Coding Mutations in Vacuolar Protein-Sorting 4 AAA+ ATPase Endosomal Sorting Complexes Required for Transport Protein Homologs Underlie bc-2 and New bc-4 Gene Conferring Resistance to Bean Common Mosaic Virus in Common Bean

Bean common mosaic virus (BCMV) is a major disease in common bean (Phaseolus vulgaris L.). Host plant resistance is the most effective strategy to minimize crop damage against BCMV and the related Bean common mosaic necrosis virus (BCMNV). To facilitate breeding for resistance, we sought to identify candidate genes and develop markers for the bc-2 gene and the unknown gene with which it interacts. Genome-wide association study (GWAS) of the Durango Diversity Panel (DDP) identified a peak region for bc-2 on chromosome Pv11. Haplotype mapping narrowed the bc-2 genomic interval and identified Phvul.011G092700, a vacuolar protein-sorting 4 (Vps4) AAA+ ATPase endosomal sorting complexes required for transport (ESCRT) protein, as the bc-2 candidate gene. The race Durango Phvul.011G092700 gene model, bc-2[UI111], contains a 10-kb deletion, while the race Mesoamerican bc-2[Robust] consists of a single nucleotide polymorphism (SNP) deletion. Each mutation introduces a premature stop codon, and they exhibit the same interaction with the pathogroups (PGs) tested. Phvul.005G125100, another Vps4 AAA+ ATPase ESCRT protein, was identified as the candidate gene for the new recessive bc-4 gene, and the recessive allele is likely an amino acid substitution in the microtubule interacting and transport (MIT) domain. The two Vps4 AAA+ ATPase ESCRT proteins exhibit high similarity to the Zym Cucsa.385040 candidate gene associated with recessive resistance to Zucchini yellow mosaic virus in cucumber. bc-2 alone has no resistance effect but, when combined with bc-4, provides resistance to BCMV (except PG-V) but not BCMNV, and, when combined with bc-ud, provides resistance to BCMV (except BCMV PG-VII) and BCMNV. So instead of different resistance alleles (i.e., bc-2 and bc-22), there is only bc-2 with a differential reaction based on whether it is combined with bc-4 or bc-ud, which are tightly linked in repulsion. The new tools and enhanced understanding of this host-virus pathogen interaction will facilitate breeding common beans for resistance to BCMV and BCMNV.

Phenotypic and molecular screening of dry bean (Phaseolus vulgaris L.) breeding lines for resistance to bean common mosaic virus and bean common mosaic necrosis virus

Bean common mosaic virus (BCMV) and bean common mosaic necrosis virus (BCMNV) are among the most economically important virus species infecting common bean. The use of resistant plant cultivars is the most effective way to control these viruses. National dry bean breeding studies have been conducted by seven different governmental agricultural research institutes in Turkey, and advanced breeding lines have been developed by using the selected local dry bean populations and crossing studies. In this study, 204 breeding lines were tested for resistance levels to BCMV and BCMNV. Initially, BCMNV NL-3 and BCMV NL-4 strains were individually sap-inoculated onto the leaves of bean plants belonging to each breeding lines with 10 replications, and the reactions of plants were evaluated for symptomatic appearance of virus infection 21 days after inoculation. Additionally, phenotypic evaluation was confirmed by molecular markers linked to resistance genes. As a result of the study, 153 breeding lines were found to involve the dominant I gene whereas four and five of the tested lines had the recessive genes bc-1² and bc-2², respectively. In conclusion, it was emphasized that these breeding lines could be registered after evaluating them in terms of yield and quality. Also, the use of seeds of the resistant lines to supply the source of virus-resistance in breeding studies and maintaining their seeds at the national genebank were recommended.

