1. 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

2. 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

3. 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

4. BDM Samples Examined 4 4

5. Microbe Isolation and BDM Biodegradation Assay 1 2 3 4 56 5 5 0.5 g mulch in PBS Serial dilutions Multipipetter Dilution plates After incubation Minimal media with BDM sample

6. 4: TN/IN/BT #9 Bacteria 75X,growing on mulch 2: TN/IN/BT #9 Bacteria 1000X, ˽ = 3.0 µm 3: TX/OUT/BB #2 Fungi 20X, growing on mulch 1: WA/IN/SB Fungi 1 st BDM Test 6 6

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8. 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

9. 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 9 9

10. 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) 10

11. Culturing Fungal Organisms from Symptomatic Tissues ITS-PCR sequencing Morphology ID Morphology ID 11 Ulocladium spp. Botrytis spp.Fulvia fulva 11

12. AUDPC for Physiological Leaf Roll MulchHigh TunnelOpen Field Biobag25291954 c BioTelo23812011 c Cellulose control 24091543 b Non-BDM23342146 c Non-mulch2053488 a Spunbond PLA 2170637 a P ValueNSD<.0001 12 BT BB SB NM

13. Pathogens in Nearby Plots Lettuce is susceptible to Verticillium wilt, caused by V. dahliae; we isolated and confirmed (ITS) V. tricorpus. 13

14. Source of Verticillium sp. on Lettuce? Seedborne? Soilborne? 14 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

15. Lettuce: Verticillium Assays From the seed?From the soil? CultivarV. dahliaeV. tricorpus Coastal Star 00 Jericho Star 00 Plot V. dahliae CFU/g soil V. tricorpus CFU/g soil IN71 OUT40 15

16. Completing Koch’s Postulates on Lettuce 16 Grow inoculum Inoculate seedling Evaluate disease Culture and characterize

17. 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

18. Special Thanks Dr. Debra Ann Inglis Babette Gunderson Jeremy Cowen Dr. Marion Brodhagen Ashley Florence Carl Evans 18