Advancing common bean (Phaseolus vulgaris L.) disease detection with YOLO driven deep learning to enhance agricultural AI.

Common beans (CB), a vital source for high protein content, plays a crucial role in ensuring both nutrition and economic stability in diverse communities, particularly in Africa and Latin America. However, CB cultivation poses a significant threat to diseases that can drastically reduce yield and quality. Detecting these diseases solely based on visual symptoms is challenging, due to the variability across different pathogens and similar symptoms caused by distinct pathogens, further complicating the detection process. Traditional methods relying solely on farmers' ability to detect diseases is inadequate, and while engaging expert pathologists and advanced laboratories is necessary, it can also be resource intensive. To address this challenge, we present a AI-driven system for rapid and cost-effective CB disease detection, leveraging state-of-the-art deep learning and object detection technologies. We utilized an extensive image dataset collected from disease hotspots in Africa and Colombia, focusing on five major diseases: Angular Leaf Spot (ALS), Common Bacterial Blight (CBB), Common Bean Mosaic Virus (CBMV), Bean Rust, and Anthracnose, covering both leaf and pod samples in real-field settings. However, pod images are only available for Angular Leaf Spot disease. The study employed data augmentation techniques and annotation at both whole and micro levels for comprehensive analysis. To train the model, we utilized three advanced YOLO architectures: YOLOv7, YOLOv8, and YOLO-NAS. Particularly for whole leaf annotations, the YOLO-NAS model achieves the highest mAP value of up to 97.9% and a recall of 98.8%, indicating superior detection accuracy. In contrast, for whole pod disease detection, YOLOv7 and YOLOv8 outperformed YOLO-NAS, with mAP values exceeding 95% and 93% recall. However, micro annotation consistently yields lower performance than whole annotation across all disease classes and plant parts, as examined by all YOLO models, highlighting an unexpected discrepancy in detection accuracy. Furthermore, we successfully deployed YOLO-NAS annotation models into an Android app, validating their effectiveness on unseen data from disease hotspots with high classification accuracy (90%). This accomplishment showcases the integration of deep learning into our production pipeline, a process known as DLOps. This innovative approach significantly reduces diagnosis time, enabling farmers to take prompt management interventions. The potential benefits extend beyond rapid diagnosis serving as an early warning system to enhance common bean productivity and quality.

Reduction in vertical transmission rate of bean common mosaic virus in bee-pollinated common bean plants.

Vertical transmission, the transfer of pathogens across generations, is a critical mechanism for the persistence of plant viruses. The transmission mechanisms are diverse, involving direct invasion through the suspensor and virus entry into developing gametes before achieving symplastic isolation. Despite the progress in understanding vertical virus transmission, the environmental factors influencing this process remain largely unexplored. We investigated the complex interplay between vertical transmission of plant viruses and pollination dynamics, focusing on common bean (Phaseolus vulgaris). The intricate relationship between plants and pollinators, especially bees, is essential for global ecosystems and crop productivity. We explored the impact of virus infection on seed transmission rates, with a particular emphasis on bean common mosaic virus (BCMV), bean common mosaic necrosis virus (BCMNV), and cucumber mosaic virus (CMV). Under controlled growth conditions, BCMNV exhibited the highest seed transmission rate, followed by BCMV and CMV. Notably, in the field, bee-pollinated BCMV-infected plants showed a reduced transmission rate compared to self-pollinated plants. This highlights the influence of pollinators on virus transmission dynamics. The findings demonstrate the virus-specific nature of seed transmission and underscore the importance of considering environmental factors, such as pollination, in understanding and managing plant virus spread.

Assessment of Prickly Sida as a Potential Inoculum Source for Sida Golden Mosaic Virus in Commercial Snap Bean Farms in Georgia, United States.

