Pathogenic Fungi in Garlic Seed Cloves from the United States and China, and Efficacy of Fungicides Against Pathogens in Garlic Germplasm in Washington State

Commercially distributed garlic (Allium sativum) seed cloves from six states of the United States and mainland China were surveyed for the presence of fungi recorded as pathogenic to garlic in the literature. Aspergillus niger, A. ochraceus, Botrytis porri, Embellisia allii, Fusarium oxysporum f. sp. cepae, F. proliferatum and Penicillium hirsutum, were each recovered from one or more of these commercial sources, as was F. verticillioides, not previously reported as pathogenic to garlic, but here demonstrated to be a pathogen. Seed garlic distributed from public germplasm collections may also contain fungal pathogens: E. allii, F. oxysporum f. sp. cepae and/or F. proliferatum caused severe losses in 2002–2003 and 2005–2006 during germplasm regeneration and storage for the National Plant Germplasm System in Pullman, Washington. Use of fludioxonil, thiophanate methyl and/or benomyl (the latter withdrawn from the market, but used here as a standard) at label rates against E. allii and Fusarium species promoted plant health, but not when infections were located deep within tissues nor under some situations involving high disease pressure. Introduction In 2002–2003, significant losses were documented during routine maintenance of the USDA-ARS National Plant Germplasm System (NPGS) clonal garlic (Allium sativum L.) collection in Pullman, Washington. Most losses appeared subsequent to harvest after detached bulbs were hanging in the drying shed prior to storage. Further losses from the same material were recorded after storage and just prior to fall planting of seed cloves. Significant losses again occurred in 2005–2006. The pathogens primarily responsible were Fusarium proliferatum (Matsushima) Nirenberg, recently reported on garlic in North America (Dugan et al., 2003), Embellisa allii (Campanile) Simmons and Fusarium oxysporum Schlechtend.: Fr. f. sp. cepae (H. N. Hans.) W. C. Snyder & H. N. Hans. Symptoms of rot from Fusarium spp. were frequently observed along the longitudinal axis (innermost leaf scales) of affected cloves from the 2002 harvest. Fungal growth could sometimes be discerned along this axis even when exterior tissues appeared healthy, an aetiology also reproduced in artificial inoculations (Dugan et al., 2003). In the 2003 harvest, symptoms were concentrated on bulb exteriors, probably because dry, hard soil was abrasive at lifting, presumably creating infection courts at harvest. Because of our experiences with fungal infection of NPGS garlic germplasm, and because of analogous infection in some garlic obtained commercially for experimental purposes, we suspected that commercially distributed seed garlic might commonly contain miscellaneous fungi pathogenic to garlic. We resolved to survey commercially distributed seed garlic grown from various locations in the United States, and from mainland China, now a major exporter of garlic. We also conducted bioassays of our own field soil, to confirm presence of Fusarium pathogens in the soil. Limited guidance is available for fungicides previously or currently labelled for garlic and potentially able to control Fusarium species. Crowe (1995) reported that benomyl is effective against Fusarium culmorum (W. G. Sm.) Saccardo, as well as Penicillium. Thiophanate methyl is labelled for Penicillium on garlic seed cloves in Washington State. Azoxystrobin fungicides are labelled for foliar diseases of garlic, but not for seed cloves in Washington State. Fludioxonil is labelled in Washington State for garlic, and according to the label controls Fusarium species, but it is not systemic. There are occasional reports of chemicals (systemic or otherwise) used against E. allii. Portela et al. (1996)) reported from Argentina that of thiram, quintozene (PCNB) and benomyl, the degree of control www.blackwell-synergy.com J. Phytopathology 155, 437–445 (2007) doi: 10.1111/j.1439-0434.2007.01255.x Journal compilation 2007 Blackwell Verlag, Berlin No claim to original US government works was greatest with thiram and least with benomyl. An old experimental treatment in Italy used immersion of bulbs in 3% formalin before planting (Anon, 1925, citing Campanile, 1924). In Bulgaria, best results were with mercury chloride and thiram, followed by carboxin, chinosol, captafol, propineb, ziram, chlorothalonil, mancozeb and captan (Anon, 1980, citing Nakov et al., 1979). Depending on regulatory status, some of these applications (most notably mercury chloride and formalin) are no longer allowable. In addition to conducting the survey of commercial seed garlic, we decided to test efficacies of thiophanate methyl and