Identification of Genes for Resistance to Bean Common Mosaic Virus and Bean Common Mosaic Necrosis Virus in Snap Bean (Phaseolus vulgaris L.) Breeding Lines Using Conventional and Molecular Methods

Bean common mosaic virus (BCMV) and Bean common mosaic necrosis virus (BCMNV) are among the biggest threats for snap bean production in Bulgaria due to their seed, aphid and mechanical transmission. Old valuable Bulgarian snap bean varieties are being neglected, because of the high percentage of virus‐infected seeds. Breeding resistant cultivars is the best way to solve the problem. The genetic control towards both viruses is assured by one dominant I gene and a number of recessive (bc‐u, bc‐1, bc‐1², bc‐2, bc‐2² and bc‐3) genes. Our aim was to identify resistance gene combinations in advanced F8 breeding lines, derived from two crosses (A‐8‐40‐7‐2‐1 × IVT 7214) and (Zaria × RH 26D), by the application of conventional and molecular approaches. Four methods were applied for the characterization of their resistance gene makeup: (i) leaf‐abscission infection test designed to identify I gene by direct inoculation with NL3 strain of BCMNV; (ii) intact‐plant infection test with strain NY15 of BCMV to separate immune genotypes, possessing bc‐ubc‐1², bc‐ubc‐2^2,bc‐ubc‐2bc‐3, I, Ibc‐1², Ibc‐2² or Ibc‐3; (iii) PCR analysis with the following markers: SCAR – SW13 (for I gene), SBD5 (for bc‐1²), ROC11 (for bc‐3) and CAPS – eIFE4 (for bc‐3); and 4) high‐temperature (more than 30°C) infection test with NL3 of BCMNV to provoke systemic necrosis in I, Ibc‐1, Ibc‐1², Ibc‐1²bc‐2² or Ibc‐3. The four methods applied worked properly and complemented each other. Valuable gene combination (Ibc‐3) was established in seven breeding lines with immune reaction to BCMNV. They will be included in the snap bean breeding programme for virus resistance.     

The Response of Lettuce Cultivars against Two Lettuce mosaic virus isolates,One Able to Systemically Infect the Resistant Cultivar Salinas 88

The response of seven lettuce cultivars to two geographically different Lettuce mosaic virus (LMV) isolates (LMV‐A, LMV‐T) was statistically evaluated based on infection rate, virus accumulation and symptom severity in different time trials. LMV‐A is characterized by the ability to systemically infect cv. Salinas 88 (mo1²‐carrying resistant cultivar), and inducing mild mosaic symptoms. Among lettuce cultivars, Varamin (a native cultivar) similar to cv. Salinas showed the most susceptibility to both LMV isolates, whereas another native cultivar, Varesh, was tolerant to the virus with minimal viral accumulation and symptom scores, significantly different from other cultivars at P < 0.05. LMV‐A systemically infects all susceptible lettuce cultivars more rapidly and at a higher rate than LMV‐T. This isolate accumulated in lettuce cultivars at a significantly higher level, determined by semiquantitative ELISA and induced more severe symptoms than LMV‐T isolate at 21 dpi. This is the first evidence for a LMV isolate with ability to systemically infect mo1²‐carrying resistant cultivar of lettuce from Iran. In this study, accumulation level of LMV showed statistically meaningful positive correlation with symptom severity on lettuce plants. Based on the results, three evaluated parameters differed considerably by lettuce cultivar and virus isolate.     

Distribution and impact of virus associated diseases of common bean (Phaseolus vulgaris L.) in northern Iran

Different viral diseases infect common bean crops in Iran. A total of 248 symptomatic samples were collected from common bean fields throughout main growing fields of Guilan province in Iran during the summer of 2006. Eight viruses were detected using double antibody-sandwich – enzyme-linked immunosorbent assay (DAS-ELISA). Bean common mosaic virus – BCMV (1%), Bean leaf roll virus – BLRV (9%), Cowpea mild mottle virus – CpMMV (6%), Southern bean mosaic virus – SBMV (3%), Cucumber mosaic virus – CMV (15%), Bean golden mosaic virus – BGMV (2%), Bean common mosaic necrosis virus – BCMNV (1%) and Bean yellow mosaic virus – BYMV (1%) were detected. Comparatively CMV (15%) was found to be more prevalent in Guilan province. Multiple infections of viruses were recorded in many samples. Weed species belonging to Chenopodiaceae, Solanaceae, Malvaceae and Amaranthaceae families were also found to be infected with the viruses.     