Sida golden mosaic virus (SiGMV), an obligate pathogen that infects snap beans (Phaseolus vulgaris), is known to infect prickly sida (Sida spinosa L.), which is a common weed in agricultural farms in Georgia. Prickly sida has also been reported as a suitable host of sweetpotato whitefly (Bemisia tabaci), the vector of SiGMV. Despite being a host for both SiGMV and its vector, the role of prickly sida as a reservoir and inoculum source for SiGMV in snap bean farms has not been evaluated. This study was conducted to document the occurrence of SiGMV-infected prickly sida plants and to assess its potential role as a source of SiGMV inoculum in snap bean farms. A survey of 17 commercial snap bean farms conducted in spring 2021 confirmed the presence of SiGMV-infected prickly sida in southern Georgia. In fall 2021 and 2022, on-farm field trials were conducted in four commercial farms where SiGMV-infected prickly sida plants were documented earlier as a part of survey in spring 2021. The spatial distribution and temporal patterns of adult whiteflies and SiGMV on snap bean were compared between macroplots (13.7 × 30.5 m) "with prickly sida" or "without prickly sida" that were at least 232 m apart from each other. We did not observe any consistent differences in counts of adult whiteflies between macroplots with or without prickly sida in the four commercial farms. SiGMV infection was detected earlier and with higher incidences in snap bean macroplots "with prickly sida" compared with macroplots "without prickly sida." An apparent disease gradient was observed in two of the four farms assessed. Higher SiGMV incidences were observed on the edges of macroplots "with prickly sida." These findings indicate prickly sida as a potential natural reservoir and a source for SiGMV spread in snap bean farms in southern Georgia.

A viral RNA hijacks host machinery using dynamic conformational changes of a tRNA-like structure.

Viruses require multifunctional structured RNAs to hijack their host's biochemistry, but their mechanisms can be obscured by the difficulty of solving conformationally dynamic RNA structures. Using cryo­electron microscopy (cryo-EM), we visualized the structure of the mysterious viral transfer RNA (tRNA)­like structure (TLS) from the brome mosaic virus, which affects replication, translation, and genome encapsidation. Structures in isolation and those bound to tyrosyl-tRNA synthetase (TyrRS) show that this ~55-kilodalton purported tRNA mimic undergoes large conformational rearrangements to bind TyrRS in a form that differs substantially from that of tRNA. Our study reveals how viral RNAs can use a combination of static and dynamic RNA structures to bind host machinery through highly noncanonical interactions, and we highlight the utility of cryo-EM for visualizing small, conformationally dynamic structured RNAs.

R-BPMV-Mediated Resistance to Bean pod mottle virus in Phaseolus vulgaris L. Is Heat-Stable but Elevated Temperatures Boost Viral Infection in Susceptible Genotypes.

In the context of climate change, elevated temperature is a major concern due to the impact on plant-pathogen interactions. Although atmospheric temperature is predicted to increase in the next century, heat waves during summer seasons have already become a current problem. Elevated temperatures strongly influence plant-virus interactions, the most drastic effect being a breakdown of plant viral resistance conferred by some major resistance genes. In this work, we focused on the R-BPMV gene, a major resistance gene against Bean pod mottle virus in Phaseolus vulgaris. We inoculated different BPMV constructs in order to study the behavior of the R-BPMV-mediated resistance at normal (20 °C) and elevated temperatures (constant 25, 30, and 35 °C). Our results show that R-BPMV mediates a temperature-dependent phenotype of resistance from hypersensitive reaction at 20 °C to chlorotic lesions at 35 °C in the resistant genotype BAT93. BPMV is detected in inoculated leaves but not in systemic ones, suggesting that the resistance remains heat-stable up to 35 °C. R-BPMV segregates as an incompletely dominant gene in an F2 population. We also investigated the impact of elevated temperature on BPMV infection in susceptible genotypes, and our results reveal that elevated temperatures boost BPMV infection both locally and systemically in susceptible genotypes.

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.

Weed-infecting viruses in a tropical agroecosystem present different threats to crops and evolutionary histories.

In the Caribbean Basin, malvaceous weeds commonly show striking golden/yellow mosaic symptoms. Leaf samples from Malachra sp. and Abutilon sp. plants with these symptoms were collected in Hispaniola from 2014 to 2020. PCR tests with degenerate primers revealed that all samples were infected with a bipartite begomovirus, and sequence analyses showed that Malachra sp. plants were infected with tobacco leaf curl Cuba virus (TbLCuCV), whereas the Abutilon sp. plants were infected with a new bipartite begomovirus, tentatively named Abutilon golden yellow mosaic virus (AbGYMV). Phylogenetic analyses showed that TbLCuCV and AbGYMV are distinct but closely related species, which are most closely related to bipartite begomoviruses infecting weeds in the Caribbean Basin. Infectious cloned DNA-A and DNA-B components were used to fulfilled Koch's postulates for these diseases of Malachra sp. and Abutilon sp. In host range studies, TbLCuCV also induced severe symptoms in Nicotiana benthamiana, tobacco and common bean plants; whereas AbGYMV induced few or no symptoms in plants of these species. Pseudorecombinants generated with the infectious clones of these viruses were highly infectious and induced severe symptoms in N. benthamiana and Malachra sp., and both viruses coinfected Malachra sp., and possibly facilitating virus evolution via recombination and pseudorecombination. Together, our results suggest that TbLCuCV primarily infects Malachra sp. in the Caribbean Basin, and occasionally spills over to infect and cause disease in crops; whereas AbGYMV is well-adapted to an Abutilon sp. in the Dominican Republic and has not been reported infecting crops.