fludioxonil as preplanting dips for seed cloves, and to relate results to those obtained with benomyl, now withdrawn from the market, but previously found effective against Fusarium infections when used as a dip (Crowe, 1995). We also wished to test thiophanate methyl and to compare it to benomyl when these fungicides were used as postharvest dips. In each instance, our strategy was to conduct a minimum of one field experiment with natural levels of inoculum, plus one or more analogous experiments in the greenhouse or laboratory, in which inoculum levels could be precisely quantified. Manufacture of benomyl has been discontinued, with use allowed only under specified conditions (MacArthur, 2001; Damicone, 2002). Although application of thiophanate methyl on garlic replaces benomyl primarily as means to control Penicillium in seed cloves, it has the potential to reduce losses from Fusarium (e.g., Nan, 1995). Our objectives focus on germplasm management. Cost-effective treatment of bulbs destined for consumption is not addressed. Materials and Methods Survey of marketed seed garlic for fungal pathogens Seed garlic cloves grown in California, Idaho, Oregon, New York, North Dakota, Washington State and People’s Republic of China (province unknown) were purchased from commercial distributors in spring 2005. From each location, 25 bulbs were individually loosely wrapped in aluminium foil, placed inside a perforated plastic box and incubated at 28 C until symptoms of rot appeared. Tissues excised from margins of rotted areas were surface-disinfested for 2–3 min in 0.5% NaOCl with a distilled water rinse, and transferred to half strength (1⁄2) V8 agar (Stevens, 1981) amended with 50 lg/ml streptomycin sulphate and 50 lg/ml tetracycline hydrochloride for recovery of fungi. Fungi were maintained on slants of 1/2V8 agar, stored in glycerol at )80 C and identified as described below. Isolation of fungi from USDA germplasm In addition to fungi isolated as described above, fungi were recovered by closely analogous procedures from symptomatic garlic bulbs produced on the USDA Western Regional Plant Introduction Station (WRPIS) farm in 2001, 2002–2003 and 2005–2006. Fungi were stored in glycerol at )80 C or in liquid nitrogen vapour. Fusarium proliferatum was isolated, identified and reported for the first time as a cause of bulb rot of garlic in North American (Dugan et al., 2003). Confirmation of pathogenicity To verify pathogenicity of taxa identified as pathogens of garlic in the literature and/or isolated by us (Table 1), we selected a minimum of one (but usually two or more) isolates of a given taxon from each source of seed garlic in which it was detected, and conducted artificial inoculations. Five to six cloves, disinfested in 0.5% NaOCl for 45 s, rinsed with sterile water, were injured to a depth of 4.5 mm with a probe of 1 mm diameter, and the wound filled with 1⁄2V8 agar colonized by the isolate. Controls used sterile agar in place of colonized agar. Cloves were placed individually in single wells of 6-well plates, and incubated on the laboratory bench top at 25–28 C for 31⁄2– 5 weeks for symptom development. We also tested two isolates of Fusarium verticillioides (Sacc.) Nirenberg, a species which, to our knowledge, has not been described as a pathogen of garlic, but which was repeatedly isolated by us. That the fungi so inoculated were responsible for the observed pathogenesis was confirmed by examination of sporulation within the lesions or, especially for F. verticillioides, by re-isolation back into pure culture. Identification of pathogens Some Fusarium isolates recovered from garlic rotting in the field or drying shed were provisionally identified as F. oxysporum f. sp. cepae and tested by inoculation of representative isolate PI540346#3 into onion (A. cepa L.) bulbs and garlic cloves, using FOC-8 Table 1 Fungi pathogenic to garlic and isolated from commercial seed garlic Geographic source Cultivar Pathogenic fungi California Silver Rose, softneck Aspergillus niger, Embellisia allii, F. oxysporum f. sp. cepae, Fusarium proliferatum Idaho Inchelium Red, softneck Botrytis porri, E. allii, Penicillium hirsutum Oregon Siberian, hardneck A. ochraceus, F. proliferatum, P. hirsutum New York Music, hardneck E. allii, F. oxysporum f. sp. cepae, F. verticillioides, P. hirsutum North Dakota Montana Roja, hardneck E. allii, F. oxysporum f. sp. cepae, F. proliferatum, P. hirsutum Washington State Russian Giant, hardneck A. ochraceus, E. allii, F. oxysporum f. sp. cepae, P. hirsutum China Spanish Roja, hardneck F. proliferatum, F. verticillioides, P. hirsutum F. verticillioides from China lost some aggressiveness in consecutive trials, but F. verticillioides from New York remained aggressive. 438 Dugan et al.