Occurrence,distribution and seasonal changes of viruses infecting common bean in northwestern Iran

Many surveys were conducted during 2003–2005 to study the identity, prevalence and fluctuation of bean infecting viruses in northwestern Iran. In total, 649 bean samples with virus- like symptoms were collected and analysed by double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) and tissue-print immunoassay to detect infectious viruses. Serological tests revealed the presence of Bean common mosaic virus (BCMV), Bean common mosaic necrosis virus (BCMNV), Bean yellow mosaic virus (BYMV), Cucumber mosaic virus (CMV), Alfalfa mosaic virus (AMV), Bean leaf roll virus (BLRV), Bean pod mottle virus (BPMV) and Southern bean mosaic virus (SBMV), with some co-infection occurred, with prevalence of BCMV, BCMNV and BYMV (17–29% infection rate). The incidence of viruses showed variation in over 3 years of research including more than double increase in CMV from 2004 to 2005 and obvious one-third decrease in AMV from 2003 to 2005. SBMV and BPMV were detected sporadically in the fields and the response of some differential test plants was analysed by mechanical inoculation. Western immunoblotting analysis of SBMV infected bean leaf total proteins using SBMV-specific polyclonal antibody revealed viral CP with molecular mass of 28.5 kDa which confirmed the presence of SBMV as a new threat for bean production.     

A short motif in the N‐terminal part of the coat protein is a host‐specific determinant of systemic infectivity for two potyviruses

Although the biological variability of Watermelon mosaic virus is limited, isolates from the three main molecular groups differ in their ability to infect systemically Chenopodium quinoa. Mutations were introduced in a motif of three or five amino acids located in the N‐terminal part of the coat protein, and differing in isolates from group 1 (motif: lysine‐glutamic acid‐alanine (Lys‐Glu‐Ala) or KEA, systemic on C. quinoa), group 2 (Lys‐Glu‐Thr or KET, not systemic on C. quinoa) and group 3 (KEKET, not systemic on C. quinoa). Mutagenesis of KEKET in an isolate from group 3 to KEA or KEKEA was sufficient to make the virus systemic on C. quinoa, whereas mutagenesis to KET had no effect. Introduction of a KEA motif in Zucchini yellow mosaic virus coat protein also resulted in systemic infection on C. quinoa. These mutations had no obvious effect on the disorder profile or potential post‐translational modifications of the coat protein as determined in silico.     

BCMV-ukr: isolate of bean common mosaic virus revealed in Ukraine

SUMMARY

Bean common mosaic virus (BCMV) is distributed worldwide and causes a serious disease in bean reducing growth and crops yield. The aim of this study was to investigate the occurrence of BCMV and Bean common mosaic necrotic virus (BCMNV) in Ukraine, to characterise host range and reactions of indicator plants to mechanical inoculation with the isolate and to differentiate it by using Drijfhout’s differentials. Leaf samples were positive for BCMV infection in RT-PCR assay employing specific primers with amplification of a 340-bp product. Based on a biological test on bean differentials, the isolate was assigned to pathogroup VII despite the fact that strain differed markedly from the standard strains in symptoms producing on differential groups IV and V. Partial sequence data of the coat protein region show 100% identity with BCMV 125 sequences tested. To our knowledge, this is the first attempt to characterise the BCMV circulating in Ukraine.     

Interaction of Cucumber mosaic virus and Bean yellow mosaic virus in co-infected plants of bean and broad bean

After evaluation of the responses of bean and broad bean common cultivars against an isolate of Cucumber mosaic virus (CMV-K) and Bean yellow mosaic virus (BYMV-K), interaction of isolates was statistically studied on co-infected plants of bean cv. Bountiful and broad bean cv. Lahijan at two trials. Based on viral relative concentration determined by quantitative enzyme-linked immunosorbent assay, BYMV interacts synergistically with CMV in bean at 14 days post inoculation, while in co-infection with BYMV, CMV interacts antagonistically in both host plants at least in one of the two trials. This suggests that CMV/BYMV interaction is dependent on host species and developmental stage of plant. Co-infection like single infection with CMV in bean plants led to significantly decrease in plants’ height and fresh weight than BYMV singly infected and healthy plants, while viral infection of broad bean plants did not significantly affect growth parameters. Decline effect of viral infection (especially co-infection) on chlorophyll and carotenoids value of bean plants was greater than those of broad bean. Viral infection (singly or doubly) caused irregular changes in nutrient elements values of both hosts compared with healthy ones.     