Biological and molecular characterization of bean bushy stunt virus, a novel bipartite begomovirus infecting common bean in northwestern Argentina.

Common bean plants (Phaseolus vulgaris L.) showing different virus-like symptoms were collected in northwestern Argentina. Dot-blot hybridization tests showed that the begomoviruses bean golden mosaic virus and tomato yellow vein streak virus were the most prevalent, but they also revealed the presence of unknown begomoviruses. The complete genome sequence of one of these unknown begomoviruses was determined. Sequence analysis showed that the virus is a typical New World begomovirus, for which the name "bean bushy stunt virus" (BBSV) is proposed. Biological assays based on biolistic inoculations showed that BBSV induced leaf roll and stunting symptoms similar to those observed in the field-collected common bean sample.

Antiviral Activity of PD-L1 and PD-L4, Type 1 Ribosome Inactivating Proteins from Leaves of Phytolacca dioica L. in the Pathosystem Phaseolus vulgaris-Tobacco Necrosis Virus (TNV).

Using the pathosystem Phaseolus vulgaris-tobacco necrosis virus (TNV), we demonstrated that PD-L1 and PD-L4, type-1 ribosome inactivating proteins (RIPs) from leaves of Phytolacca dioica L., possess a strong antiviral activity. This activity was exerted both when the RIPs and the virus were inoculated together in the same leaf and when they were inoculated or applied separately in the adaxial and abaxial leaf surfaces. This suggests that virus inhibition would mainly occur inside plant cells at the onset of infection. Histochemical studies showed that both PD-L1 and PD-L4 were not able to induce oxidative burst and cell death in treated leaves, which were instead elicited by inoculation of the virus alone. Furthermore, when RIPs and TNV were inoculated together, no sign of H2O2 deposits and cell death were detectable, indicating that the virus could have been inactivated in a very early stage of infection, before the elicitation of a hypersensitivity reaction. In conclusion, the strong antiviral activity is likely exerted inside host cells as soon the virus disassembles to start translation of the viral genome. This activity is likely directed towards both viral and ribosomal RNA, explaining the almost complete abolition of infection when virus and RIP enter together into the cells.

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.

Complete genome sequence of a novel comovirus infecting common bean.

The complete genome sequence of a novel comovirus identified in Guanajuato, Mexico, in a common bean plant (Phaseolus vulgaris L.) coinfected with Phaseolus vulgaris alphaendornavirus 1 (PvEV-1) and Phaseolus vulgaris alphaendornavirus 2 (PvEV-2) is presented. According to the current ICTV taxonomic criteria, this comovirus corresponds to a new species, and the name "Phaseolus vulgaris severe mosaic virus" (PvSMV) is proposed for this virus based on the observed symptoms of "severe mosaic" syndrome caused by comoviruses in common bean. PvSMV is closely related to bean pod mosaic virus (BPMV), and its genome consists of two polyadenylated RNAs. RNA-1 (GenBank accession number MN837498) is 5969 nucleotides (nt) long and encodes a single polyprotein of 1856 amino acids (aa), with an estimated molecular weight (MW) of 210 kDa, that contains putative proteins responsible for viral replication and proteolytic processing. RNA-2 (GenBank accession number MN837499) is 3762 nt long and encodes a single polyprotein of 1024 aa, with an estimated MW of 114 kDa, that contains putative movement and coat proteins. Cleavage sites were predicted based on similarities in size and homology to aa sequences of other comoviruses available in the GenBank database. Symptoms associated with PvSMV include mosaic, local necrotic lesions, and apical necrosis. This is the first report of a comovirus infecting common bean in Mexico.

Epidemiology and control of emerging criniviruses in bean.