Evaluation of Bean Common Mosaic Disease and Associated Aphid Vector, Aphis fabae L., on Common Bean (Phaseolus vulgaris L.) Production in Lower Eastern Kenya

Production of common bean in Kenya is constrained by pests and diseases and to improve bean yields amongst majority small-scale farmers, appropriate management strategies should be adopted. Bean common mosaic disease (BCMD) caused by bean common mosaic virus and vectored by bean aphids and infected seeds, substantially inhibit common bean production in Kenya. An extensive and diagnostic field survey was conducted in six agro ecological zones (AEZs) of lower eastern Kenya during the long and short rains of 2018 to determine BCMD incidence (BCMD-I), severity (BCMD-S), bean aphid abundance (BAA), bean aphid incidence (BAI) and the management strategies applied by farmers. Significant (P≤0.001) variations observed for these traits between bean varieties, rainy seasons and AEZs implied that farmers could select and grow a tolerant bean variety or grow a variety either in a season or an AEZ with low BCMD and bean aphid pressure. Such included AEZ-UMSA with least mean BCMD-I (42%), BCMD-S (1.9) and BAI (11%) compared to two AEZs (LHSH & LM4) that showed BCMD-I of >70%, BCMD-S >3.0 and BAI >50%. The AEZs differences could be attributed to variations in altitudes, temperature and humidity that influences vector (aphid) movement. Of the nine bean varieties identified during the survey, Selian 14 was the most preferred by farmers (at ~35%) with relatively lower BCMD-I (~49%) and BAI (~35%) compared to the least (<5%) farmer-preferred variety Wairimu that showed higher BCMD-I (56%) and BAI (~68%). Therefore variety Selian 14 was considered tolerant to BCMD and bean aphid. Significant (P≤0.001) and positive correlations (r = 0.67) between BAI and BCMD-I implied an effective control of bean aphids could reduce the impact of BCMD on bean production. On visual diagnostics, >75% of farmers could generally identify diseased or pest-infested bean crops and stage of growth of the crop most affected. None (0%) could however identify BCMD symptoms although ~40% identified the vector bean aphids with ~26% implementing some form of aphid or pest management strategy. On management, season-driven early planting and bean intercropping were the most applied strategies (>80%), crop rotation and weed control accounted for ~71%, certified seeds at 1% and non-chemical or pesticide applications (0%). Both low adoption of certified seeds and no chemical aphid control were attributed to high costs, despite the possibility the two factors could have contributed to higher incidences and severity of BCMD in the study area as the disease is both seed and vector-borne. In summary, lack of knowledge and training among farmers on diagnosis and management of aphid-pests and BCMD, were cited as the main constraints for low bean cultivation. This study therefore recommends provision of adequate extension services and farmer training in lower eastern Kenya for improved bean yield and subsequent better family livelihoods and income.

First report of bean common mosaic virus infecting heavenly bamboo (Nandina domestica) in China

Heavenly bamboo (Nandina domestica) is an evergreen ornamental plant with worldwide distribution. In May 2018, seven out of twenty N. domestica plants showing virus-like symptoms, such as yellow mosaic and curling, were observed in Lin’an, Zhejiang province. To determine the causal agent, a small RNA library was constructed using the Small RNA v1.5 Sample Prep Kit (Illumina, San Diego, USA) with total RNA extracted from leaves of a symptomatic plant. The library was sequenced by the Solexa platform at BGI Genomics (Shenzhen, China). A total number of 21,071,675 high-quality reads of 17-28 nucleotides (nt) in length remained after trimming adapter sequences and quality control. Reads were assembled using Velvet 0.7.31 and Oases 0.2.07 with the k-mer value of 17 (Schulz et al. 2012). BlastN and BlastX search against the GenBank viral nonredundant sequence databases revealed fifty-six contigs homologous to bean common mosaic virus (BCMV; genus Potyvirus; family Potyviridae). No contig homologous to the genomic sequence of other plant-infecting viruses was identified. These contigs were further assembled into a 9,315-nt fragment by SeqMan Pro 7.1.0 in Lasergene package (DNASTAR, Madison, WI), which covered 92.68% of the genome of BCMV strain CT (BCMV-CT; GenBank accession no. KM076650). The genome of this BCMV isolate (BCMV-NTZ1) was amplified by reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) using primers designed based on assembled contigs with the Phusion® High-Fidelity DNA Polymerase (New England Biolabs, Beijing, China) and the FirstChoice® RLM-RACE Kit (Invitrogen, Carlsbad, USA), respectively. Amplicons were cloned and Sanger sequenced with three independent clones per amplicon. The genome is 10,052 nt in length excluding the poly-A tail (Genbank accession no. MZ670770) and shared the highest nt sequence identities with BCMV-CT (88.46%). The putative polyprotein shared 93.36% amino acid (aa) sequence identity with that of BCMV-CT. BCMV-NTZ1 also clustered with BCMV-CT in phylogenetic trees based on BCMV full genomes and aa sequences of coat protein. Five-leaf-stage seedlings of Nicotiana tabacum, N. benthamiana, Glycine max (Linn.) Merr., and Capsicum frutescens were mechanically inoculated with sap of BCMV-infected N. domestica leaves at fifteen plants per species. Seedlings of G. max developed virus-like (mosaic and leaf deformity) symptoms (7/15) at 15 days post-inoculation, while other plants remained symptomless throughout the experiment. Subsequent RT-PCR on all the plants using primers 27F1/14Rter and sequencing confirmed the presence and absence of BCMV-NTZ1 in all symptomatic G. max seedlings and other asymptomatic indicator plants, respectively. Subsequent RT-PCR survey further confirmed the association of BCMV with symptomatic heavenly bamboo samples but not asymptomatic plants (7/20). To the best of our knowledge, this is the first report of BCMV naturally infecting heavenly bamboo in China. N. domestica is susceptible to many viruses, e.g., cucumber mosaic virus, plantago asiatica mosaic virus, nandina stem pitting virus, apple stem grooving virus, and alternanthera mosaic virus (Barnett et al. 1973; Ahmed et al. 1983; Hughes et al. 2002, 2005; Tang et al. 2010; Wei et al. 2015). Our results indicate that N. domestica can also serve as an overwinter reservoir for BCMV and special attention should be paid to the damage it may cause.

