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White Rot of Garlic and Onion (Causal Agent, Sclerotium cepivorum): A Status Report from the Pacific Northwest
Shari L. Lupien, Barbara C. Hellier, and Frank M. Dugan, USDA-ARS Western Regional Plant Introduction Station, Washington State University, Pullman, WA 99164; Linnea G. Skoglund, Schutter Diagnostic Laboratory, Department of Plant Sciences and Plant Pathology, Montana State University, Bozeman, MT 59717; and Karen F. Ward, Plant Diagnostic Clinic, Department of Plant Pathology, Washington State University, Pullman, WA 99164
Lupien, S. L., Hellier, B. C., Dugan, F. M., Skoglund, L. G., and Ward, K. F. 2013. White rot of garlic and onion (causal agent, Sclerotium cepivorum): A status report from the Pacific Northwest. Online. Plant Health Progress doi:10.1094/PHP-2013-0619-01-RV.
There is evidence from literature, state department of agriculture documents, and recent diagnoses that Sclerotium cepivorum, causal agent of white rot of garlic and onion, is spreading and/or becoming more established in the Pacific Northwest. Previously documented distributions are summarized and the fungus is reported for the first time from Latah Co., ID; Pend Oreille Co., WA; and Lake, Sanders, and Missoula counties, MT. Although known from a tightly quarantined prior occurrence in the Idaho portion of Treasure Valley (southwest Idaho), the pathogen has not been formally reported from that state nor from the state of Montana. Latah Co. has commercial production of seed garlic and borders adjacent Whitman Co., WA, where the National Plant Germplasm System (NPGS) maintains North Americas largest non-commercial collection of garlic and wild/ornamental onion. Strict phytosanitary protocols have been implemented on the NPGS farm. Various areas within the Pacific Northwest have long been important for commercial Allium production, and a list of state departments of agriculture regulations addressing white rot is presented for Idaho, Oregon, and Washington.
Sclerotium cepivorum, causal agent of white rot of Allium species, "is distributed worldwide and is increasing in prevalence" (4). The disease has long been of concern in the Pacific Northwest (8,12). As discussed below, several states and Canada regard it as a pathogen of quarantine significance. We present a synopsis of published prior occurrences and distribution in the Pacific Northwest, some recent novel records, regulations of various state departments of agriculture, and we briefly comment on various challenges and prospects for successful management. Although diagnosis of the agent via symptoms and characteristic sclerotia is relatively reliable, morphologically similar, closely related species exist, so we also present a protocol for identification via ITS sequence analysis.
Synopsis of White Rot in the Pacific Northwest
Sclerotium cepivorum has been formally reported on onion and garlic in Oregon and British Columbia, and on onion in Washington (6). The fungus was first confirmed as causing white rot in the United States in La Grande, Union Co., OR, in 1918 (5). It was subsequently documented in various locations in California, and by 1957-1958 had been observed in the areas of Walla Walla, WA, and Milton-Freewater and Klamath Falls, OR, with further infestations also observed in California. Benton, and Linn counties in the Willamette Valley, and Jefferson Co., OR, were later known to have infestations of white rot (5). Other older records in Oregon document white rot in Clackamas, Multnomah, Polk, Tillamook, Washington, Lane, Douglas, Jackson, Hood River, Wasco, and Baker counties; and in 2004, a diagnosis of white rot was made for a commercial grower in Marion Co., OR (M. Putnam and M. Serdani, Oregon State University Plant Clinic, personal communications). In 2008 white rot was detected in Crook Co. (17). In Washington, additional known areas of infestation are the Yakima Valley and in non-commercial situations west of the Cascades (5,13). White rot was confirmed in Benton Co., WA, in 2002 (J. Eggers, Hermiston Agricultural Research and Extension Center, personal communication). Consultation of clinic records for prior diagnoses from eastern Washington was not feasible due to closure of the diagnostic laboratory in Prosser in 2003, but records of diagnoses over the past decade in western Washington State document instances of white rot from Thurston Co. north to Skagit, Jefferson and Island counties (J. Glass, WSU Puyallup Insect and Disease Diagnostic Laboratory, personal communication). The first confirmed (but as yet not published in scientific literature) instances in Idaho date from 1986 in the Treasure Valley (5), although there is a clinic record for an instance in Idaho as early as 1973 but without designation of within-state locale (M. Putnam, personal communication). Thirty-seven incidents of the disease were indicated for Idaho statewide in 2010, but these were interceptions of imported material, not involving material produced in Idaho [(11), E. Vavricka, Idaho Department of Agriculture, personal communication]. White rot has been said to occur in home gardens in Montana (5). In British Columbia, white rot has been reported since the early1960s, with most reports concentrated in coastal British Columbia and the Okanogan and Frasier Valleys (3,7). Additional reports include West Kootenay and the Sunshine Coast (V. Joshi, British Columbia Ministry of Agriculture, personal communication).