During the last two decades, new criniviruses emerged in green bean crops in the south-east of Spain. Bean yellow disorder virus (BnYDV) was first detected in 2003 and caused major economic damage in crops grown in greenhouses. It was characterized as the first crinivirus to infect a member species of the Leguminosae family. Symptoms induced during BnYDV infection include interveinal chlorosis and yellowing on leaves, and reduced fruit yield and quality. Similar symptoms, although more severe, were observed in bean crops in the same region during the fall of 2011. From that moment on, BnYDV was not detected anymore in diseased plants, but instead lettuce chlorosis virus (LCV) was associated with the diseased plants. Previously, LCV was detected only in California, USA, infecting lettuce and sugarbeets. The host range and partial genomic sequences lead to the description of the new strain, LCV-SP. The complete sequence of its genome revealed the virus as a recombinant of BnYDV and LCV, in which the latter had lost two ORFs in the RNA1 of the bipartite genome and had acquired two homologue ORFs from BnYDV. Both viruses are transmitted by the whitefly Bemisia tabaci. When compared with other crinivirus pathosystems, the transmission efficiency of BnYDV to its primary host bean, is among the highest, and its persistence in the vector among the longest, up to 9 days. The host range of BnYDV s restricted to several crop species of the Leguminosae: common bean (Phaseolus vulgaris), pea (Pisum sativum), tirabeque (P. sativum subsp. sativum var. macrocarpon), lentil (Lens culinaris) and faba bean (Vicia faba). LCV-SP is also able to infect green bean plants but not lettuce, its original host, probably following its recombinant nature. Symptoms and epidemiology of the bean criniviruses are compared with similar pathosystems that occur in the same region and that involve cucurbit yellow stunting disorder virus and tomato chlorosis virus, infecting cucurbitaceous and solanaceous crops, respectively. Control of the criniviruses in bean crops will depend on efficient control of the vector. Physical control with greenhouses that prevent viruliferous whiteflies from gaining access to crops reduces BnYDV infection in plants and loss of production. Integrated pest management in beans would be preferred and the use of natural enemies to reduce secondary spread within greenhouses must be investigated.

Seed transmission of a distinct soybean yellow mottle mosaic virus strain identified from India in natural and experimental hosts.

Soybean yellow mottle mosaic virus (SYMMV) is a newly identified member of the genus Gammacarmovirus from grain legumes in India. As the modes of transmission of this virus have not been described, we assessed the possibility of SYMMV to be transmitted through seed collected from field infected mungbean plants and mechanically sap inoculated French bean plants using serological and molecular techniques followed by progeny assays. Direct antigen coated enzyme linked immunosorbent assay (DAC-ELISA) and reverse transcription polymerase chain reaction (RT-PCR) results are inconsistent with field infected mungbean seed tissues to ensure seed transmissibility irrespective of seed number used. Seed from mechanical sap inoculated French bean showed higher absorbance values in DAC-ELISA and amplification corresponding to replicase, movement and coat protein regions of SYMMV genome. The relative accumulation of SYMMV was higher in pod walls, immature seed and stamens and stigma of mechanical sap inoculated French bean. Progeny assays with infected seed revealed the seed transmissibility of SYMMV at the rate of 63.33% in mungbeanand 73.33% in French bean. Mechanical sap inoculation of mungbean progeny seedlings on French bean cv. Pusa Parvati produced characteristic symptoms of SYMMV. The results obtained from this study demonstrate that SYMMV is seed borne in nature and can be transmitted to next generation seedlings. This is the first report of seed transmission of SYMMV in mungbean and French bean.

Molecular characterization of common bean curly stunt virus: a novel recombinant geminivirus in China.

A new geminivirus was identified in common bean (Phaseolus vulgaris) showing severe stunt and leaf curling symptoms in Heilongjiang province of China, via sequencing and assembly of small RNAs. The genome of this geminivirus comprises 2,959 nucleotides (nt) and shares 21.77-54.97% nt sequence identity with other geminiviruses. The coat protein (CP) shares the highest amino acid (aa) sequence identity (23.5%) with that of sesame curly top virus (SeYMV; genus Turncurtovirus), whereas the C1 (Rep) shares the highest aa sequence identity (66.5%) with that of beet severe curly top virus (BSCTV; genus Curtovirus). This geminivirus neighbors the turncurtoviruses in phylogenetic trees based on the full genome sequence or the amino acid sequence of the Rep protein, but it forms a distinct clade in the phylogenetic tree based on the coat protein. Recombination analysis showed that parts of the C1 coding region of this geminivirus were recombined from a curtovirus or turncurtovirus. Based on these results, the name "common bean curly stunt virus" (CBCSV) is proposed for this virus.

