Alfalfa Mosaic Virus Research Articles

Exploring the hemp virome and assessing hemp germplasm for resistance to an emerging pathogen

Hemp (Cannabis sativa L., <0.3% tetrahydrocannabidiol, THC) as a crop has been widely adopted throughout the United States. However, there is little support or guidance on disease identification and management for growers. This study aimed to describe the diversity and distribution of viruses infecting hemp in Colorado and determine if there is genetic resistance to pathogens in hemp. Throughout 2021 and 2022, leaf tissues from symptomatic and asymptomatic plants were sampled during three phenological stages (early vegetative, late vegetative, and mature flowering) in four regions of Colorado. High Throughput Sequencing (HTS) revealed a total of seven viruses across both years from 48 pooled samples: alfalfa mosaic virus, beet curly top virus (BCTV), cannabis cryptic virus, cannabis sativa mitovirus 1, grapevine line pattern virus, opuntia umbra-like virus, and tomato bushy stunt virus. These viruses had >97% nucleotide identity to their nearest GenBank accessions. As the growing season progressed, viral incidence and the number of viral species increased. We observed similar and unique viromes between individual cultivars from the same field. Given the prevalence of BCTV in the virome and its prevalence across the western United States, 13 genotypes of hemp were screened for resistance to this pathogen. Two genotypes (4587 and 4710) had a lower BCTV copy number and disease index than others. Our study provides initial evidence of the diversity of viral communities in hemp across Colorado and provides impetus for developing accurate detection methods and screening for host resistance.

Alfalfa Mosaic Virus and White Clover Mosaic Virus Combined Infection Leads to Chloroplast Destruction and Alterations in Photosynthetic Characteristics of Nicotiana benthamiana.

Alfalfa mosaic virus (AMV) is one of the most widely distributed viruses; it often exhibits combined infection with white clover mosaic virus (WCMV). Even so, little is known about the effects of co-infection with AMV and WCMV on plants. To determine whether there is a synergistic effect of AMV and WCMV co-infection, virus co-infection was studied by electron microscopy, the double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA), and real-time fluorescence quantitative PCR (RT-qPCR) of AMV and WCMV co-infection in Nicotiana benthamiana. Meanwhile, measurements were carried out on the photosynthetic pigments, photosynthetic gas exchange parameters, and chlorophyll fluorescence parameters. The results showed that the most severe disease development was induced by AMV and WCMV co-infection, and the disease grade was scale 7. N. benthamiana leaves induced mottled yellow-green alternating patterns, leaf wrinkling, and chlorosis, and chloroplasts were observed to be on the verge of disintegration. The relative accumulation of AMV CP and WCMV CP was significantly increased by 15.44-fold and 10.04-fold upon co-infection compared to that with AMV and WCMV single infection at 21 dpi. In addition, chlorophyll a, chlorophyll b, total chlorophyll, the net photosynthetic rate, the water use efficiency, the apparent electron transport rate, the PSII maximum photochemical efficiency, the actual photochemical quantum yield, and photochemical quenching were significantly reduced in leaves co-infected with AMV and WCMV compared to AMV- or WCMV-infected leaves and CK. On the contrary, the carotenoid content, transpiration rate, stomatal conductance, intercellular CO2 concentration, minimal fluorescence value, and non-photochemical quenching were significantly increased. These findings suggest that there was a synergistic effect between AMV and WCMV, and AMV and WCMV co-infection severely impacted the normal function of photosynthesis in N. benthamiana.

Investigation of Tissue-Specific Distribution and Genetic Variation of Alfalfa Mosaic Virus and Chinese Artichoke Mosaic Virus in Chinese Artichoke (Stachys affinis miq.).

