e-Xtra* A Strain of yellow vein 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. (Aphis glycines) outbreaks occurring since 2000 have been associated with severe (ClYVV) (family virus epidemics in snap bean () production in the Great Lakes region. Our , genus ) was sus- objective was to identify specific 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 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 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 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. qui- BYMV-R, 5′-CTCGCTCTACAAAGA followed by a final extension step for 7 noa, Sutter Pink, and Hystyle. TCAG-3′. min at 72°C. Reaction products were re- Cultures of ClYVV determined to be Total nucleic acid preparation and solved by electrophoresis through 1.5% free of other viruses were inoculated onto RT-PCR. From bean plants determined agarose gels containing 1× Tris-acetate- UI-34, Hystyle, 92US-1006, and snap bean not to be infected with CMV or AMV EDTA buffer (24). DNA was purified from breeding line PS00-221A from the USDA- based on ELISA results but suspected to be the gel bands using GeneCapsule (Geno ARS, Prosser, WA, bean-breeding pro- infected with ClYVV or BYMV, total nu- Technology, Inc. St. Louis) and cloned into gram; all were determined to be suscepti- cleic acid was extracted using the method the vector pCR4 using the TOPO TA clon- ble to ClYVV. Inoculated plants were described by Dellaporta (4) as modified by ing kit (Invitrogen, Carlsbad, CA) accord- maintained in the greenhouse and observed Presting et al. (20), except that 100 mg of ing to the manufacturer’s instructions. for symptoms of necrosis on developing tissue was macerated in buffer without the Plasmid DNA was purified from Es- pods. use of liquid nitrogen. After washing with cherichia coli DH 10β using alkaline lysis Primer design. To differentiate ClYVV 70% ethanol, the final pellet was resus- (24) and clones containing the RT-PCR from BYMV by reverse-transcription po- pended in 300 µl of sterile water. Reverse product were sequenced using the dide- lymerase chain reaction (RT-PCR), DNA transcription was performed in reactions oxy-chain termination method. primers were designed using available containing 50 mM Tris, pH 8.0; 75 mM Evaluation of common bean for resis- sequences in the National Center for Bio- potassium chloride; 3 mM magnesium tance to ClYVV. Once involvement of

Table 1. Reaction of genotypes within Bean common mosaic virus (BCMV) and Bean common mosaic necrosis virus (BCMNV) host groups to Clover yel- low vein virus (ClYVV)a Symptomsb Primary Secondary Host group Host differential Resistance genes 11 dpi 21 dpi 50 dpi 0 Sutter Pink None VN TN, D D 1 Stringless Green Refugee i, bc-u VN D D 2 Redlands Greenleaf C i, bc-u, bc-1 CLL sst, sM, D D 2 Imunac i, bc-u, bc-1 NS sst, sM sst, sM 3 Redlands Greenleaf B i, bc-u, bc-12 CLL sst, sM, D D 3 UI-59 i, bc-u, bc-12 NS NS M 3 US1140c i, bc-u, bc-12 NS NS NS 4 Sanilac i, bc-u, bc-2 VN D D 5 UI-114-8 Pinto i, bc-u, bc-1, bc-2 NS NS M 6 UI-31c i, bc-u, bc-12, bc-22 NS NS NS 6 Othello i, bc-u, bc-22 VN TN D 7 IVT 7214 i, bc-u, bc-2, bc-3 NLL sM, st sM, st, TN 8 Black Turtle I I NLL, VN D D 9 Jubila I, bc-1 NLL M, D D 9 Jolandac I, bc-1 NS sst, sM sst, sM 10 Red Kloud I, bc-12 CLL sst, sM, Bl sst, sM, Bl 11 92US-1006 I, bc-u, bc-22 NS NS TN, pod lesions 12 TARS-VR-1s I, bc-3 NS NS NS a All lines were evaluated using ClYVV-WI and ClYVV-OR (strains from Wisconsin and Oregon, respectively) in separate experiments. Host plants reacted similarly with each strain, except symptoms caused by ClYVV-WI were more severe. Only symptoms caused by ClYVV-WI are listed in the table. b Symptom legend: Bl = blistering, CLL = chlorotic local lesions, M = mosaic, mM = mild mosaic, NLL = necrotic local lesions, NS = no symptoms, D = plant death, sM = severe mosaic, st = stunted growth, sst = severe stunting, TN = top (systemic) necrosis, and VN = vein necrosis; dpi = days post inocula- tion. c Lines and cultivars with resistance to ClYVV reported in the literature.