A New Strain of Bean Common Mosaic Virus From Lima Bean (Phaseolus lunatus): Biological and Molecular Characterization.

Lima bean (Phaseolus lunatus) is a popular cultivated legume vegetable grown in the United States for dry bean or canned bean production. In 2017, two symptomatic P. lunatus plants exhibiting mosaic, vein banding, and growth retardation were collected in a public garden in Honolulu, HI. Both samples contained bean common mosaic virus (BCMV), and the two BCMV isolates were subjected to biological characterization on a panel of 11 differential cultivars of common bean (P. vulgaris), and to molecular characterization through whole genome sequencing. Both samples contained nearly identical BCMV sequences, named BCMV-A1, which, in turn, were 93% identical to the peanut stripe virus strain of BCMV. BCMV-A1 induced an unusually severe systemic necrosis in cultivar 'Dubbele Witte', and pronounced necrotic or chlorotic reaction in inoculated leaves of five other bean differentials. BCMV-A1 was able to partially overcome resistance alleles bc-1 and bc-2 expressed singly in common bean, inducing no systemic symptoms. Phylogenetic analysis of the BCMV-A1 sequence, and distinct biological reactions in common bean differentials suggested that BCMV-A1 represented a new lima bean strain of BCMV. In 2017, two BCMV isolates were collected in Idaho from common bean, and based on partial genome sequences were found 99% identical to the BCMV-A1 sequence. The data suggest that the lima bean strain of BCMV may have a wider circulation, including common bean as a host. This new strain of BCMV may thus pose a significant threat to common bean production.

Double-Stranded RNA High-Throughput Sequencing Reveals a New Cytorhabdovirus in a Bean Golden Mosaic Virus-Resistant Common Bean Transgenic Line.

Using double-strand RNA (dsRNA) high-throughput sequencing, we identified five RNA viruses in a bean golden mosaic virus (BGMV)-resistant common bean transgenic line with symptoms of viral infection. Four of the identified viruses had already been described as infecting common bean (cowpea mild mottle virus, bean rugose mosaic virus, Phaseolus vulgaris alphaendornavirus 1, and Phaseolus vulgaris alphaendornavirus 2) and one is a putative new plant rhabdovirus (genus Cytorhabdovirus), tentatively named bean-associated cytorhabdovirus (BaCV). The BaCV genome presented all five open reading frames (ORFs) found in most rhabdoviruses: nucleoprotein (N) (ORF1) (451 amino acids, aa), phosphoprotein (P) (ORF2) (445 aa), matrix (M) (ORF4) (287 aa), glycoprotein (G) (ORF5) (520 aa), and an RNA-dependent RNA polymerase (L) (ORF6) (114 aa), as well as a putative movement protein (P3) (ORF3) (189 aa) and the hypothetical small protein P4. The predicted BaCV proteins were compared to homologous proteins from the closest cytorhabdoviruses, and a low level of sequence identity (15⁻39%) was observed. The phylogenetic analysis shows that BaCV clustered with yerba mate chlorosis-associated virus (YmCaV) and rice stripe mosaic virus (RSMV). Overall, our results provide strong evidence that BaCV is indeed a new virus species in the genus Cytorhabdovirus (family Rhabdoviridae), the first rhabdovirus to be identified infecting common bean.

Development of Soybean Yellow Mottle Mosaic Virus-Based Expression Vector for Heterologous Protein Expression in French Bean.

Plant virus-based vectors provide attractive and valuable tools for rapid production of recombinant protein in large quantities as they produce systemic infections in differentiated plant tissues. In the present study, we engineered the Soybean yellow mottle mosaic virus (SYMMV) as a gene expression vector which is a promising candidate for systemic expression of foreign proteins in French bean plants. Full virus vector strategy was exploited for insertion of foreign gene by inserting MCS through PCR in the circular pJET-SYMMV clone. To examine the ability of the SYMMV vector system, GFP gene was cloned after the start codon of coat protein (CP) so that its expression was driven by the SYMMV-CP subgenomic promoter. When in vitro run off SYMMV-GFP transcript was mechanically inoculated to French bean leaves, good level of GFP expression was observed through confocal microscopy up to 40 dpi. Expression of heterologous protein was also confirmed through ISEM, DAC-ELISA and RT-PCR with specific primers at 20 dpi. The recombinant SYMMV construct was stable in in vitro runoff transcript inoculated plants but the inserted GFP was lost in progeny virion inoculated plants. The system developed here will be useful for further studies of SYMMV gene functions and exploitation of SYMMV as a gene expression vector.