The Chinese artichoke (Stachys affinis syn. S. sieboldii) is a widely cultivated crop, and its rhizome is used as a medicinal vegetable. To investigate the causes of viral diseases in Chinese artichokes, the infection rates of four virus species infecting Chinese artichoke were investigated. Since the Chinese artichoke propagates through its tuber, this study aimed to determine whether viral transmission to the progeny is possible through the tuber, by identifying the virus present in the tuber and investigating its accumulation. First, reverse transcription polymerase chain reaction analysis was performed to detect viruses using total RNA extracted from the flowers, leaves, and tubers of Chinese artichoke plants. Alfalfa mosaic virus (AMV) and Chinese artichoke mosaic virus (ChAMV) had high infectivity in Chinese artichoke and most plants were simultaneously infected with AMV and ChAMV. These viruses were present in all tissues, but their detection frequency and accumulation rates varied across different tissues of the Chinese artichoke. Also, we sequenced the coat protein (CP) genes of AMV and ChAMV to investigate genetic variations of virus between the leaf and tuber. It provides information on CP gene sequences and genetic diversity of isolates identified from new hosts of AMV and ChAMV. This study offers valuable insights into the distribution and spread of the ChAMV and AMV within Chinese artichoke plants, which have implications for the management and control of viral infections in crops.

Crop age is the main driver affecting alfalfa mosaic virus: The predominant virus in the alfalfa virome

AbstractThe alfalfa virome has been understudied. Existing research dealing with viral incidences within the crop primarily concentrate on local factors, rather than considering a broader perspective. In this comprehensive 2‐year study, we define the alfalfa virome and the main local and landscape factors affecting the incidence and annual increase of the Alfalfa mosaic virus (AMV), the most prevalent virus in alfalfa. The study was conducted in commercial alfalfa fields located along the highly productive northeast region of the Iberian Peninsula. For the first time in Europe, next‐generation sequencing revealed the presence of 14 different viruses representing the genera Cytorhabdovirus, Alphapartitivirus, Amalgavirus, Alfamovirus, Luteovirus, Enamovirus and Flavivirus. AMV was the most prevalent species, accounting for 89% of the identified viral contigs. Enzyme‐linked immunosorbent assays showed that the incidence of AMV varied between fields, with the average incidence doubling from 34% in 2019 to 65% in 2020. To assess the effect of local and landscape characteristics on the incidence of AMV and the observed annual increase, we selected different local variables and recorded landscape structure at three different buffer radii (250, 500 and 1000 m) from the centre of each field. Both the incidence and annual increase in AMV were driven mainly by local characteristics. The incidence of AMV showed a significant relationship with crop age and field area, whereas the annual increase was mainly influenced by crop age and the cumulative number of alate morphs of the aphid Acyrthosiphon pisum. Only one landscape composition variable, the percentage of alfalfa, showed a significant relationship with AMV incidence at the 250‐m scale. These results confirm the effect of local variables on the population structure of generalist viruses.

Transmission from seed to seedling and elimination of alfalfa viruses.

Viral diseases have become a vital factor limiting the development of the alfalfa (Medicago sativa) industry. Six viruses infecting alfalfa with a high incidence rate are Alfalfa mosaic virus (AMV), Medicago sativa alphapartitivirus 1 (MsAPV1), Medicago sativa alphapartitivirus 2 (MsAPV2), Medicago sativa deltapartitivirus 1 (MsDPV1), Medicago sativa amalgavirus 1 (MsAV1), and Cnidium vein yellowing virus 1 (CnVYV1). The purpose of this study was to develop preventive measures against these viruses by investigating their transmission through alfalfa seeds. In this study, we investigated the transmission rate of alfalfa viruses from seed to seedling by PCR, determined the location of viruses in seed by dissecting seed embryos and seed coat, tracked the changes of viruses in seedlings, and finally discover effective elimination measures for alfalfa viruses from 16 measures. Our results demonstrated that all these six viruses could be transmitted from alfalfa seeds to seedlings with the transmission rate ranging from 44.44% to 88.89%. For AMV, MsAPV2, and MsAV1, the viral load was significantly higher in the seed coats than in the seed embryos; however, it did not show significant differences between these two parts of the seeds for MsAPV1, MsDPV1, and CnVYV1. Dynamic accumulation analysis of AMV and MsAPV2 indicated that the viral load in plants increased continuously in the early growth stage, making it important to inactivate these viruses prior to their seed-to-seedling transmission. Sixteen treatments including physical, chemical, and combinations of physical and chemical measures were compared in terms of their elimination efficiency on AMV and MsAPV2 and impacts on seed germination. The results showed that soaking alfalfa seeds in sterile distilled water for 2h + 2% NaClO for 1h or 2% NaClO for 1h were more promisingly applicable because it could significantly reduce AMV and MsAPV2 particles in both seeds and seedlings. Our data revealed a route of virus transmission in alfalfa and shed light on the discovery of a highly efficient method for the management of alfalfa viral diseases.