White Rot Confirmed in the Palouse Region of Northern Idaho
Whitman Co., WA, plus Latah Co., ID, and parts of adjacent counties in Idaho and Washington are commonly referred to as the Palouse region, and garlic (Allium sativum) is often grown, sometimes commercially, in gardens and farms.
In September 2010, a sample of diseased garlic from a home garden in Latah Co. was brought to the USDA-ARS Western Regional Plant Introduction Station (WRPIS) for diagnosis. WRPIS is a unit of the USDA-ARS National Plant Germplasm System (NPGS) in Whitman Co. Immediately adjacent to Latah Co., WRPIS maintains the largest non-commercial collection of garlic, wild garlic relatives, and wild and ornamental onion (Allium spp.) germplasm in North America, so the sample captured our attention. Strict phytosanitary protocols were immediately implemented on the NPGS farm even prior to definitive identification of the agent.
The symptomatic plants brought to WRPIS were representative of plants in the home garden. The plants exhibited severe bulb and neck rot with gray mycelium colonizing the entire bulb, neck, and spaces between cloves. Small (~0.5 mm) black globose to subglobose sclerotia were present in the neck of one plant and between cloves of the second plant.
Mycelium and individual sclerotia were directly transferred from symptomatic plants to Difco potato dextrose agar (PDA) and half strength V8 agar (½V8) (18). Sclerotia transferred from diseased plants did not germinate in culture, but hyphal tips transferred to ½V8 readily generated sclerotia indistinguishable from those on symptomatic garlic plants (Fig. 1). In addition to sclerotia, a phialidic spermatial state was observed in the fast-growing, grayish white, flat mycelium.
Symptoms, colonies, sclerotia, and spermatia matched characters in standard published descriptions of colonies of S. cepivorum (4,14). Definitive identification to species was based on the internal transcribed spacer (ITS) sequences. Isolates originating from hyphal tips (Latah ht-4 and Latah ht-5) were grown in Difco potato dextrose broth on an orbital shaker (125 RPM) for seven days at ambient lab temperatures (22-24°C), after which mycelium was aseptically removed and washed three times with sterile diH2O, lyophilized, and stored at -80°C until DNA extraction. Lyophilized mycelium (10 mg) was disrupted while frozen with liquid nitrogen in the presence of MP Biomedicals Lysing Matrix A (Saloon, OH) in a Fast Prep 120 cell disruptor (speed 4 for 30 sec). Genomic DNA was isolated immediately following tissue disruption using Qiagen DNeasy Plant Mini Kit (Qiagen, Valencia, CA) following the manufacturers instructions using twice the recommended volume of lysis (AP1) and AP2 buffers. The lysate was cleared by centrifugation at 50,000 × g prior to loading on to the QIAshredder column. (Modifications were required for successful DNA extraction; viscous lysate interfered with extraction under manufacturer conditions.) DNA was eluted from the column with 100 µl of sterile water. Amplification of the ITS and 5.8S rDNA region was accomplished using primers ITS5 and ITS4 (22). DNA amplification was performed in an Applied Biosystems Gene Amp 9700 thermalcyler. Reaction conditions consisted of a single cycle of 10 min at 95°C followed by 35 cycles (of 35 sec at 94°C, 60 sec at 52°C, and 2 min at 72°C) plus a final extension cycle of 10 min at 72°C. Amplified PCR products were purified with Qiagen QIAquick spin columns. The 1210 nucleotide ITS amplicon was sequenced on