Plant Disease / July 2008 1027 ClYVV in the disease complex was deter- each of 12 host groups developed by lated from (Pisum sativum L.) and a mined and verified, biological assays were Drijfhout et al. (6) for identification of strain from New York (ClYVV-NY) de- initiated to identify potential genes for strains of BCMV and BCMNV. The differ- scribed by Provvidentti et al. (23). resistance to ClYVV isolated from in- entials represent eight resistance genes or Three individual plants of each line or fected snap bean from Wisconsin, hereon combinations thereof and are listed in Ta- cultivar were mechanically inoculated at referred to as ClYVV-WI. Because ble 1. Inoculated plants were maintained in the primary leaf stage with each of the US1140, UI-31, ‘Jolanda,’ and ‘Imuna’ a greenhouse at 18 and 30°C (night and strains. The procedure was repeated 2 days have been reported as resistant to ClYVV day, respectively) under natural light for following the first inoculation to reduce (23,25–27), we evaluated their resistance symptom development. A collection of 63 the possibility of escapes. Plants were to the newly obtained strain of ClYVV commercial snap bean cultivars and breed- maintained under greenhouse conditions from Wisconsin. Seedlings of each line in ing lines also was screened for resistance and evaluated based on symptom response. several repeated experiments were inocu- to ClYVV-WI with the intent of identify- A disease severity index (dsi) was assigned lated at the primary leaf stage and main- ing a resistance source of immediate use to as follows: 1 = no disease symptoms; 2 = tained in the greenhouse for at least 50 the snap bean industry. To compare the mild mosaic on secondary leaves and no days. biological effects of ClYVV-WI with other observable stunting; 3 = mosaic, leaf roll, The limited diagnostic host range de- known strains of ClYVV, the lines were stunting; 4 = severe mosaic, severe stunt- scribed above was expanded to include a screened additionally with a mild strain of ing, leaf roll, blistering; and 5 = severe set of bean differentials representative of ClYVV from Oregon (ClYVV-OR) iso- mosaic, systemic vein necrosis, apical necrosis, plant death.

RESULTS Field observations and host response. Snap bean samples obtained from the Great Lakes region between 2000 and 2005 with pods showing varying levels of necrosis were evaluated for the presence of viruses, with focus on ClYVV. All pods determined by RT-PCR to be infected with ClYVV from Wisconsin (and other neighboring states) exhibited extensive brown to rust-colored pod necrosis that often affected the entire pod (Fig. 1A). Pods frequently showed internal necrotic areas (Fig. 1B). By comparison, CMV and AMV were detected as a coinfection in pods exhibiting necrosis that was less se- vere, more localized, and somewhat darker in color (Fig. 2). Reactions on bean plants in grower’s fields infected with either ClYVV or BYMV consisted of foliar symptoms typical of these viruses, includ- ing severe stunting and mosaic. Apical (top) necrosis occurred with infection by ClYVV in field-grown Hystyle and on greenhouse-grown Sutter Pink, Black Tur- tle II, UI-34, and Hystyle inoculated with ClYVV-WI. Top necrosis on these hosts never was observed in the field or green- house when infected with BYMV only; however, severe mosaic symptoms were typical on leaves and mottling was often observed on the pods. ClYVV-WI caused local lesions on C. quinoa and C. amaranticolor. Virus symp- toms became systemic on both hosts, caus- ing severe vein and leaf necrosis, malfor- mation, and stunting. Symptoms caused by BYMV in C. amaranticolor included local chlorotic lesions followed by systemic vein yellowing and slightly malformed leaves. Serology