Identification and characterization of differentially expressed Phaseolus vulgaris miRNAs and their targets during mungbean yellow mosaic India virus infection reveals new insight into Phaseolus-MYMIV interaction.

Phaseolus vulgaris is an economically important legume in tropical and subtropical regions of Asia, Africa, Latin-America and parts of USA and Europe. However, its production gets severely affected by mungbean yellow mosaic India virus (MYMIV). We aim to identify and characterize differentially expressed miRNAs during MYMIV-infection in P. vulgaris. A total of 422 miRNAs are identified of which 292 are expressed in both MYMIV-treated and mock-treated samples, 109 are expressed only in MYMIV-treated and 21 are expressed only in mock-treated samples. Selected up- and down-regulated miRNAs are validated by RT-qPCR. 3367 target ORFs are identified for 270 miRNAs. Selected targets are validated by 5' RLM-RACE. Differentially expressed miRNAs regulate transcription factors and are involved in improving stress tolerance to MYMIV. These findings will provide an insight into the role of miRNAs during MYMIV infection in P. vulgaris in particular and during any biotic stress conditions in Leguminosae family in general.

New approach for the construction of infectious clones of a circular DNA plant virus using Gibson Assembly.

Viruses belonging to the genus Begomovirus (family Geminiviridae) have circular single-strand DNA genomes encapsidated into quasi-icosahedral particles, and are transmitted by whiteflies of the Bemisia tabaci complex. Biological and molecular properties of begomoviruses have been studied efficiently with infectious clones containing dimeric genomic components. However, current approaches employing enzymatic digestion and ligation to binary vectors are laborious, mostly due to many cloning steps or partial digestion by restriction enzyme. Here, an infectious clone of the bipartite begomovirus Bean golden mosaic virus (BGMV) was obtained using PCR and Gibson Assembly (GA). Common bean (Phaseolus vulgaris) seedlings displayed severe yellow mosaic and stunt symptoms 15 days after agroinoculation with DNA-A and DNA-B of BGMV. The approach based on PCR-GA protocol is a fast and useful tool to obtain infectious clones of a circular DNA plant virus.

First natural crossover recombination between two distinct species of the family Closteroviridae leads to the emergence of a new disease.

Lettuce chlorosis virus-SP (LCV-SP) (family Closteroviridae, genus Crinivirus), is a new strain of LCV which is able to infect green bean plants but not lettuce. In the present study, high-throughput and Sanger sequencing of RNA was used to obtain the LCV-SP full-length sequence. The LCV-SP genome comprises 8825 nt and 8672 nt long RNA1 and RNA2 respectively. RNA1 of LCV-SP contains four ORFs, the proteins encoded by the ORF1a and ORF1b are closely related to LCV RNA1 from California (FJ380118) whereas the 3´ end encodes proteins which share high amino acid sequence identity with RNA1 of Bean yellow disorder virus (BnYDV; EU191904). The genomic sequence of RNA2 consists of 8 ORFs, instead of 10 ORFs contained in LCV-California isolate. The distribution of vsiRNA (virus-derived small interfering RNA) along the LCV-SP genome suggested the presence of subgenomic RNAs corresponding with HSP70, P6.4 and P60. Results of the analysis using RDP4 and Simplot programs are the proof of the evidence that LCV-SP is the first recombinant of the family Closteroviridae by crossover recombination of intact ORFs, being the LCV RNA1 (FJ380118) and BnYDV RNA1 (EU191904) the origin of the new LCV strain. Genetic diversity values of virus isolates in the recombinant region obtained after sampling LCV-SP infected green bean between 2011 and 2017 might suggest that the recombinant virus event occurred in the area before this period. The presence of LCV-SP shows the role of recombination as a driving force of evolution within the genus Crinivirus, a globally distributed, emergent genus.