Lavender Harbors More Viruses than Previously Thought: First Report of Raspberry Ringspot Virus and Phlox Virus M in Lavandula × intermedia.

Plants of the genus Lavandula are thought to be rarely infected by viruses. To date, only alfalfa mosaic virus, cucumber mosaic virus, tobacco mosaic virus, and tomato spotted wilt virus have been reported in this host. In this study, we identified for the first time raspberry ringspot virus (RpRSV) and phlox virus M (PhlVM) in lavender using herbaceous indexing, enzyme-linked immunosorbent assay, and high-throughput sequencing. Nearly complete genome sequences for both viruses were determined. Phylogenetic and serological characterizations suggest that the obtained RpRSV isolate is a raspberry strain. A preliminary survey of 166 samples indicated RpRSV was spread only in the lavender cultivar 'Grosso', while PhlVM was detected in multiple lavender cultivars. Although RpRSV raspberry strain may have spread throughout Auckland and nearby areas in New Zealand, it is very likely restricted to the genus Lavandula or even to the cultivar 'Grosso' due to the absence or limited occurrence of the nematode vector. Interestingly, all infected lavender plants, regardless of their infection status (by RpRSV, PhlVM, or both) were asymptomatic. RpRSV is an important virus that infects horticultural crops including grapevine, cherry, berry fruits, and rose. It remains on the list of regulated pests in New Zealand. RpRSV testing is mandatory for imported Fragaria, Prunus, Ribes, Rosa, Rubus, and Vitis nursery stock and seeds for sowing, while this is not required for Lavandula importation. Our study revealed that lavender could play a role not only as a reservoir but also as an uncontrolled import pathway of viruses that pose a threat to New Zealand's primary industries.

Coat protein of alfalfa mosaic alfamovirus (AMV) from Türkiye: genetic inference and in silico docking analysis for potential antiphytoviral purposes

In 2021, a study was conducted in the Denizli region of Türkiye to investigate the phylogenetic relationship and presence of alfalfa mosaic alfamovirus (AMV) infecting pepper plants exhibiting viral disease symptoms. A total of 57 samples were collected, of which twenty-four tested positive for AMV with reverse transcriptase polymerase chain reaction (RT-PCR) assays. Samples from pepper plants displaying virus symptoms gave positive bands on the agarose gel, while healthy plants yielded negative results. One of the positive samples was randomly selected, cloned and sequenced. This sequence of the Denizli AMV isolate (753 bp) was recorded in the GenBank database with accession number OQ845956. The nucleotide sequence showed a high nucleotide consensus of 97%-99% compared with the nucleotide sequences of the same variants from different origins in GenBank. According to the phylogenetic tree generated through the Neighbour Joining (NJ) method, this AMV isolate belongs to the same group as Iranian isolates from various of hosts. Furthermore, in silico docking analysis of the coat protein (CP) of the AMV isolate and promising 12 essential oil compounds was performed to enable potential antiviral drug development. Docking study showed that eucalyptol, eugenol and carvacrol can make important contributions to the advancement of drug-based strategies for the managing of plant viruses by interacting with the virus coat protein of high binding energies, -5.3, -5.2 and -5.0 kcal mol-1, respectively. Although the presence of AMV in Denizli province has been reported previously, this study reports the phylogenetic relationships and docking analysis of the new AMV isolate in pepper crops.

