Diversity, Molecular Characterization, and Fungicide Sensitivity of Phytophthora ssp. causing damping off blight and root rot in floriculture crops

Dr. Michael Benson North Carolina State University

1. Executive Summary

   A collaborative project between NC State University (Mike Benson) and Michigan State University (Mary Hausbeck) as well as researchers at other universities has been established to characterize the diversity of P/iytophi/zora spp. in the floriculture industry, validate the use of AFLP markers as a molecular tool to track sources of Phytophthora spp., and determine fungicide resistance management strategies. A collection of Phylophihoraspp. isolates will be made from floriculture crops in the United States by cooperators in NY, SC, MI, PA as well as NC. Isolates will be sent to NC State University for species level identification and mating type determination. Isolates will be compared to type cultures from major Phyiophthora repositories such as UC-Riverside, and CMI, UK. Isolates will be tested for sensitivity to mefenoxam, the most widely used fungicide for Phytophihora control in the floriculture industry to evaluate the usefulness of current disease management recommendations. Populations of isolates from specific cooperators will be fingerprinted by AFLP analysis for genetic diversity and origin.

   After the work is completed we will have a much better picture of the relative importance of different Phzytophthora species affecting specific floriculture crops. The use of AFLP fingerprinting as a tool to trackpopulations of Phyiophthora strains in the industry should be a major contribution to understanding the genetic variation and occurrence of Phytophthora species important in the industry. The movement of plant material in the floriculture industry is intense, by understanding the occurrence and importance of introduced strains into greenhouse operations, better disease management programs can be targeted where they will have the most impact on crop health. In regions of the US where floriculture, tobacco and fruit crop production
   co-exist, the ability to differentiate strains of Phytophihora spp. should enhance our understanding of the need to be good stewards of crop health in the floriculture industry.

2. Introduction & Literature Review.

   Phytophthora diseases including damping-off; blight, and root and crown rot are a serious problem in floriculture crops. At the recent SAF Research Initiative Symposium in Chicago, research on root pathogens including Phytophthora spp. received the highest priority for funding. Although several species of Phytophthora attack nursery crops, only P. cryptogea and P. parasitica have been reported on floriculture crops (Farr et al, 1989). Phytophihora cryptogea, first reported on Transvaal daisy (Gerbera jamesonii) in 1937 in California (Tompkins and Tucker, 1937), is now known on over 26 floriculture and cut flowers crops including African violets, mums, snaps, and gloxinia (Erwin and Ribeiro, 1996). This heterothallic fungus is very similar morphologically to P. drechsleri and survives as sporangia or as oospores. Efforts to separate isolates previously designated as P. cryptogea or P. drechsleri by isozyme and mtDNA RFLP analysis were incomplete due to species variability (Mills et. al. 1991). Bewley and Buddin (1921) recovered P. cryptogea in greenhouse water supplies 80 years ago and this source of inoculum is no doubt important today even though growers have moved away from soil-based potting mixes.

   Phytophihoraparasitica
   Dastur (1913) [syn. P. nicotianae Breda de Haan] has the largest host range of any species of Phytophihora yet described that includes poinsettia and several bedding plants (Erwin and Ribeiro, 1996, Fan et al. 1989). Clinic records in North Carolina indicate that P. parasitica is the most frequently encountered species of Phytophihora associated with floriculture crops. Abundant sporangium production and zoospore release by isolates ofF. parasitica as well as a high temperature optimum for growth are associated with the success of this pathogen in ornamental crops.

   AFLP fingerprinting (Vos et al, 1995) provides a powerful tool for investigating genetic diversity and hence, population dynamics of Phyiophthora spp. AFLP fingerprinting has been used on a variety of organisms including P. infestans and the procedure has been shown to generate a large number of reproducible markers (Van der Lee, et al, 1995). In conjunction with adequate sampling it allows for the tracking of an epidemic and in many cases can be used to determine an inoculum source. Identification of the species involved and AFLP fringerprinting of populations of Phzytophthora spp. at a given greenhouse will aid in our understanding of the distribution of Phytophthora within the industry as well as provide information for development of durable prevention strategies.

   In the past 15 years, a limited number of fungicides have been registered for control of Phytophthora diseases, but metalaxy! (Subdue 2E) and an improved version, mefenoxam (Subdue Maxx) have been the most widely used. In 1991, Ferrin and Kabashima found isolates ofF. citricola and P. parasilica from ornamental crops that were insensitive to metalaxyl, but since that time few additional. reports of insensitivity have appeared. However, resistance ofF. infestans to mefenoxam in the USA, Canada, Europe and Middle East has lead to a serious epidemic of late blight in potato that continues to threaten the potato industry (Fry and Goodwin, 1997). Within individual species of .Phytophthora that attack ornamentals, little is known about the genetic diversity and hence response of isolates to currently used fungicides. Monitoring of Phytophihora spp. sensitivity to the commonly used fungicide, mefenoxam is important to the floriculture industry because shifts in baseline sensitivity and selection of insensitive isolates could result in failed disease control with this fungicide. Since the floriculture industry has growers who specialize in propagating stock and/or growing seedlings, the potential exists for wide scale distribution of mefenoxam-insensitive strains of Phytophthora with infected plant material shipped to other growers who finish crops.