both strands using primers ITS2, ITS3, ITS4, and ITS5 (22). Sequencing reactions and primer synthesis were performed by Eurofins MWG/Operon (Huntsville, AL). Sequence data was assembled and aligned using Sequencher 4.9 software (Gene Codes Corp. Ann Arbor, MI). The resulting sequences were compared to the GenBank nucleotide data base (blast.ncbi.nlm.nih.gov), published sequences of Sclerotinia and Sclerotium species included in a phylogenetic analysis of plant pathogenetic Sclerotium species (23), and an isolate of S. cepivorum causing white rot of garlic in Hungary (1) using BLASTN 2.2.21 software (24).
Latah ht-4 and ht-5 ITS sequences shared 99-100% identity with two S. cepivorum strains present in GenBank including CBS276.93 (isolated from Allium) which was part of a phylogenetic study (23). Our strains from Latah Co. shared 100% identity with S. cepivorum causing white rot of garlic in Hungary (1) and CBS321.65 isolated from an Allium cepa bulb. ITS sequences of closely related fungi such as Sclerotium perniciosum, Sclerotinia trifoliorum, and Sclerotinia sclerotiorum included in the phylogenetic study (23) have 100% identity within these individual species but only share 98% identity with our S. cepivorum strains. On the basis of the above morphological, cultural, and molecular characters, our Palouse isolates were conclusively identified as S. cepivorum.
White Rot Confirmed in Lake, Sanders, and Missoula Counties, MT, and in Pend Oreille Co., WA
The Montana specimens were submitted to the Schutter Diagnostic Laboratory at Montana State University. The causal agent was identified by symptomatology and microscopic examination. White mycelium was noted on bulbs and abundant small (~0.5 mm diameter) sclerotia were present on outer scales of the basal plate (Fig. 2), with signs and symptoms matching descriptions and illustrations (4,13).
Specimens of commercially grown garlic from Pend Oreille Co., WA, were sent to the Plant Diagnostic Clinic at Washington State University. The causal agent, S. cepivorum, was suspected on the basis of symptoms (dying and dead leaves, plus cortical rot of roots) and presence of small sclerotia. The fungus was isolated by transfer of mycelium to acidified PDA. Resultant growth produced numerous black sclerotia (~0.5mm diameter) amid appressed white mycelium, matching the description (14).
White Rot in Legislation, Literature, and Agricultural Practice in the Pacific Northwest
We could find no report of the fungus on garlic or onion (Allium cepa) in Idaho in any scientific research publication, nor did the largest database of comprehensive documentation (6) contain such records. Nonetheless, we know of a prior finding of white rot in Idaho, consisting of "one to three occurrences tightly quarantined" (15) and there are repeated references to interceptions of white rot in Idaho State Department of Agriculture summary reports, such as indicated above. White rot is "a key disease of garlic in Washington … although not currently present in all parts of the state" (8). We report the new instances of white rot in Idaho, Montana and Washington because (i) seed garlic is commercially distributed in quantity from Latah Co. and because the locales in Montana and Washington are also sources of commercial distribution, (ii) because seed garlic is distributed non-commercially by WRPIS from Whitman Co., WA, immediately adjacent to Latah Co., and (iii) to rectify the lack of formal reports in scientific literature for S. cepivorum in Idaho and Montana.