Turkish isolates of alfalfa mosaic virus belong to a distinct lineage among global population

Complete RNA3 sequences of 15 alfalfa mosaic virus (AMV) isolates sampled from Afyonkarahisar, Ankara, Konya, Tokat, Kayseri, and Nevşehir Provinces of Türkiye were obtained using RT-PCR with two primer pairs designed in this study. Molecular and population analyses were performed on them together with 63 global isolates which full genome sequences available in NCBI GenBank. Phylogenetic analysis based on movement protein (MP) and coat protein (CP) regions showed unrelated isolates clustering between the constructed trees, and the new Turkish isolates were dispersed in different phylogroups. Particularly, three new Turkish potato isolates: B6, K1, and K2 occupied a divergent MP lineage, together with one alfalfa isolate from China: ZM1 NX3. S, η, and k parameters of genetic diversity confirmed that CP were more conserved than MP sequences. The 15 new Turkish isolates shared 95.4–100% nucleotide and 94–100% amino acid, and 92.8–100% nucleotide and 93.3–100% amino acid identities among themselves at MP and CP regions, respectively. All tested populations were found to be under very strict purifying constraints (dN/dS ratios of 0.061–0.219), and had undergone recent expansion, probably due to new mutations in their genome, according to diversity and neutrality tests. Fixation index (FST) values obtained by pairwise comparisons of phylogroups were all above 0.33, suggested that the isolates clustering has been done convincingly.

Evaluation of in situ and ex situ forage germplasm collections reveals the first occurrence and seed-transmission of alfalfa mosaic virus and southern bean mosaic virus in Urochloa species

Grasses of the genus Urochloa (Urochloa species syn. Brachiaria species) are among the most important tropical forage grasses of African origin. The International Livestock Research Institute (ILRI) Forage Genebank maintains 671 Urochloa grass accessions, belonging to 28 species, of which 261 (39%) accessions are Urochloa brizantha (A.Rich.) R.D.Webster. The susceptibility of Urochloa grasses to disease and insect pests is a key challenge to the sustainable production of these grasses in Africa. In this study, using a dot blot assay and RT-PCR, we report for the first time the association of Alfalfa Mosaic Virus (AMV) and Southern Bean Mosaic Virus (SBMV) with 92% of the tested accessions conserved in situ and ex situ in the ILRI forage genebank. In addition, virus transmission from seed to plant and from legume to grass was confirmed through the bioassay test. The detection of these two viruses in Urochloa species presents a new challenge to germplasm conservation, distribution, and their sustainable production in the region, and demands immediate attention towards developing an effective method of cleaning viruses from the conserved germplasm. Furthermore, there is an urgent need to assess the economic importance of diseases in the grasses, and for the selection of germplasm that shows resistance to diseases.

Australian Cool-Season Pulse Seed-Borne Virus Research: 1. Alfalfa and Cucumber Mosaic Viruses and Less Important Viruses.

Here, we review the research undertaken since the 1950s in Australia's grain cropping regions on seed-borne virus diseases of cool-season pulses caused by alfalfa mosaic virus (AMV) and cucumber mosaic virus (CMV). We present brief background information about the continent's pulse industry, virus epidemiology, management principles and future threats to virus disease management. We then take a historical approach towards all past investigations with these two seed-borne pulse viruses in the principal cool-season pulse crops grown: chickpea, faba bean, field pea, lentil, narrow-leafed lupin and white lupin. With each pathosystem, the main focus is on its biology, epidemiology and management, placing particular emphasis on describing field and glasshouse experimentation that enabled the development of effective phytosanitary, cultural and host resistance control strategies. Past Australian cool-season pulse investigations with AMV and CMV in the less commonly grown species (vetches, narbon bean, fenugreek, yellow and pearl lupin, grass pea and other Lathyrus species) and those with the five less important seed-borne pulse viruses also present (broad bean stain virus, broad bean true mosaic virus, broad bean wilt virus, cowpea mild mottle virus and peanut mottle virus) are also summarized. The need for future research is emphasized, and recommendations are made regarding what is required.

