus.

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Source of Verticillium sp. on Lettuce?

Seedborne? Soilborne?

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Tested seeds from lettuce seed lot for 2010 planting

Soil dilution plates from plots with diseased lettuce

Counting V. spp.V. Colonies growing on NP-10

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Lettuce: Verticillium Assays

From the seed? From the soil?Cultivar V. dahliae V. tricorpus

Coastal Star

0 0

Jericho Star

0 0

Plot V. dahliaeCFU/g soil

V. tricorpusCFU/g soil

IN 7 1

OUT 4 0

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Completing Koch’s Postulates on Lettuce

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Grow inoculum Inoculate seedling

Evaluate disease Culture and characterize

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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).

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Special Thanks

• Dr. Debra Ann Inglis• Babette Gunderson• Jeremy Cowen

• Dr. Marion Brodhagen• Ashley Florence• Carl Evans