and RT-PCR. Bean samples exhibiting the characteristic pod necrosis disease or foliar symptoms, and infected with ClYVV but not BYMV, did not react positively with the Potyvirus group- specific PTY 1 MAb. In contrast, samples Fig. 1. A, Extensive external pod necrosis symptoms on snap bean caused by infection with the Clover exhibiting symptoms typical of BYMV yellow vein virus isolate from Wisconsin. B, Snap bean pods cut longitudinally showing internal and such as leaf mosaic or yellow mosaic re- external necrotic areas resulting from infection with the Clover yellow vein virus isolate from Wiscon- acted positively with the PTY 1 Potyvirus sin. Uppermost pod is healthy. MAb. For the purpose of determining the

1028 Plant Disease / Vol. 92 No. 7 effect of ClYVV-WI alone, samples testing strain ClYVV no. 30 (accession no. different strains of ClYVV (-OR, -NY, and positive for BYMV, CMV, or AMV were NP734172), a strain not reported to cause -WI) exhibited typical symptoms of infec- eliminated from further testing. pod necrosis (26,34), only four amino acid tion by ClYVV except ‘Roma 442,’ a In all cases where extracts from symp- substitutions occurred in the CP sequence, Romano type bean that remained symp- tomatic leaves of plants exhibited pod corresponding to 98% identity level (Fig. 3). tomless for up to 21 dpi (Table 2). At 50 necrosis but tested negative by ELISA with Evaluation of common bean lines for dpi, this cultivar had developed leaf mosaic the Potyvirus MAb PTY 1, the plant ex- resistance to ClYVV. Susceptible dry but did not exhibit stunting symptoms, tract subsequently yielded a positive reac- bean lines expressed typical symptoms apical necrosis, or pod necrosis. Infection tion by RT-PCR using DNA primers de- resulting from infection with ClYVV. All in Roma 442 was verified by RT-PCR and signed for the detection of ClYVV. No BCMV host differentials screened were by back-inoculation to indicator hosts. In amplicons were produced with the BYMV susceptible to ClYVV-WI and ClYVV-OR additional experiments, Roma 442 was primers from any samples with pod necro- except UI-31 and US1140, and TARS-VR- inoculated with the NL3-D strain of sis that did not react with MAb PTY 1. In 1s with I + bc-3 genes (Table 1). Lines BCMNV. Inoculated plants showed epi- contrast, preparations from samples that 92US-1006 and UI-114-8 Pinto initially nasty and vein necrosis on inoculated pri- exhibited symptoms typical of BYMV appeared to be resistant but subsequently mary leaves followed by severe stunting tested positive with the Potyvirus MAb and showed characteristic virus symptoms by and top necrosis, a response indicating I produced amplified products (1,113 bp) by 50 days post inoculation (dpi). Reactions gene presence but not bc-3. All snap bean RT-PCR using the BYMV-specific primers of the host differentials to inoculations lines except Roma 442 were rated suscep- (data not shown). Neither ClYVV nor with ClYVV-WI and ClYVV-OR were tible to highly susceptible to ClYVV. All BYMV amplicons were produced from consistent across resistant and susceptible plants with a dsi = 5 rating at 7 dpi had leaf samples of bean plants that did not lines, except that symptoms in susceptible responded with vein necrosis on inoculated exhibit visible foliar symptoms. Extracts lines were considerably more severe with primary leaves. This was followed by se- from symptomless plants did not yield any ClYVV-WI than with ClYVV-OR. ClYVV vere stunting, apical necrosis, and death, detectable infection of host range plants was not detected by RT-PCR in bean lines usually within 14 to 21 dpi. Similar to after mechanical inoculation. that did not develop symptoms; thus, these observation of symptoms in dry bean, In some