The incidence of viruses in wild Brassica nigra in Dorset (UK)

Using enzyme‐linked immunosorbent assays, the frequency of occurrence of six viruses was determined in Brassica nigra collected from five coastal sites in Dorset, spanning approximately 24 km. During 1998–2000, the viruses detected were: Turnip mosaic virus (genus Potyvirus) (TuMV), Turnip yellow mosaic virus (genus Tymovirus) (TYMV), Turnip crinkle virus (genus Carmovirus) (TCV), Turnip rosette virus (genus Sobemovirus) (TRoV), Beet western yellows virus (genus Polerovirus) (BWYV) and Cauliflower mosaic virus (genus Caulimovirus) (CaMV). Multiple infections were detected in some individuals (48/447). TuMV was detected infrequently over the three‐year period (5/597). A representative isolate of each virus was tested for its effects on glasshouse‐grown individuals from different half‐sib families of B. nigra from four of the sites. Whether inoculated manually or via aphids (Myzus persicae), TuMV caused a rapid (within 10 days) lethal systemic necrosis in the B. nigra seedlings except when they were near flowering at the time of inoculation. Each of the other viruses invaded systemically but were not lethal. Indeed, BWYV systemically invaded 13/19 glasshouse‐grown B. nigra seedlings but did not produce any visible symptoms. Otherwise, the isolates tested differed in their pathogenicity and in the symptoms they produced in infected B. nigra. With TYMV or TCV viral antigen concentration was closely linked to pathogenicity; for TRoV or CaMV, there was little or no difference in virus concentration between plants with and without symptoms. Substantial and reproducible differences were observed in sensitivity/susceptibility among B. nigra genotypes from different sites in Dorset challenged with the same virus isolate.     

Genetic diversity of legume yellow mosaic begomoviruses in Indonesia and Vietnam

High incidences of yellow mosaic symptoms were observed in soybean and yard‐long bean crops in Indonesia in 2009 and in mungbean crops in Vietnam in 2011. All five soybean and 20 yard‐long bean samples from Java, Indonesia, and 15 mungbean samples from Vietnam with symptoms tested positive for begomovirus infection by polymerase chain reaction (PCR) with primer pair PAL1v1978B/PAR1c715H. On the basis of collection location and the nucleotide sequence comparisons of the 1.5 kb begomoviral DNA‐A components amplified, a subset of samples comprising two soybean and six yard‐long bean isolates from Indonesia and five mungbean isolates from Vietnam were taken forward for more detailed examination. Sequence comparison and phylogenetic analysis of the full‐length sequences of all Indonesian and Vietnam isolates alongside other legume‐infecting begomoviruses revealed that all the isolates from Indonesia were Mungbean yellow mosaic India virus (MYMIV) strain‐A, and all from Vietnam were Mungbean yellow mosaic virus (MYMV) strain‐B. To the best of our knowledge, this is the first identification of MYMIV and MYMV associated with yellow mosaic of legumes in Indonesia and Vietnam, respectively. The epidemiological implications and potential consequences of the emergence of legume‐infecting begomoviruses on legume production in these areas of Southeast Asia are discussed.     

Identification and Characterization of a Potyvirus Isolated from Siratro Plants

The present work describes the identification and characterization of a potyvirus isolated from siratro (Macroptilium atropurpureum Urb.) in the north‐west region of the State of São Paulo, Brazil. The virus was transmitted by mechanical inoculation. Its host range was restricted mainly to members of the Fabaceae. A cDNA fragment of about 930 bp was amplified by RT/PCR, cloned and sequenced. The fragment, which included the coat protein gene, had amino acid identity percentages between 88 and 98% with isolates of Bean common mosaic virus (BCMV). Phylogenetic analysis grouped the siratro potyvirus and BCMV isolates in 99% of the replicates, including Azuki mosaic virus, Dendrobium mosaic virus, Blackeye cowpea mosaic virus and Peanut stripe virus, which have been classified as BCMV strains. This is the first citation on the presence of BCMV in siratro plants in Brazil.     

