e-Xtra* A Strain of Clover yellow vein virus that Causes Severe Pod Necrosis Disease in Snap Bean Richard C. Larsen and Phillip N. Miklas, Unites States Department of Agriculture–Agricultural Research Service, Prosser, WA 99350; Kenneth C. Eastwell, Department of Plant Pathology, Washington State University, IAREC, Prosser 99350; and Craig R. Grau, Department of Plant Pathology, University of Wisconsin, Madison 53706 plants in fields were observed showing ABSTRACT extensive external and internal pod necro- Larsen, R. C., Miklas, P. N., Eastwell, K. C., and Grau, C. R. 2008. A strain of Clover yellow sis, a disease termed “chocolate pod” by vein virus that causes severe pod necrosis disease in snap bean. Plant Dis. 92:1026-1032. local growers. The necrosis frequently affected 75 to 100% of the pod surface. Soybean aphid (Aphis glycines) outbreaks occurring since 2000 have been associated with severe Clover yellow vein virus (ClYVV) (family virus epidemics in snap bean (Phaseolus vulgaris) production in the Great Lakes region. Our Potyviridae, genus Potyvirus) was sus- objective was to identify specific viruses associated with the disease complex observed in the pected as the causal agent based on pre- region and to survey bean germplasm for sources of resistance to the causal agents. The principle liminary host range response; however, causal agent of the disease complex associated with extensive pod necrosis was identified as Clover yellow vein virus (ClYVV), designated ClYVV-WI. The virus alone caused severe mo- identity of the pathogen was not immedi- saic, apical necrosis, and stunting. Putative coat protein amino acid sequence from clones of ately confirmed. ClYVV causes mild to amplicons generated by reverse-transcription polymerase chain reaction was 98% identical to severe mosaic, leaf malformation and vein ClYVV strain no. 30 identified in Japan that has not been reported to cause pod necrosis. necrosis, stunting of the plant, and apical ClYVV-WI amplicons were 96% identical to a mild strain of ClYVV from Oregon. A distin- necrosis in highly susceptible cultivars guishing feature of this new strain is that it does not react with Potyvirus broad-spectrum mono- (22). Pods often are mottled, twisted, or clonal antibody PTY 1. A survey of common bean lines and cultivars revealed that, in addition to malformed. Symptoms in bean plants in- UI-31 and US1140 with known resistance to ClYVV, lines with the bc-3 gene for resistance to fected with ClYVV can be highly variable Bean common mosaic necrosis virus also were resistant to ClYVV-WI. An evaluation of 63 snap due to the numerous strains of the virus bean cultivars and breeding lines revealed just one, Roma 442, with a moderate level of toler- and their interaction with different bean ance to ClYVV-WI. Introgression of the bc-3 gene and resistances from UI-31 and US1140 into cultivars. Symptoms frequently resemble snap bean may offer a high level of resistance to extensive pod necrosis disease caused by those caused by the closely related Potyvi- ClYVV in the Great Lakes region. rus BYMV, making visual distinction be- tween the two viruses difficult. Genes for resistance to ClYVV have Snap bean (Phaseolus vulgaris L.) is a viruses that may constrain production in been reported in the literature. The cyv major crop in the Great Lakes region of the the absence of resistance. The potential gene present in US1140 (23) was trans- United States. The most significant pro- negative effect of viruses on snap bean ferred to ‘Black Knight’ (27), UI-31 pos- duction occurs in Wisconsin, Michigan, production was fully evident in 2000 when sesses two recessive genes (32), and the New York, Illinois, and Pennsylvania. Snap a severe virus outbreak occurred in Wis- desc gene discovered in snap bean cv. bean crops also are produced in the Pacific consin and other neighboring snap bean Jolanda also was present in snap bean Northwest, Georgia, Florida, and Ontario, production states. The most frequently cultivars Imuna and Evolutie (26). The Canada. In 2005, 124,367 ha of snap bean detected viruses included Cucumber mo- latter three cultivars are not currently were harvested with a farm gate value of saic virus (CMV) and many distinct strains grown in the United States. The reactions approximately $400 million (35). Because of Alfalfa mosaic virus (AMV) (8,13). The of these resistant sources against isolates most snap bean pods are produced for outbreaks were directly associated with of ClYVV from the Great Lakes region are fresh market, freezing, or canning, pods high populations of the soybean aphid unknown. To our knowledge, no other harvested with imperfections due to dis- (Aphis glycines) in adjacent soybean fields commercial snap bean cultivars have been ease or mechanical injury are discarded, (9). A high incidence of mixed infections evaluated for resistance or tolerance to effectively reducing the growers’ yield. of CMV and AMV was associated with ClYVV. The objectives of this work were Pods harvested with imperfections that necrotic lesions or line patterns on pods to validate whether ClYVV was responsi- exceed allowable thresholds result in rejec- (8). Other viruses detected included Bean ble for the chocolate pod disease in snap tion by processors of the entire harvest. common mosaic virus (BCMV), Bean bean, and to identify sources of resistance Snap bean cultivars in the United States yellow mosaic virus (BYMV), Clover yel- to the virus. are susceptible to many important plant low mosaic virus, Tobacco streak virus, and White clover mosaic virus. Although MATERIALS AND METHODS Corresponding author: Richard Larsen BCMV was detected, Bean common mo- Field isolates, host plants, and serol- E-mail: [email protected] saic necrosis virus (BCMNV) never was ogy. Snap bean plants exhibiting leaf mo- detected in any samples evaluated. saic, apical stem necrosis, and pod necrosis * The e-Xtra logo stands for “electronic extra” and Significant virus outbreaks again oc- symptoms were collected from fields in indicates that Figures 1 and 2 appear in color in the curred in 2001 (13) and 2003 to 2005 (R. Wisconsin, Michigan, Minnesota, Ohio, online edition. Larsen, unpublished) in Iowa, Illinois, and New York during the 2001–05 growing Accepted for publication 14 February 2008. Kentucky, Michigan, Minnesota, New seasons. Samples were tested routinely by York, Wisconsin, and the province of On- indirect enzyme-linked immunosorbent tario, Canada. The same complex of vi- assays (ELISA) (2) for AMV using a poly- doi:10.1094/ PDIS-92-7-1026 ruses resulted in yield losses up to 100%, clonal antiserum produced at the United This article is in the public domain and not copy- especially in fields of late-season process- States Department of Agriculture– rightable. It may be freely reprinted with custom- ary crediting of the source. The American Phyto- ing and fresh-market snap bean crops. Agricultural Research Service (USDA- pathological Society, 2008. During these growing seasons, snap bean ARS) laboratory in Prosser, WA, and for 1026 Plant Disease / Vol. 92 No. 7 CMV using commercially available poly- technology Information GenBank database chloride; 5 mM dithiothreitol; 500 mM clonal antiserum (Agdia, Inc., Elkhart, IN). for each virus. Target areas for forward and each dATP, dCTP, dGTP, and dTTP; and 1 The group-specific ELISA for potyviruses reverse primers were selected from con- µM reverse primer. After the addition of 5 used monoclonal antibody (MAb) PTY 1 served regions in nucleotide sequence µl of total nucleic acid, the final reaction developed by Jordan (10) and commer- alignments of nine different accessions was brought to 20 µl with sterile water. cially available (Agdia, Inc.). Assays were (AB003308, AB011819, AF185959, After 3 min at 70°C followed by cooling to performed according to the manufacturer’s AF203536, AY169801, D86044, D89539, 4°C, 200 units of Moloney murine leuke- protocols. Reactions were evaluated 30 D89540, and S77521) of ClYVV and eight mia virus reverse transcriptase (Promega min and 24 h after the addition of sub- accessions (AB029436, AB029436, Corps., Madison, WI) was added to the strate. Leaf tissue exhibiting mosaic symp- AY178050, AY192568, D28819, D83749, reaction and incubated at 42°C for 1 h. The toms was ground using a mortar and pestle DQ641248, and U47033) of BYMV. The subsequent PCR amplification consisted of in 50 mM potassium phosphate buffer, pH forward (F) primers for ClYVV and 25-µl reactions containing 2 µl of first- 7.4, containing10 mM sodium sulfite. The BYMV were located in the NIb region of strand cDNA template; 10 mM Tris, pH extract then was used to inoculate a limited the viral polyprotein. The reverse (R) 8.0; 50 mM potassium chloride; 0.1% selection of diagnostic bean host plants, primer for ClYVV was located at the 3′ Triton X-100; 1.5 mM MgCl2; 150 mM including ‘Hystyle,’ ‘Sutter Pink,’ ‘Black terminus of the coat protein (CP) gene, and each dATP, dCTP, dGTP, and dTTP; 500 Turtle II,’ and UI-34. Chenopodium quinoa the reverse primer for BYMV was located nM each forward and reverse primer; and 1 Willd. and C. amaranticolor Coste & at the 3′ terminus of the nontranslated unit of Taq DNA polymerase (Promega Reynier were used as local lesion hosts. region. The primer sequences are as fol- Corp.). Thermocycling parameters were When other viruses were suspected based lows: ClYVV-F, 5′-TTGATGACAGCC optimized and a final profile was employed on visual diagnosis or detection by ELISA, AGATG-3′; ClYVV-R, 5′-GAATCGTGC that consisted of a single cycle of 2 min at local lesions were carefully excised from TCCAGCAATG-3′; BYMV-F, 5′-GAT 95°C; followed by 35 cycles of 1 min at C. quinoa as soon as they became visible, GGAGAGGAGCAGGTGCAC-3′; and 94°C, 1 min at 58°C, and 1 min at 72°C; ground as above, and inoculated to C.