cases, ClYVV amplicons were were considered resistant. Additional dry ClYVV-OR produced milder symptoms on produced from chocolate pod tissue origi- bean lines with I + bc-3 (USLK-1, USLK- 46 of 63 snap bean lines relative to those nating from field samples where foliar 2, USLK-3, USDK-4 USDK-5, USWK-6, symptoms produced by ClYVV-WI or symptoms were absent. When test plants USCR-7, USCR-9, and Raven; 15–19), ClYVV-NY (Table 2). were inoculated with leaf and pod tissue and with bc-3 alone (USCR-8; (17) re- from these samples, infection was estab- mained symptomless after 50 dpi and DISCUSSION lished only from pod tissue. Infection with tested negative for ClYVV by RT-PCR . ClYVV isolated from snap bean in Wis- the virus subsequently was verified by RT- Evaluation of commercial snap bean consin and other areas of the Upper Mid- PCR. Interestingly, no symptoms were cultivars. Sixty-three snap bean cultivars west and New York is a previously unre- produced in test plants inoculated with and breeding lines inoculated with three ported strain that causes extensive pod expressed sap from the symptomless leaves, and no amplicons were produced from total nucleic acid extracted from these samples. Nucleotide sequence of amplicons (835 bp) was determined for two clones each of the ClYVV-WI RT-PCR products obtained from four different snap bean samples after local lesion transfer. Nucleic acid sequence identity was greater than 98% within each pair of clones. The sequence encoding the CP of ClYVV-WI was deposited in Gen- Bank (accession no. EF591473). Nucleic acid sequence identities ranged from 83 to 96% with other ClYVV CP sequences available in GenBank whereas amino acid identities ranged from 91 to 98%. The most common amino acid change between samples was substitution of the proline residue with serine at position 19 in the motif SKDKEP, and the glycine residue with glutamic acid at position 94 in the motif TQEQLG (Fig. 3). Compared with ClYVV-OR, 5 amino acid substitutions occurred within the first 50 residues of ClYVV-WI at positions 6, 19, 26, 34, and 47 (Fig. 3). ClYVV-WI contained the NAG aphid transmission motif located seven residues downstream of the CP 5′ N-terminus, which is consis- tent with all other known isolates of ClYVV. The amino acid sequence align- ment of ClYVV-WI was 96% identical with the CP gene sequence of ClYVV-OR Fig. 2. Pod symptoms on a commercial snap bean cultivar co-infected with Cucumber mosaic virus (Fig. 3). When compared with Japanese and Alfalfa mosaic virus.

Plant Disease / July 2008 1029 necrosis. The necrosis was typically more ruses, BCMV, Lettuce mosaic virus, and widely grown in regions where snap bean severe than that caused by the complex of Pea seedborne mosaic virus have been crops are produced and is a known host of CMV and AMV, and is referred to as reported to be pollen transmitted (30) but ClYVV (22). However, during 2002 and chocolate pod by local growers (8,31). ClYVV has not. Common bean is self- 2003 in New York, Shah et al. (29) investi- These pod symptoms have not been ob- pollinated and cross-pollination in the field gated potential movement of CMV, AMV, served or reported on snap bean as a result rarely occurs except under high popula- and ClYVV or BYMV into snap bean of infection by previously identified strains tions of insect pollinators or by wind. Ad- fields from local and remote alfalfa fields. of ClYVV. Generally, infection with ditional studies using ClYVV-WI are re- They found no evidence for an increased ClYVV produces distortion and mottling quired to test this hypothesis. Other risk of virus infection in snap bean adja- in pods without necrosis (22). Depending pathogens also have been reported to cause cent to alfalfa, and no