The Idaho State Department of Agriculture has long been aware of the potential impact of white rot on the onion-growing region in and near Treasure Valley (southwest Idaho and eastern Oregon). Sclerotium cepivorum is denoted a regulated pest in onion and garlic for multiple counties of Idaho: Ada, Bingham, Blaine, Boise, Bonneville, Canyon, Cassia, Elmore, Gem, Gooding, Jefferson, Jerome, Lincoln, Madison, Minidoka, Owyhee, Payette, Power, Twin Falls, and Washington counties (IDAPA 02.06.07). Specific rules apply to shipping of "bulbs, sets or seedlings of onion, garlic, leek, chives, shallots or other Allium species" (IDAPA 02.06.07). Washington State treats S. cepivorum as subject to quarantine in Adams, Franklin and Grant counties (21). Oregon (16) has regulations on Malheur Co., coordinated with those of Idaho with respect to the growing area of Treasure Valley. A search of the website for Montana Department of Agriculture with "white rot" or "cepivorum" did not return any records, although "It has occurred in home gardens in Montana" (5) as noted above. An analogous search of the British Columbia Ministry of Agriculture website returned records for the Ministrys production guide for garlic, but no records for regulatory activities targeting white rot. However, S. cepivorum is a regulated pest by rules of the Canadian Food Inspection Service (2).
Latah Co., ID, is not amongst the counties in which white rot is addressed in IDAPA 02.06.07 (10), but the Idaho Department of Agriculture maintains an interest in the status of white rot in that county (E. Vavricka, personal communication). Whitman Co. is not amongst the counties addressed in WAC 16-470-300 but as noted, Whitman Co., WA, is immediately adjacent to Latah, and is the site of the WRPIS farm on which accessions of garlic, garlic relatives, and wild and ornamental onion are propagated for the USDA-ARS NPGS. More strict sanitation practices, including disinfestations of footwear, tools and equipment with 10% household bleach, have been implemented on the WRPIS farm subsequent to the discovery of white rot in Latah Co. Growers in the white rot quarantine area of Idaho may receive material from WRPIS if the material receives prior screening by the Potato Tissue Culture Laboratory at the University of Idaho.
Challenges and Prospects
Cumulative reports summarized above, and new reports herein, clearly indicate that white rot is spreading and/or increasingly well documented throughout the Pacific Northwest (Fig. 3). While some spread via table onion or table garlic is possible, spread via distribution of infested seed garlic (or of infested table garlic used for seed) is most plausible. Other sources of contamination might be infested equipment, tools, footwear, or soil. Sclerotia are long viable in soil and no species of Allium is known to be resistant (4,20). In onion, infected plants can be rogued to limit spread and fumigants can be used for spot treatments. In areas with cool summers, short-season crops such as scallion can minimize sclerotial populations (4). Increasing planting spacing (to reduce plant-to-plant spread) can assist in managing the disease. Avoidance of planting infested areas requires accurate mapping. Optimum management is based on integrating methods such as those just mentioned with germination stimulants ("tricking" sclerotia into germinating) and biological control agents (fungi parasitic on S. cepivorum). Where white rot is established, disease development is temperature-dependent, essentially restricted or negligible at summer soil temperatures of 22°C or higher (4). Although exclusion via awareness and quarantine is highly desirable, commercial production may ultimately depend on integrated control methods. Details of existing and experimental management techniques, including biological control and the potential for resistance, are provided or referenced (4,9,19,20).
The authors acknowledge Gwen Pentecost for technical assistance, Wayne Olson for obtaining the Latah Co. specimens, Vippen Joshi, Jordan Eggers, Fred Crowe, Elizabeth Vavricka, Jenny Glass, Melodie Putnam, and Maryna Serdani for numerous helpful communications on distribution of the agent and regulatory aspects. We also thank Elizabeth Vavricka and Krishna Mohan for constructive comments on the manuscript.
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