Diagnosis and Characterization of Plant Viruses Using HTS to Support Virus Management and Tomato Breeding.

Viral diseases pose a significant threat to tomato crops (Solanum lycopersicum L.), one of the world's most economically important vegetable crops. The limited genetic diversity of cultivated tomatoes contributes to their high susceptibility to viral infections. To address this challenge, tomato breeding programs must harness the genetic resources found in native populations and wild relatives. Breeding efforts may aim to develop broad-spectrum resistance against the virome. To identify the viruses naturally infecting 19 advanced lines, derived from native tomatoes, high-throughput sequencing (HTS) of small RNAs and confirmation with PCR and RT-PCR were used. Single and mixed infections with tomato mosaic virus (ToMV), tomato golden mosaic virus (ToGMoV), and pepper huasteco yellow vein virus (PHYVV) were detected. The complete consensus genomes of three variants of Mexican ToMV isolates were reconstructed, potentially forming a new ToMV clade with a distinct 3' UTR. The absence of reported mutations associated with resistance-breaking to ToMV suggests that the Tm-1, Tm-2, and Tm-22 genes could theoretically be used to confer resistance. However, the high mutation rates and a 63 nucleotide insertion in the 3' UTR, as well as amino acid mutations in the ORFs encoding 126 KDa, 183 KDa, and MP of Mexican ToMV isolates, suggest that it is necessary to evaluate the capacity of these variants to overcome Tm-1, Tm-2, and Tm-22 resistance genes. This evaluation, along with the characterization of advanced lines using molecular markers linked to these resistant genes, will be addressed in future studies as part of the breeding strategy. This study emphasizes the importance of using HTS for accurate identification and characterization of plant viruses that naturally infect tomato germplasm based on the consensus genome sequences. This study provides crucial insights to select appropriate disease management strategies and resistance genes and guide breeding efforts toward the development of virus-resistant tomato varieties.

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.

A new begomovirus isolated from a potyvirus-infected bean plant causes asymptomatic infections in bean and N. benthamiana.

In this work, a begomovirus isolated from a bean plant coinfected with the potyviruses bean common mosaic virus and bean common mosaic necrosis virus was characterized. The three viruses were detected by high-throughput sequencing and assembly of total small RNAs, but the begomovirus-related contigs did not allow precise identification. Molecular analysis based on standard DNA amplification techniques revealed the presence of a single bipartite virus, which is a novel begomovirus according to the current taxonomic criteria. Infectious clones were generated and agroinoculated into Phaseolus vulgaris and Nicotiana benthamiana plants. In all cases, viral DNA-A and DNA-B were detected in new growths, but no symptoms were observed, thus indicating that this virus produces asymptomatic infections in both host species.

Simultaneous Detection of Both RNA and DNA Viruses Infecting Dry Bean and Occurrence of Mixed Infections by BGYMV, BCMV and BCMNV in the Central-West Region of Mexico.

A multiplex reverse transcription polymerase chain reaction (RT-PCR) assay was developed to simultaneously detect bean common mosaic virus (BCMV), bean common mosaic necrotic virus (BCMNV), and bean golden yellow mosaic virus (BGYMV) from common bean leaves dried with silica gel using a single total nucleic acid extraction cetyl trimethyl ammonium bromide (CTAB) method. A mixture of five specific primers was used to amplify three distinct fragments corresponding to 272 bp from the AC1 gene of BGYMV as well as 469 bp and 746 bp from the CP gene of BCMV and BCMNV, respectively. The three viruses were detected in a single plant or in a bulk of five plants. The multiplex RT-PCR was successfully applied to detect these three viruses from 187 field samples collected from 23 municipalities from the states of Guanajuato, Nayarit and Jalisco, Mexico. Rates of single infections were 14/187 (7.5%), 41/187 (21.9%), and 35/187 (18.7%), for BGYMV, BCMV, and BCMNV, respectively; 29/187 (15.5%) samples were co-infected with two of these viruses and 10/187 (5.3%) with the three viruses. This multiplex RT-PCR assay is a simple, rapid, sensitive, and cost-effective method for detecting these viruses in the common bean and can be used for routine molecular diagnosis and epidemiological studies.

A new allele of γ-kafirin gene coding for a protein with high lysine content in Mexican white sorghum germplasm.

BACKGROUND: Low protein digestibility and lysine content of white sorghum grain limit its use as a foodstuff. The increase in γ-kafirin cross-linking, has an important role in the reduction of protein digestibility. The objective of this study was to characterize the γ-kafirin gene in 12 Mexican tannin-free white sorghum genotypes and its relationship with protein digestibility and lysine content. RESULTS: Two alleles of γ-kafirin gene were identified: alleles 1 and 7. The predicted amino acid sequence of allele 7 showed seven point mutations; six were silent, and one missense (C235G), causing the substitution P79A in the deduced amino acid sequence. In silico analysis showed that γ-kafirin codified by allele 1 has five α-helixes without disulfide bonds, while γ-kafirin coding by allele 7 has four α-helixes and three disulfide bonds. Genotypes with allele 7 had higher lysine content than those with allele 1, showing no differences in the kafirin electrophoretic profile, neither a correlation with the protein content nor the in vitro pepsin digestibility. CONCLUSIONS: Mexican tannin-free white sorghum genotypes showed two γ-kafirin alleles, 1 and 7. Allele 7 was associated with higher lysine content; in silico analysis showed that the substitution of P79A in this allele could modify γ-kafirin secondary structure. © 2015 Society of Chemical Industry.