correlation was on the virus strain, environment, cultivar, necrosis on bean pods. A russet disease on found between virus incidence in alfalfa and vector populations, ClYVV may or snap bean caused by Plectosporium taba- and adjacent snap bean. may not have a significant effect on har- cinum described by Dillard et al. (5) was Unlike other reported common strains of vest yields in processing bean. For exam- reported recently that produces symptoms the virus (10–12), ClYVV-WI was unde- ple, a severe outbreak of ClYVV was re- on pods similar to those caused by ClYVV tectable by the Potyvirus broad-spectrum- ported recently in a bean field in or CMV+AMV. However, no pathogenic reacting PTY 1 MAb. The inability of PTY California, but pods apparently were not fungi were isolated from any of the pods 1 to detect ClYVV causing pod necrosis affected (3). we examined that were infected with has been problematic with regard to identi- Several samples of snap bean plants ClYVV-WI. fying the virus in snap bean each year in were received in different years that Other than its isolation from snap bean the affected regions. PTY 1 has been re- showed pod necrosis symptoms associated grown in the Great Lakes region, the ulti- ported to react to at least 48 different with ClYVV-WI; however, no symptoms mate source of the ClYVV strain causing aphid-transmissible potyviruses, including were present in leaves and the virus could chocolate pod is unknown. The strain does all strains of the BYMV subgroup, includ- not be detected in leaf extracts using RT- appear to be specifically associated with ing BYMV, Pea mosaic virus, White lupin PCR. Infection by movement of infected high populations of soybean aphid. Alfalfa mosaic virus, and ClYVV (7,11,12). How- pollen has been considered as a hypothesis (Medicago sativa L.) was considered a ever, in the absence of BYMV, none of the for this occurrence. Among the potyvi- potential source of the virus because it is samples collected that were exhibiting the

Fig. 3. Amino acid sequence comparison of the coat protein region of Clover yellow vein virus strain from Wisconsin (ClYVV-WI), Oregon (ClYVV-OR), and strain no. 30 (ClYVV No. 30) (26,34) (accession no. NP734172). Amino acids of each strain that differ from those of ClYVV-WI are indicated.

1030 Plant Disease / Vol. 92 No. 7 Table 2. Reaction of commercial snap bean chocolate pod disease caused by ClYVV- termined by these genes alone that confer cultivars and breeding lines to inoculation with WI and related isolates in 2000–05 from resistance to BCMV. three strains of Clover yellow vein virus Wisconsin or other states, including Jolanda and Imuna both possess the desc (ClYVV) under greenhouse conditions Michigan, Minnesota, Ohio, or New York, gene for resistance to ClYVV as well as Disease ratinga resulted in positive reactions to this anti- gene bc-1 for resistance to BCMV. The body by ELISA (data not shown). Like- two genes are unrelated because other lines Cultivar WI OR NY wise, the Oregon and New York strains with bc-1 were susceptible to ClYVV, as Angers 4 3 4 used in our evaluations also were not de- determined by Sato et al (26). Jolanda and Arena 5 4 5 tected. It is not immediately clear why Imuna both were susceptible to ClYVV- Banga 5 5 5 ClYVV-WI or other isolates evaluated WI, but exhibited symptoms less severe Benton 5 4 4 Booster 4 4 3 from this region did not react with the than their BCMV host group counterparts Bronco 5 4 4 PTY1 MAb, although similar results have in this study. The susceptibility of lines Carlo 4 4 3 been reported for other Potyvirus isolates. with reported resistance to ClYVV (28) Castano 5 4 5 For example, PTY1 did not detect 3 of 15 provides additional evidence that ClYVV- Eden 5 3 5 