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Characterization of Isolates of Phytophthora Infestans from Four

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Characterization of Isolates of Phytophthora Infestans from Four JOBNAME: horts 41#7 2006 PAGE: 1 OUTPUT: October 28 12:42:13 2006 tsp/horts/127877/01691R HORTSCIENCE 41(7):1635–1639. 2006. been reported as being more genetically diverse on tomato than on potato (Wangsomboondee et al., 2002). Epidemics occurred in 2004 Characterization of Isolates of when over 50% of the commercial crop was lost in eastern states from New York to Phytophthora infestans from Four Florida; 80% to 90% of early seedbeds in Florida were a complete loss from Nov. 2004 Solanaceous Hosts Growing in to Apr. 2005. Most growers reported that no available fungicides would control the rapid spread of the epidemic. Heavy losses oc- Association With Late Blight-infected curred in transit; symptoms developed on infected but symptomless fruit within 5 d of Commercial Potato Crops harvest. 1 Late blight affects both the leaves and Kenneth L. Deahl , Richard W. Jones, and Frances M. Perez fruit of tomatoes, spreading rapidly. On Vegetable Laboratory, USDA, ARS, Beltsville, MD 20705-2350 leaves, greasy-looking, irregularly shaped gray spots appear around which a ring of David S. Shaw white mycelium may develop, especially in School of Biological Sciences, University of Wales, Bangor, U.K., and Sa´rva´ri wet weather. The spots eventually turn dry Research Trust, Siambra Gwynion, Llandygai, Bangor, LL57 4BG, U.K. and papery. Blackened areas may appear on the stems. The fruit also develop large, irreg- Louise R. Cooke ularly shaped, greasy gray spots. P. infestans Department of Applied Plant Science, School of Agriculture & Food Science, can overwinter in frost-free areas in dead Queen’s University, Newforge Lane, Belfast, BT9 5PX, U.K. tomato plants and diseased debris (Peterson, 1947). Because it spreads to potatoes, it also Additional index words. tomato, petunia, nightshade, Solanum tuberosum, Lycopersicon overwinters in potato tubers. For transplant esculentum, disease susceptibility tomatoes, the initial source of disease may be infected transplants. Other potential sources Abstract. The oomycete, Phytophthora infestans, is a devastating pathogen of potato of inoculum are potato cull piles and volun- worldwide. Several genotypes of P. infestans are able to infect other cultivated and weed teer potatoes and tomatoes. species of the family Solanaceae and cause symptoms similar to late blight. Changes in Solanaceous weed and ornamental spe- P. infestans populations have stimulated investigations to determine if potato strains cies may also be infected with late blight and, from new immigrant populations infect nonpotato hosts more often than those from the where they grow in proximity to potato and older population. Expansion of the effective host range may be one of the mechanisms tomato crops, may act as reservoirs of in- involved in pathogenic changes in natural populations of P. infestans and to determine its oculum or aid generation of diversity if they significance, it is necessary to establish if the pathogen strains on nonpotato hosts permit contact between crop-specialized gen- represent distinct genotypes/populations or are freely exchanging with those on potato. otypes. Furthermore, late blight has also been This article reports characterization of P. infestans isolates from four solanaceous hosts reported on weed species (e.g., nightshade) in (black nightshade, hairy nightshade, petunia, and tomato) growing within and around the United States and Canada where many fields of blighted potatoes in four U.S. locations and one U.K. location and their potato and tomato production areas are di- comparison with isolates collected from adjacent infected potatoes. Isolates were rectly linked geographically or by crop mar- characterized for mitochondrial DNA haplotype, mating type, metalaxyl resistance, keting. Species reported to be involved allozymes of glucose-6-phosphate isomerase and peptidase, and DNA fingerprint with the include petunia, black nightshade, and hairy RG57 probe. Analysis showed close similarity of the petunia, hairy and black nightshade nightshade. Although late blight is a highly isolates to potato isolates. However, tomatoes from New Jersey and Pennsylvania, significant, well-studied disease of potato and respectively, were infected by two distinct and previously unreported pathogen tomato, relatively little is known about this genotoypes, which had quite different fingerprints from P. infestans isolates recovered disease incited by the same organism on other from nearby infected potatoes. Potato growers should be aware that both weed and solanaceous hosts. cultivated solanaceous species can be infected with P. infestans and may serve as clandestine reservoirs of inoculum. Because some of these plants do not show conspicuous symptoms, they may escape detection and fail to be either removed or treated and so may Materials and Methods play a major role in the introduction and spread of pathogens to new locations. Collection of isolates. Samples were col- lected from solanaceous hosts with lesions similar