3. Objectives & Anticipated Benefits

   The proposed project will address three objectives: 1) To collect and characterize Phytophthora isolates from floriculture crops in the US; 2) To validate AFLP fingerprinting of Fhytophthora spp. as a tool to track populations; and 3) To determine sensitivity of Phytophthora isolates to mefenoxam and to compare the baseline sensitivity for isolates collected since the introduction of mefenoxam to that pre-existing in Phytophihora populations.

   The proposed project will be the first in the USA to look at the importance of Phytophthora diseases in floriculture crops on an industry-wide basis. After the work is completed we will have a much better picture of the relative importance of different Phytophthora species affecting specific floriculture crops. The use of AFLP fingerprinting as a tool to track populations of Phytophihora strains in the industry should be a major contribution to understanding the genetic variation and occurrence of Phytophihora species important in the industry. In regions of the US where floriculture, tobacco and fruit crop production co-exist, the ability to differentiate strains of Phytophthora spp. should enhance our understanding of the need to be good stewards of our crop health. The movement of plant material in the floriculture industry is intense; by understanding the occurrence and importance of introduced strains into greenhouse operations, better disease management programs can be targeted where they will have the most impact on crop health. The INSV epidemic that swept the floriculture industry in the 1980’s is an excellent example where research on occurrence and detection of the pathogen resulted in effective disease management programs for the industry. Although fungicide use is just one part of disease management programs for Phytophthora, many growers rely heavily on this tactic. For durable fungicide use, information generated on fungicide sensitivity will be invaluable in guiding deployment of a fungicide strategy in the future.

4. Materials & Methods: Isolate collection

   Commercial greenhouse operations in North Carolina will be surveyed for Phytophthora diseases in potted plants such as Transvaal daisy, gloxinia, African violet, poinsettia and bedding plants. Multiple samples of infected plant material will be collected from each greenhouse where available. For AFLP analysis a minimum of 50 samples of infected plant material will be collected from a single crop at a given greenhouse operation. On-site surveys will also be conducted to determine disease incidence in specific crops in a given operation. Isolates will also be collected from floriculture crop samples submitted to North Carolina Plant Disease and Insect Clinic. In addition, cooperators in New York, Michigan, Pennsylvania and South Carolina will provide Phytophthora isolates from floriculture crops collected locally. Once isolates have been identified (see below) representative cultures of those Phytophthora species will be obtained from international repositories such as UC Riverside and CMI, Kew Gardens, England as well as from our other cooperators for comparison of morphological characteristics, sensitivity to mefenoxam, and AFLP analysis. (Benson lab)

   Isolate identification and mating type. Isolates of Phytophihora spp. collected from floriculture crops will be identified to species by comparison of morphological and cultural characteristics of known species as described in several excellent taxonomic keys (Waterhouse, Stamps and Waterhouse, Newhook et al., Erwin and Ribeiro, 1996). Mating type of heterothallic species recovered will be determined by inducing oospore formation in paired cultures with tester isolates of known mating type on media such as V8, hemp, lima bean, carrot or
   oatmeal agar (Erwin and Riberio, 1996). (Benson lab)

   AFLP analysis

   Phytophihora isolates recovered from single locations, which exhibit phenotypic diversity, will be further analyzed using the AFLP procedure. Isolates will be grown on water agar and single hyphal tips transferred to RA (50 ppm rifamipicin, 150 ppm ampicillin) amended UCV8 (840 ml distilled water, 163 ml unclarified V8 juice, 3 g of calcium carbonate, and 16 g Bacto agar) plates. Plugs of actively expanding mycelium will be transferred to approximately 20 ml of RA-UCV8 broth and incubated for three days. Mycelial mats will be washed, filter dried, frozen to ¬ó20C, and lyophilized. Lyophilized mats will be ground with a sterile mortar and pestle and DNA extracted using a QU1AGEN DNeasy small plant genome kit according to manufacturer directions. Approximately 100 ng of DNA will be subjected to restriction with EcoRl and Msel while concomittantly ligating adaptor sequences using the adaptors and protocols outlined in the Perkin-Elmer Microbial Fingerprinting kit (Perkin Elmer Applied Biosystems henceforth referred to as PE/ABI). Subsets of the resulting DNA fragments will be PCR amplified using the fluorescently labeled selective primers in the PE/ABI fingerprinting kit. AFLP fragments will be resolved using a PE/ABI 377 DNA sequencer (MSU sequencing facility). The fluorescing fragments emit light upon laser excitation and the emission peaks, which correspond to discrete fragments, are recorded as an electropherogram. The resulting electropherogram data will be analyzed using the GenoTyper software (PE/ABI). (Hausbeck lab)