isolates of ringspot virus in Florida WI may be a unique strain of virus. Envy 5 4 4 (1) and mottle virus isolates M and Except for IVT 7214, genotypes with Espada 5 4 4 VS were not detected by Li et al. but an- the bc-3 gene for resistance to BCMV and Evergreen 4 3 3 Excalibur 4 4 4 other isolate of the virus was detected by BCMNV were resistant to ClYVV-WI. Firstmate 5 5 5 the antibody (11,14). A possible reason for These results suggest that bc-3, or a gene Flo 5 4 5 lack of detection by PTY 1 may involve tightly linked with bc-3, may be associated Foremost 4 3 3 weakened avidity due to a difference of with resistance to ClYVV-WI. Gold Crop 4 3 4 one or more of the target amino acids in Because ClYVV is transmitted by Goldmine 5 3 3 the core region of the ClYVV-WI CP. in a nonpersistent manner, the use HB 1745 4 4 3 Recent reports have focused on place- of pesticides to manage the spread of the Hercules 5 4 5 ment of ClYVV isolates into subsets or virus is generally ineffective. Hence, the Hialeah 4 3 5 Hystyle 5 4 5 strains based on serology, host response, most effective management strategy is to IDMV 19 5 4 4 and sequence comparisons (25,33). Sa- develop cultivars with resistance or toler- IDMV 20 5 4 4 saya et al. (25) described variabilities in ance to ClYVV. Unfortunately, no snap IDMV 40 5 3 4 10 isolates of ClYVV collected in Japan bean cultivars currently in production ex- Igloo 5 4 5 that allowed them to be divided into two hibit resistance to the virus. It is likely that Improved Tendergreen 5 5 5 subsets based on serology, host response, Roma 442 may be grown successfully in Kentucky Blue 4 4 4 and sequence comparisons. The predicted areas with a high incidence of ClYVV, Labrador 5 4 5 Lynx 4 3 3 CP amino acid sequence of ClYVV-WI although evaluation under field conditions Masai 5 4 5 was 98% identical with seven isolates in is a prerequisite for this recommendation. Medinah 5 3 5 subset 1 and ranged from 91 to 93% iden- In addition, because it is a flat-pod proc- Minuette 4 3 4 tity with three isolates composing subset essing Romano type, production is limited Moncayo 4 3 3 2. ClYVV-WI also produced systemic compared with the more popular round- MV 185 5 4 5 symptoms of mosaic, severely malformed podded snap bean types. Introgression of Nelson 5 4 4 leaves, and necrosis in C. amaranticolor the bc-3 gene into current snap bean culti- Newton 4 4 4 and C. quinoa, which was consistent with vars may offer resistance against ClYVV, Nicelo 5 4 4 NY 6020-4 5 4 5 host responses for the strains reported in but further investigation is warranted to Opera 5 5 5 subset 1. Although we were not able to verify that bc-3 confers resistance to Orion 4 4 4 compare serological reactions with the ClYVV. PLS 87 3 3 4 MAbs produced by Sasaya et al. (25), the PLS 84 5 3 5 biological data and molecular sequence ACKNOWLEDGMENTS PLS 553 5 4 5 comparisons suggest that ClYVV-WI are ClYVV-NY was kindly supplied by A. Taylor, Cornell University. PLS 118 5 3 3 similar to their isolates in subset 1. It is Primo 5 4 5 Rambo 4 3 4 unknown if any of the Japanese isolates LITERATURE CITED Roma 442 2 2 2 produced symptoms on snap bean compa- 1. Baker, C. A., Lecoq, H., and Purcifull, D. E. Romano Gold 5 5 5 rable with those caused by ClYVV-WI 1991. Serological and biological variability Sag 5 5 5 because pod necrosis was not cited as a among Papaya ringspot virus type-W isolates Saurus 4 3 3 symptom (25). in Florida. Phytopathology. 81:722-728. 