to late blight in four locations in Late blight, incited by the oomycete path- mating type (US-8) and an A1 mating type major potato production areas in the United ogen Phytophthora infestans (Mont.) de Bary, (US-11), both resistant to the fungicide met- States and one location in Wales (see sub- has become an increasingly important prob- alaxyl, are present (Daayf et al., 2000; sequently). Blighted potato material was lem to agriculture in the United States and Dorrance et al., 1999; Gavino et al., 2000; collected from the same locations by coop- many other countries in the past decade. More Goodwin et al., 1998). The US-8 clonal erators during crop inspections of naturally aggressive fungicide-resistant and host- lineage is the most common on potatoes, infected fields. Each sample consisted of specialized isolates have appeared attacking but US-11 has been particularly troublesome infected leaves and stems from one or more potato (Solanum tuberosum L.) and tomato on tomatoes (Gavino et al., 2000). Although plants within a single crop. Information on (Lycopersicon esculentum Mill.) crops. these two pathogen genotypes predominate, site, fungicide use, potato cultivar, and blight In Canada and the United States, two the infrequent occurrence of other genotypes incidence was collected for each sample. major genotypes of the pathogen, an A2 (Goodwin et al., 1998) indicates that sexually Single lesions were incubated under high reproducing population pockets or intro- humidity for 24 to 48 h to encourage sporu- duced pathogen genotypes pose a continuing lation, then isolates were obtained by collect- Received for publication 16 June 2006. Accepted threat. ing sporangia from infected foliage and for publication 4 Aug. 2006. Late blight has suddenly reemerged as initially maintained on detached glasshouse- 1To whom reprint requests should be addressed; a major concern in most tomato-producing grown potato leaflets or tuber slices of sus- e-mail [email protected]. areas of the United States. The pathogen has ceptible cultivars free from R-genes. HORTSCIENCE VOL. 41(7) DECEMBER 2006 1635 JOBNAME: horts 41#7 2006 PAGE: 2 OUTPUT: October 28 12:42:15 2006 tsp/horts/127877/01691R Tissue pieces 3 to 4 mm2 were cut from transplants at a glasshouse in central Mary- diameters measured once average growth the margins of lesions on the detached leaf- land. Infected plants had extensive light gray, diameters reached 15 mm on untreated plates lets, surface sterilized, and transferred to irregularly shaped, slightly sunken lesions up (typically after 5 to 6 d for faster-growing Petri plates containing rye A agar (Caten to 3 cm long on the upper leaves and isolates) and again 2 to 3 d later. The and Jinks, 1968) amended with antibiotics branches. Stems were distorted or dead above percentage reduction in growth on 10 mgÁL–1 (100 mgÁmL–1 ampicillin, 100 mgÁmL–1 nys- points where lesions had coalesced to cause metalaxyl compared with growth on the un- tatin, 50 mgÁmL–1 rifampicin). The plates total or almost complete stem girdling. The amended control plates was calculated. Each were incubated at 20 °C for 5 to 7 d to allow disease initially occurred as scattered foci, isolate was tested at least twice. Isolates were mycelia to grow into the medium. Small agar but rapidly spread through the crop causing designated as metalaxyl-resistant if growth blocks containing hyphal tips were cut from significant losses. The disease reappeared in was >60% of the control, -intermediate if the colony margins and transferred to un- the same and adjacent greenhouses in 2002 growth was 10% to 60% of the control, or amended rye A agar for growth and sporula- and 2003, producing severe damage in sev- -sensitive if growth was <10% of the control tion at 20 °C. Stock cultures were maintained eral crops. Three isolates were obtained from using the criteria of Shattock (1988). on unamended rye A agar and transferred at sporulating lesions on the leaves and stems. Allozyme assays. Genotypes at two poly- 6- to 12-month intervals. Late blight was observed on potato plants in morphic allozyme loci, Gpi-1 (glucose- Alternate hosts of Phytophthora infestans. fields in close proximity to the petunia glass- 6-phosphate isomerase, GPI, E.C. 5.3.1.9.) Lesions similar to those incited by P. infes- houses and P. infestans isolated. and Pep-1 (peptidase, PEP, E.C. 3.4.3.1.), tans were found on other cultivated and weed Tomato. Samples of tomato foliage and were determined using the protocols of species of the family Solanaceae and isolates fruit with late blight lesions were collected Goodwin et al. (1995a). Chilled supernatants were obtained from these (Table 1) as de- from commercial field crops in New Jersey in containing protein released from mycelial tailed subsequently. 2003 and Pennsylvania in 2004. Three iso- fragments in sterile distilled water were Black nightshade. In 1999, brown necrotic lates were obtained from New Jersey and five loaded onto cellulose acetate plates equili- leaf lesions with pale green margins were isolates from Pennsylvania all from different brated in the appropriate buffer: Tris-glycine found on black nightshade (Solanum nigrum crops.
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