   Fungicide screening. Phytophthora isolates will be screened in vitro for sensitivity to mefenoxam (Benson, 1979, Benson and Grand, 2000, Lamour and Hausbeck, 2000). Base line sensitivities will be compared from recently collected isolates exposed to mefenoxam with isolates collected before introduction of mefenoxam to determine if any shift in baseline sensitivity has occurred. Standard microbiological methods that involve the use of mycelial growth rate in agar incorporated with mefenoxam at various concentrations will be used (Benson, 1979). Comparison of growth rate in unamended agar will be used to assess inhibition at various concentrations of mefenoxarn. In addition, insensitivity of isolates to mefenoxam will be tested by exposing isolates to a 100-fold EC concentration of mefenoxam. (Benson Lab)

   Timeline.

   Objective	Year 1	Year 2	Year 3
   1a) isolate collection	————————	–Benson Lab–	————————
   1b) Isolate comparsion/characterization	——————–	–Benson Lab–	—————-
   2) AFLP analysis		–Hausbeck lab–	—————-
   3) Fungicide sensitivity		–Benson Lab–	—————-

5. Literature Cited.

   -Benson, D.M. 1979. Efficacy and in vitro activity of two systemic acylalanizies and ethazole for control of Phytophthora cinnurnoini root rot of azalea. Phytopathology 69:174-178.

   -Benson, D. M., and Jones, R. K. 1980. Etiology of rhododendron dieback caused by four species of Phytophthora Plant Dis. 64:687-691.

   -Benson, D. M. and Grand, L. F. 2000. Incidence of Phytophthora root rot of Fraser fir in North Carolina and sensitivity of isolates of Phytophthora cinnainomi to metalaxyl. Plant Dis. 84: 66 1-664.

   -Bewley, \V. F. and Buddin, W. 1921. ON the fungus flora of glasshouse water supplies in relation to plant disease. Ann. Appl. Bio. 8:10-19.

   -Dastur. J. F. 1913. Phytophthora parasitic n. sp., a new disease of the castor oil plant. Mem. Dep. Agric. Inida, Bot. Ser. 5(4):177-231.

   -Erwin, D. C. and Ribeiro, 0. K. 1996. Phytophthora Diseases Worldwide APS Press, St. Paul, MN, 562 pp.

   -Farr, D. F., Bill, G. F., Chamuris, G. P. and Rossman, A. Y. 1989. Fungi onplants and plant products in the United States. APS Press, St. Paul, MN 1252 pp.

   -Ferrin, D. M. and Kabashima, J. N. 1991. In vitro insensitivity to metalaxyl of isolates of Phytophihora citricola and P. parasitica from ornamental hosts in southern California. Plant Dis. 1041-1044.

   -Fry \V E., and Goodwin, S. B. 1997. Resurgence of the Irish potato famine fungus Bioscience 47:303-372.

   -Lamour, K. H., and Hausbeck \1 K. 2000. Mefenoxam insensitivity and the sexual stage of Phytophthora capsici Michigan cucurbit fields Phytopathology 90:396-400.

   -Mills. S. D., Forster, H., and Coffey, M. D. 1991. Taxonornic structure of Phytophihora cryptogea and P. drechsleri based on isozyme and mitochondrial DNA analysis. Mycol. Res. 95:31-45.

   -Ristaino, J.B., Madritch, M., Trout, C.L., and Parra, G. 1998. PCR amplification of ribosomal DNA for species identification in the plant pathogen genus Phytophihora. Appl Environ. Microbiol 64:948-954..

   -Stanghellini, ME., Kim, D.H., Rasmussen, S.L., and Rorabough, P.A. 1996. Control of root rot of reppers caused by Phytuplithora capsici with a nonionic surfactant Plant Disease 80:1113-1116.

   -Thompkins, C. M., and Tucker, C. NI. 1937. Foot rot of China-aster, annual stock, and Transvaal daisy caused by Phytophihora cryptogea J. Agric. Res. 55:563-574.

   -Van der Lee, T., De Witte, I., Drenth, A., Alfonso, C., and Govers, F. 1997. AFLP linkage map of the oomycete Phi’ophthoru infestans Fzingal Genetics and Biology 21: 278-291. Non Broembsen S.L. 1984. Distribution of Phytophihora cinnamomi in rivers of the south-western Cape Province. Phytophylactica 16:227-229.

   -Von Broembsen, S.L. and Deacon, J.W. 1997. Calcium interference with zoospore biology and infectivity of Phytophthora parasitica in nutrient irrigation ion solutions. Phytopathology 87:522-528.

   -Vos, P: et al. 1995. AFLP: a new technique for Dna fingerprinting. Nucleic Acids J?esearch 23. 4407-4414

   -Waterhouse, G. 1963, Key to the species of Phytophthora de Barv. Mycol. Pap. 92. 22 pp. Commonw. Mycol Inst. Kew, U.K.

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