2. Converse, R., and Martin, R. 1990. Pages 179- SB 4243 5 4 4 Bean differential hosts UI-31 and 196 in: Serological Methods for Detection and Sea Biscuit 5 4 4 US1140 did not develop symptoms in re- Identification of Viral and Bacterial Plant Serin 5 4 4 sponse to inoculation with ClYVV-WI. Pathogens. R. Hampton, E. Ball, and S. De Sirio 4 4 3 Boer, eds. American Phytopathological Soci- Spartacus 5 3 5 Provvidenti and Schroeder (23) proposed a single recessive gene, by-3, the symbol of ety, St. Paul, MN. Summit 4 3 5 3. Crnov, R. and Gilbertson, R. L. 2001. Out- Tapia 3 3 3 which was later changed to cyv (21), that break of Clover yellow vein virus in a bean Titan 4 3 5 conferred a high level of resistance to field in Colusa County, California. Plant Dis. Top Crop 5 4 5 ClYVV in US1140. The gene has been 85:444. Ulysess 4 3 4 introgressed into some dry bean cultivars 4. Dellaporta, S. L. 1993. Plant DNA miniprep Valentino 4 4 4 (27) but it is not known to be present in and microprep: version 2.1-2.3. Pages 522-525 VR Romano 5 4 4 in: The Maize Handbook. M. Freeling and V. any snap bean breeding lines or cultivars. Walbot, eds. Springer-Verlag, New York. a 2 ClYVV strains from Wisconsin (WI), Oregon In addition, US1140 possesses bc-1 and 5. Dillard H. R., Cobb A. C., Shah D. A., and (OR), and New York (NY). Disease severity UI-31 possesses bc-12 and bc-22, which Straight K. E. 2005. Identification and charac- index: 1= no foliar or pod symptoms; 2 = mild confer resistance to BCMV. However, the terization of russet on snap beans caused by mosaic, no stunting or apical necrosis; 3 = susceptibility of other host differentials Plectosporium tabacinum. Plant Dis. 89:700- mosaic, stunting; 4 = severe mosaic, severe 2 2 704. stunting, leafroll or apical necrosis; and 5 = with bc-1 or bc-2 resistance genes (Table 6. Drifjhout, E., Silbernagel, M. J., and Burke, vein and apical necrosis followed by death of 1) indicates that resistance to ClYVV, in D. W. 1978. Differentiation of strains of the plant. the case of UI-31 and US1140, is not de- Bean common mosaic virus. Neth. J. P1ant

Plant Disease / July 2008 1031 Pathol. 84:13-26. plasm lines USDK-4, USDK-5, and USWK-6 thology 87:1014-1019. 7. Guaragna, M. A., Jordan, R. L., and Putnam, with resistance to Bean common mosaic and 26. Sato, M., Masuta, C., and Uyeda, I. 2003. M. L. 2004. First report of Bean yellow mosaic necrosis viruses. Annu. Rep. Bean Improv. Natural resistance to Clover yellow vein virus virus (pea mosaic strain) in Verbena × hybrida. Coop. 41:229-230. in beans controlled by a single recessive locus. Plant Dis. 88:574. 17. Miklas, P. N, and Hang, A. N. 1998. Release of Mol. Plant-Microbe Interact. 16:994-1002. 8. Grau, C. R., Stevenson, W. R., and Mondjana, light red kidney dry bean germplasm lines 27. Scully, B., Provvidenti, R., Benscher, D., A. M. 2002. Viruses causing losses on process- USLK-1, -2, and -3 with resistance to Bean Halseth, D. E., Miller, J. C., and Wallace, D. ing beans in the Midwest. Proc. Wis. Fertilizer, common mosaic and necrosis viruses. Annu. H. 1995. Five multiple-virus-resistant common Aglime, Pest Manage. Conf. 41:248-256. Rep. Bean Improv. Coop. 41:231-232. bean breeding lines. HortScience 30:1320- 9. Hill, J. H., Alleman, R., Hogg, D. B., and 18. Miklas, P. N., Hang, A. N., Kelly, J. D., 1323. Grau, C.R. 2001. First report of transmission Strausbaugh, C. A., and Forster, R. L. 2002. 28. Scully, B., Provvidenti, R., Halseth, D. E., and of Soybean mosaic virus and Alfalfa mosaic vi- Registration of three kidney bean germplasm Wallace, D. H. 1991. CU-M90: a black dry rus by Aphis glycines in the New World. Plant lines resistant to Bean common mosaic and bean breeding line resistant to Clover yellow Dis. 85:561. necrosis potyviruses: USLK-2 light red kidney, vein virus. HortScience 26:435-436. 