Occurrence of three distinct begomoviruses in cassava in Madagascar

The presence of East African cassava mosaic virus in association with cassava mosaic disease in Madagascar has previously been reported. We now describe virus isolates from mosaic‐affected Madagascan cassava with epitope profiles typical of African cassava mosaic virus, and an isolate with a nucleotide sequence similar to that of South African cassava mosaic virus. Thus, three distinct begomoviruses occur in cassava in Madagascar.     

Plant Virology and Next Generation Sequencing: Experiences with a Potyvirus

Next generation sequencing is quickly emerging as the go-to tool for plant virologists when sequencing whole virus genomes, and undertaking plant metagenomic studies for new virus discoveries. This study aims to compare the genomic and biological properties of Bean yellow mosaic virus (BYMV) (genus Potyvirus), isolates from Lupinus angustifolius plants with black pod syndrome (BPS), systemic necrosis or non-necrotic symptoms, and from two other plant species. When one Clover yellow vein virus (ClYVV) (genus Potyvirus) and 22 BYMV isolates were sequenced on the Illumina HiSeq2000, one new ClYVV and 23 new BYMV sequences were obtained. When the 23 new BYMV genomes were compared with 17 other BYMV genomes available on Genbank, phylogenetic analysis provided strong support for existence of nine phylogenetic groupings. Biological studies involving seven isolates of BYMV and one of ClYVV gave no symptoms or reactions that could be used to distinguish BYMV isolates from L. angustifolius plants with black pod syndrome from other isolates. Here, we propose that the current system of nomenclature based on biological properties be replaced by numbered groups (I–IX). This is because use of whole genomes revealed that the previous phylogenetic grouping system based on partial sequences of virus genomes and original isolation hosts was unsustainable. This study also demonstrated that, where next generation sequencing is used to obtain complete plant virus genomes, consideration needs to be given to issues regarding sample preparation, adequate levels of coverage across a genome and methods of assembly. It also provided important lessons that will be helpful to other plant virologists using next generation sequencing in the future.     

First Report of Tomato infectious chlorosis virus on Tomato in Bulgaria

Virus‐like chlorotic symptoms were observed on tomato plants, cv. Velocity, grown in a greenhouse, region of Plovdiv. Samples collected from the leaves with interveinal yellowing and with initial interveinal chlorosis were tested for virus presence. Only the samples collected from the upper leaves with slight interveinal chlorosis were positive for Tomato infectious chlorosis virus (TICV) in indirect ELISA. Further, RT‐PCR analysis with specific primers for Tomato chlorosis virus (ToCV) heat shock protein 70, for TICV heat shock protein 70 and for TICV minor capsid protein was positive for TICV in all tested samples. No signals were obtained with primers for ToCV. Phylogenetic analysis showed that the Bulgarian sequence of Hsp70 and a sequence of Greek isolate clustered together having the highest resampling score. Regarding CPm, the Bulgarian isolate was more relevant to the French isolate. The obtained results from phylogenetic analysis supported the idea of a close relationship between the Bulgarian and Greek isolates.     

Incidence and diversity of viruses in cowpeas and weeds in the unmanaged farming systems of savanna zones in Nigeria

Fields surveys were conducted to assess the incidence of commonly known legume viruses on cowpeas and weed hosts within and around the cowpeas farms in nine locations across the three agro-ecological zones of Nigeria. Of 315 cowpea leaf samples collected and tested for eight viruses, 69.5% were found to be infected. Bean common mosaic virus-blackeye mosaic (BCMV-BlCM), genus Potyvirus had the highest incidence (70%) and was also the most prevalent (78%). Cowpea aphid-borne mosaic virus (CABMV, genus Potyvirus) had 64% as incidence, incidence of Southern bean mosaic virus (SBMV, genus Sobemovirus) was 21%. Bean pod mosaic virus (BPMV, genus Comovirus) was detected in 1% of the samples tested. Cowpea mosaic virus (CPMV, genus Comovirus) was undetected. Other viruses tested included Cowpea mottle virus (CPMoV, genus Carmovirus), Cucumber mosaic virus (CMV, genus Cucumovirus), and Cowpea mild mottle virus (CPMMV, genus Carlavirus). Multiple virus infections were detected in 68.0% of the infected cowpea leaf samples. The combination of BCMV-BlCM and CABMV was the most common, occurring in 76.4% of all samples. Virus incidence in weeds around the cowpea plots was 2.5% (9 out of 356) whereas 1.5% (5 out of 332) of the weeds collected within the cowpea plots were infected. Some of the weeds infected were Chromoleana odorata, Centrosema sp., Thithonia diversifolia and Talinum triangulare.     