Diversity and Distribution of Viruses Infecting Wild and Domesticated Phaseolus spp. in the Mesoamerican Center of Domestication

Viruses are an important disease source for beans. In order to evaluate the impact of virus disease on Phaseolus biodiversity, we determined the identity and distribution of viruses infecting wild and domesticated Phaseolus spp. in the Mesoamerican Center of Domestication (MCD) and the western state of Nayarit, Mexico. We used small RNA sequencing and assembly to identify complete or near-complete sequences of forty-seven genomes belonging to nine viral species of five genera, as well as partial sequences of two putative new endornaviruses and five badnavirus- and pararetrovirus-like sequences. The prevalence of viruses in domesticated beans was significantly higher than in wild beans (97% vs. 19%; p < 0.001), and all samples from domesticated beans were positive for at least one virus species. In contrast, no viruses were detected in 80–83% of the samples from wild beans. The Bean common mosaic virus and Bean common mosaic necrosis virus were the most prevalent viruses in wild and domesticated beans. Nevertheless, Cowpea mild mottle virus, transmitted by the whitefly Bemisia tabaci, has the potential to emerge as an important pathogen because it is both seed-borne and a non-persistently transmitted virus. Our results provide insights into the distribution of viruses in cultivated and wild Phaseolus spp. and will be useful for the identification of emerging viruses and the development of strategies for bean viral disease management in a center of diversity.

Exploring New Sources of Viral Resistance in Cowpea Germplasm and Its Elaboration through Multivariate Analysis

Background: Cowpea is a major food legume rich in protein but its production has been dwindling by several factors including viral infection due to various virus strains in all agro-ecological zones.Methods: Sixty eight cowpea genotypes were screened against qualitative traits (leaf shape, seed surface, twinning tendency, anthocynin pigment, plant type, fodder type and cream color) and four seed borne viruses viz. cucumber mosaic virus (CMV), cowpea aphid borne mosaic virus (CABMV), black eye cowpea mosaic virus (BlCMV) and bean common mosaic virus (BCMV) under both in situ and laboratory conditions using DAC-ELISA.Result: Based on in situ screening, 16 genotypes were found resistant to all the four viruses, whereas for ELISA, 13 genotypes (27005, 27041, 27075, 27141, 27145, 27146, 27147, 27158, 27160, 27167, 27172, IT85F-1380 and IT86D-719) were found resistant to all. Twelve clusters were obtained from UPGMA based on disease severity. Genotype 27008 (Pakistan) was present in cluster VI and was susceptible to all antisera CMV, CABMV, BICMV and BCMV. Whereas 13 genotypes were present in cluster VIII which were found resistant to all the four antisera applied. Therefore, 13 genotypes suggested for safe use in any breeding program at developing resistant cultivars. First two factors obtained through PCA with eigen-values greater than 1 contributed greater than 80 per cent variability. Twelve distinct groups were observed and these were in coordination with cluster analysis.