10. Jordan, R. 1989. Mapping of potyvirus- USDK-4 dark red kidney, and USWK-6 white 29. Shah, D. A., Dillard, H. R., Mazumdar- specific and group-common antigenic determi- kidney. Crop Sci. 42:674-675. Leighton, S., Gonsalves, D., and Nault, B. A. nants with monoclonal antibodies by western- 19. Miklas, P. N., and Kelley, J. D. 2002. Registra- 2006. Incidence, spatial patterns, and associa- blot analysis and coat protein amino acid se- tion of two cranberry bean germplasm lines re- tions among viruses in snap bean and alfalfa in quence comparisons. (Abstr.) Phytopathology sistant to Bean common mosaic and necrosis New York. Plant Dis. 90:203-210. 79:1157. potyviruses: USCR-7 and USCR-9. Crop Sci. 30. Shukla, D. D., Ward, C. W., and Brunt, A. A. 11. Jordan, R. 1992. Potyviruses, monoclonal 42:673-674. 2004. The Potyviridae. CAB International, antibodies, and antigenic sites. Arch. Virol. 20. Presting, G. G., Smith, O. P., and Brown, C. R. Wallingford, UK. (Suppl. 5):81-95. 1995. Resistance to potato leafroll virus in po- 31. Stevenson, W. R., Grau, C. R., and German, T. 12. Jordan, R., and Hammond, J. 1991. Compari- tato plants transformed with the coat protein L. 2006. Reaction of snap bean cultivars and son and differentiation of potyvirus isolates gene or with vector control constructs. Phyto- advanced breeding lines to aphid transmitted and identification of strain-, virus-, subgroup- pathology 85:436-442. viruses. Proc. Wis. Fertilizer, Aglime, Pest specific and potyvirus group-common epitopes 21. Provvidenti, R. 1987. List of genes in Phaseo- Manage. Conf. 45:249-256. using monoclonal antibodies. J. Gen. Virol. lus vulgaris for resistance to viruses. Annu. 32. Tachel, S. P., Baggett, J. R., and Hampton, R. 72:25-36. Rep. Bean Improv. Coop. 30:1-4. O. 1985. Relationship between resistance to 13. Larsen, R. C., Miklas, P. N., Eastwell, K. C., 22. Provvidenti, R., and Morales, F. J. 2005. Clo- severe and type strains of Bean yellow mo- Grau, C. R., and Mondjana, A. 2002. A virus ver yellow vein virus. Pages 75-76 in: Com- saic virus. J. Am. Soc. Hortic. Sci. 110:96- disease complex devastating late season snap pendium of Bean Diseases. H. F. Schwartz, ed. 99. bean production in the Midwest. Bean Improv. American Phytopathological Society, St. Paul, 33. Uga, H., Kobayashi, Y. O., Hagiwara, K., Coop. Proc. 45:36-37. MN. Honda, Y., Omura, T., and Sasaya, T. 2002. 14. Li, R. H., Zettler, F. W., Elliott, M. S., Peter- 23. Provvidenti, R., and Schroeder, W. T. 1973. Molecular characterization of Bean yellow mo- sen, M. A., Still, P. E., Baker, C. A., and Mink, Resistance in Phaseolus vulgaris to the severe saic virus from vegetatively propagated dwarf G. I. 1991. A strain of Peanut mottle virus strain of . Phytopa- Gentiana plants. J. Gen Plant Pathol. 68:378- seedborne in bambarra groundnut. Plant Dis. thology 63:196-197. 381. 75:130-133. 24. Sambrook, J., Fritsch, E. F., and Maniatis, T. 34. Uyeda, I., Takahashi, T., and Shikata, E. 1991. 15. Miklas, P. N., and Hang, A. N. 1998. Release 1989. Molecular Cloning: A Laboratory Man- Relatedness of the nucleotide sequence of the of cranberry dry bean germplasm lines USCR- ual, 2nd ed. Cold Spring Harbor Laboratory 3'-terminal region of Clover yellow vein poty- 7 and USCR-8 with resistance to Bean com- Press, Cold Spring Harbor, NY. virus RNA to Bean yellow mosaic potyvirus mon mosaic and necrosis viruses. Annu. Rep. 25. Sasaya, T., Shimizu, T., Nozu, Y., Nishiguchi, RNA. Intervirology 32:234-45. Bean Improv. Coop. 41:227-228. M., Inouye, N., and Koganezawa, H. 1997. 35. USDA National Agricultural Statistics Service. 16. Miklas, P. N., and Hang, A. N. 1998. Release Biological, serological, and molecular vari- 2005. Online publication. United States De- of dark red and white kidney dry bean germ- abilities of Clover yellow vein virus. Phytopa- partment of Agriculture, Washington DC.

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