TRANSMISSION OF ALFALFA MOSAIC VIRUS THROUGH SUBCOCCINELLA VIGINTIQUATUORPUNCTATA (L.) (COLEOPTERA: COCCINELLIDAE) IN ALFALFA GROWING AREAS IN TÜRKİYE

Alfalfa Medicago sativa L. (Fabaceae: Leguminosae) is an important fodder crop due to its highly nutritious animal feed and potential to adapt various environmental conditions. The average alfalfa yield in Türkiye is considerably higher than the world's average. Several diseases and pests exert negative impacts on alfalfa yield. This study investigated the status of the alfalfa mosaic virus (AMV) and the presence of insect species that could be new vectors for AMV in the alfalfa growing areas of Zonguldak and Bartın provinces in Türkiye. AMV is non-persistently transmitted by aphid species, but it is also known to be transmitted by pollen and seeds. The most prevalent harmful insect species in the studied area were Subcoccinella vigintiquatuorpunctata (L.) (Coleoptera: Coccinellidae), and Gonioctena fornicata (Brüggeman) (Coleoptera: Chrysomelidae). The AMV was prevalent in the tested plants at a rate of 85.00%, with infection rates of 80.55% in mature larvae of S. vigintiquatuorpunctate, 80.00% in pupae, and 96.77% in adults. When looking at the relationship of AMV with common pest species, AMV was not detected in all life stages of G. fornicata. In contrast, AMV was detected in all life stages of S. vigintiquatuorpunctata except eggs. This study is the first in the world to detect the presence of AMV in S. vigintiquatuorpunctata. However, further studies are needed to determine whether S. vigintiquatuorpunctata transmits AMV persistently, semi-persistently, or non-persistently. Keywords: Forage crops, 24-spot ladybird, Molecular detection, Insect Vector, Prevalence

Exploring virus presence in field-collected potato leaf samples using RNA sequencing

BackgroundThe quick and accurate identification of viruses is essential for plant disease management. Next-generation sequencing (NGS) technology may allow the discovery, detection, and identification of plant pathogens. This study adopted RNA-sequencing (RNA-Seq) technology to explore the viruses in three potato plants (S3, S4, and S6) growing under field conditions. ResultsPotato-known infecting viruses, such as alfalfa mosaic virus (AMV), potato leafroll virus (PLRV), and potato virus Y (PVY), were identified using bioinformatics programs and validated using RT-PCR. The presence of these potato viruses was also confirmed by visual inspection of host symptoms. In addition, the nearly complete genome of PLRV and the complete or partial genome sequence of multipartite virus segments have been identified. Besides the three major potato viruses that BLASTn analysis revealed were present in our samples, BLASTx analysis revealed some reads are derived from other potato viruses, such as potato virus V (PVV), Andean potato latent virus (APLV), and tomato chlorosis virus (ToCV), which are not frequently reported in potato field screenings in Egypt. Other microbial agents, such as bacteria and fungi, were also identified in the examined sample sequences. Some mycovirus sequences derived from ourmia-like viruses and Alternaria alternata chrysovirus were also identified in sample S4, confirming the complexity of the potato microbiome under field conditions. ConclusionNGS quickly and accurately identifies potato plant viruses under field conditions. Implementing this technology on a larger scale is recommended to explore potato fields and imported plants, where symptoms may be absent, unspecific, or only triggered under certain conditions.

Peppers under Siege: Revealing the Prevalence of Viruses and Discovery of a Novel Potyvirus Species in Venezuela

