Mechanisms of Broccoli-Mediated Soilborne Pathogen and Disease Suppression in Vegetable Cropping Systems K. V. Subbarao

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1 Mechanisms of roccoli-mediated Soilborne Pathogen and Disease Suppression in Vegetable Cropping Systems K. V. Subbarao Department of Plant Pathology University of California, Davis

2 Focus Genesis of the idea Proof of concept Mechanisms

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4 ackground Extensive surveys revealed the high degree of susceptibility of cauliflower and near-immunity of broccoli to Verticillium wilt. Verticillium dahliae isolates from crucifer crops were weakly pathogenic on broccoli in greenhouse tests, and others were nonpathogenic. Dry crucifer residues are better than fresh residue.

5 Reaction of broccoli to Verticillium dahliae from various hosts Disease Dry weight (g) Isolate severity Root Shoot Cauliflower Chili pepper Artichoke Cabbage Lettuce Strawberry ell pepper Eggplant Tomato Watermelon Mint Potato Cotton Alfalfa (albo-atrum) Control LSD (P 0.05) Cauliflower

6 Questions When should the broccoli crop be planted and residue incorporated? Is there a specific temperature at which propagule reduction by broccoli is maximized? Is dry broccoli better than fresh broccoli?

7 Methods Soil: Fields 1 & 2 Treatments: Fresh (8%), dry, and no broccoli in 25 g dry soil. Incubation: 10, 15, 20, 25, 30, & 35 C Assay: After 45 days incubation using the Anderson sampler technique. In a parallel set of experiments, soil sampled after 15, 30, and 45 days incubation and assayed.

8 Control D F Temperature ( o C)

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13 Are all broccoli varieties same?

14 Other Applications of this Research Efficacy of roccoli - Strawberry Rotation Conventional versus organic production systems Reductions in Sclerotinia minor sclerotia and lettuce drop incidence.

15 LL LLLL

16 Potential Mechanisms Why is broccoli resistant to V. dahliae infection? ow does broccoli result in attrition of propagules? Chemical Microbial Specific to pigmented propagules?

17 Colonization of Root Cortex roccoli and Cauliflower igh and low inoculum density soils. roccoli-amended and unamended. Periodic root sampling. Immunohistochemical staining assay

18 Verticillium dahliae colonies m No-roc roc 0 Cauliflower roccoli ost plant and broccoli amendment

19 0.14 Verticillium dahliae colonies m -1 ID -1 No-roc 0.12 roc Cauliflower roccoli ost plant and broccoli amendment

20 Cauliflower roccoli

21 Major Classes of Glucosinolates C 3 -(C 2 ) n -R alkyl- C 2 = C - (C 2 ) n -R C 2 = C - CO - (C 2 ) n -R C 3 -SO x -(C 2 ) n -R alkenyl- β-o alkenyl- methylthioalkyl- N C 2 -R indolylmethyl- (C 2 ) n -R aryl- COO - (C 2 ) n -R benzoyloxy-

22 Glucosinolates breakdown to isothiocyanates through the action of Myrosinase R C S C O N OSO O 2 - O 2 Glucose R C - S - N OSO 2 O Sulphate R N C S This can occur in the plant, and possibly in an animal s mouth or gut.

23 Glucosinolate variation among Cruciferous vegetables progoitrin glucoraphanin glucobrassicin sinigrin 0 roccoli russels Cabbage Cauliflower Kale Jeffery, U. Illinois

24 Sulforaphane (SF) O S N C S Anticarcinogenic hydrolysis product of Glucoraphanin Increases detoxification enzymes that remove carcinogens Potent upregulator of Quinone Reductase, in vitro

25 Summary of cover crop biomass and equivalent metam sodium content Treatment iomass (T/A) N (lb/a) Metam sodium Eq (gal/a) 2005 roccoli White Mustard Indian Mustard Labelled rates of metam sodium gal/a

26 Soil Microbiological Changes Significant differences between broccoli and cauliflower. roccoli residue incorporation results in 100-fold increases in bacteria and 1000-fold increase in actinomycetes. Not only these changes are quantitative but also qualitative. The diversity of these groups is highest in broccoli-amended soils compared with cauliflowerresidue amended soils.

27 Population dynam ics of m icroflora in broccoli season in 1995 J ROC CK NO-ROC J J J J J J J J J J Actinom ycetes J J J J J J acteria J J J J J J J 2.5 J 2 Fungi Days after transplanting

28 Log cfu of microorganisms per g soil Population dynam ics of m icroflora in cauliflow er season in 1995 J J J J J J J J J J J J J Days after planting Actinom ycetes J J J ROC CK J J NO-ROC acteria J 3.9 Fungi J J J

29 acterial diversity - roccoli Arthrobacter saperdae A. barkeri A. oxydans A. ramosus A. viscosus Aureobacterium esteraromaticum acillus circulans. atrocyaneus. brevis. lentus. pantothenticus. psychrophilus. pumilus Micrococcus lylae M. kristinae M. roseus Pseudomonas florescens Stephylococcos epidermis S. cohnii Stenotrophomonas maltophilia

30 acterial diversity non-broccoli acillus chlororaphis. laterosprus. linens. psychrophilus Curtobacterium inologenes C. flaccumfaciens Micrococcus halobius Pseudomonas putida

31 Myxobacteria Gram negative, gliding bacteria Produce swarms on nutrient poor media Produce characteristic fruiting bodies with dormant myxospores Lytic activity on microorganisms and nematodes

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33 Myxobacteria Myxococcus coralloides M. fulvus M. virescens M. xanthus (2) M. stipitatus M. flavascense

34 Melanin Dark pigment molecule composed of polymers with a complex structure closely related to plant lignins Occurs in microorganisms, animals, protozoa and plants Melanin is closely associated with cell wall components

35 Abiotic Stress enefits of Melanin UV radiation, temperature extremes, desiccation, free radicals, metal toxicity iotic Stress Soil microbial/host plant origin hydrolytic enzymes and free radicals Pathogenicity Appressorial penetration, virulence factor?

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37 Control plot (minus roccoli) Plot rotated with roccoli

38 Research Team Chang-Lin Xiao Li Liu Ashley ell Ravi hat Kateel Shetty Judy ubbard Steve Koike Qingming Qin Gary Vallad Cauliflower growers USDA-NRICGP USDA-CSREES USDA-SARE CA-DPR CA Lettuce Research oard CA Strawberry Commission UCIPM

39 Transcriptional Regulation by the glucosinolate ydrolysis Product sulforaphane (SF) : SF ARE ARE: 5 -TGACnnnGC-3 Antioxidant Response Element +1 Quinone Reductase Glutathione S- transferase Glucuronosyl transferase Aldehyde dehydrogenase