Occurrence and Genome Analysis of Cucurbit chlorotic yellows virus in Iran

In 2011 and 2012, several cucurbit‐growing regions of Iran were surveyed and samples with symptoms similar to those induced by Cucurbit chlorotic yellows virus (CCYV) were collected. The pathogen was transmitted to cucumber and melon under greenhouse conditions by whiteflies (Bemisia tabaci). RT‐PCR using designed CCYV‐specific primer pair (CCYV‐F/CCYV‐R) resulted in amplification of the predicted size DNA fragment (870 bp) for the coat protein (CP) gene in samples collected from Boushehr, Eyvanakay and Varamin. Nucleotide sequences of the CP of the three Iranian CCYV isolates were compared with five CCYV isolates obtained from GenBank and analysed. Phylogenetically, all CCYV isolates clustered in two groups; Group I is composed of five non‐Iranian isolates from China, Lebanon, Japan, Sudan and Taiwan, and the three Iranian isolates formed Group 2. Among Iranian isolates, the Eyvanakay isolate clustered in a distinct clade with the Boushehr and Varamin isolates. A phylogenetic tree based on amino acid identity of CP showed that CCYV was closely related to Lettuce chlorosis virus (LCV), Bean yellow disorder virus (BnYDV) and Cucurbit yellow stunting disorder virus (CYSDV). This is the first report of CCYV in Iran.     

Characterization and Complete Nucleotide Sequence of Two Isolates of Tomato mosaic virus

Two virus isolates, designated S1 and TL, were obtained from tomato and camellia root in China, respectively, and their host ranges, symptomatology, serological reactions and complete nucleotide sequences were determined. Isolate TL systemically infected Chenopodium amaranticolor causing leaf chlorosis, but the isolate S1 induced only local necrotic lesions. The complete nucleotide sequences of S1 and TL were determined and consisted of 6384 and 6383 nucleotides (Genbank accessions AJ132845 and AJ417701 ), respectively. Sequence analysis revealed that both isolates have the highest nucleotide sequence identity (over 92%) with Tomato mosaic virus (ToMV), but less (80%) with other tobamoviruses. Phylogenetic analyses based on the amino acid sequences of 30‐kD and 17.5‐kD proteins also indicated that both the isolates form a cluster with the isolates of ToMV. These data suggest that S1 and TL are isolates of ToMV. The possible reasons that TL infected C. amaranticolor systemically but S1 induced only local necrotic lesions are discussed.     

Far‐eastern strains of bean common mosaic virus and methods of their immunodiagnostics

The paper presents data of investigation on the physico‐chemical and antigenic properties of capsid proteins of the Bean common mosaic virus isolated from Phaseolus plants in the Russian Far East (BCMV‐R) and from China (BCMV‐C). A method for isolation of the virus preparation was selected. The purified preparations of two isolates BCMV have been obtained. The presence of one polypeptide in structural proteins of virions was established and their molecular masses determined (BCMV‐R ‐ 31,6 kD; BCMV‐C ‐ 32,1 kD). Polyclonal antiserum was obtained with titre 1:12800 and the indirect and “sandwich"‐variants of ELISA were developed to detect this virus. The allied relationships were established with the bean yellow mosaic virus and with the type representative of the genus Potyvirus ‐ PVY. Based on the data of physico‐chemical and antigenic properties it was concluded that isolates BCMV‐R and BCMV‐C are two independent strains of this virus. The presence of strain‐, virus‐ and genusspecific epitopes of capsid proteine was revealed as a result of comparison of antigenic characteristics of the Russian Far Eastern and Chinese strains of BCMV. A high antigenic activity of capsid protein of the Russian Far Eastern strain was observed.