Many plant virus outbreaks have been recorded in the last two decades, threatening food security around the world. During pepper production seasons in 2008, 2014, and 2022, virus outbreaks were reported from Lara (western) and Miranda (central) states in Venezuela. Three hundred seventy-three plants exhibiting virus-like symptoms were collected and tested for virus infection through reverse transcription PCR (RT-PCR). The most prevalent viruses during the 2008 surveys conducted in Lara were potato virus Y (PVY, 66.25%), cucumber mosaic virus (CMV, 57.50%), pepper mild mottle virus (PMMoV, 35%), alfalfa mosaic virus (AMV, 23.75%), and tobacco rattle virus (TRV, 17.50%). This survey revealed for the first time that pepper is a natural host of AMV and TRV in Venezuela. A further, divergent potyvirus isolate was also detected in 23.75% of pepper plants from Lara state. In 2014, a follow-up survey after virus outbreaks reported in Lara and Miranda states also detected this divergent potyvirus isolate in 21.68% of pepper plants, with tomato spotted wilt virus (TSWV) and PMMoV dominating the viral landscape (62.65 and 21.68% of tested plants, respectively). By comparison, the surveys revealed significant changes in viral community composition. The complete capsid protein (CP) sequence of the putative potyvirus was obtained from two pepper samples. According to the Potyvirus taxonomic criteria, these results suggest that the isolate represents a distinct virus species, for which we propose the name “pepper severe mottle virus” (PepSMoV). Virus outbreaks could be attributed to agricultural and environmental factors, such as climate change, the use of wastewater, the use of uncertified seeds, misuse of agricultural chemicals, transmission with food trade networks, and the development of new viral strains due to mutations and recombination and pathogen spillover. This study demonstrates the value of knowledge of the prevalence and distribution of viral species to recommend virus-resistant cultivars to replace susceptible ones, especially in virus hotspot areas.

The Identification of Viral Pathogens in a Physostegia virginiana Plant Using High-Throughput RNA Sequencing.

Physostegia virginiana is an important ornamental and cut-flower plant in China. Its commonly used method of clonal propagation leads to virus accumulation in this plant. However, which viruses can infect the Physostegia virginiana plant remains to be illuminated. In this work, five viral pathogens in a Physostegia virginiana plant with virus-like symptoms of yellow, shriveled, and curled leaves were identified using RNA-seq, bioinformatics, and molecular biological techniques. These techniques allowed us to identify five viruses comprising one known alfalfa mosaic virus (AMV) and four novel viruses. The novel viruses include a virus belonging to the genus Fabavirus, temporarily named Physostegia virginiana crinkle-associated virus 1 (PVCaV1); two viruses belonging to the genus Caulimovirus, temporarily named Physostegia virginiana caulimovirus 1 and 2 (PVCV1 and PVCV2); and a virus belonging to the genus Fijivirus, temporarily named Physostegia virginiana fijivirus (PVFV). The genome sequences of PVCaV1, PVCV1, and PVCV2, and the partial genome sequence of PVFV were identified. Genome organizations and genetic evolutionary relationships of all four novel viruses were analyzed. PVCaV1 has a relatively close evolutionary relationship with five analyzed fabiviruses. PVCV1 and PVCV2 have separately a closest evolutionary relationship with lamium leaf distortion-associated virus (LLDAV) and figwort mosaic virus (FMV), and PVFV has a close evolutionary relationship with the five analyzed fijiviruses. Additionally, PVCaV1 can infect Nicotiana benthamiana plants via friction inoculation. The findings enrich our understanding of Physostegia virginiana viruses and contribute to the prevention and control of Physostegia virginiana viral diseases.

Molecular characterization and evolution of the resident population of some alfalfa mosaic virus (AMV) isolates in Egypt

BackgroundAlfalfa mosaic virus (AMV) is an important virus affecting many vegetable crops in Egypt. In this study, virus isolates were collected from naturally infected potato, tomato, alfalfa and clover plants that showed suspected symptoms of AMV in different locations of Beheira and Alexandria governorates during the 2019–2020 growing season. The relative incidence of the virus ranged from 11–25% based on visual observations of symptoms and ELISA testing. A total of 41 samples were tested by ELISA using polyclonal antisera for AMV. Four AMV isolates collected from different host plants, named AM1 from potato, AM2 from tomato, AM3 from alfalfa and AM4 from alfalfa, were maintained on Nicotiana glutinosa plants for further characterization of AMV.ResultsElectron micrographs of the purified viral preparation showed spheroidal particles with a diameter of 18 nm and three bacilliform particles with lengths of roughly 55, 68, and 110 nm and diameters identical to those of the spheroidal particles. The CP gene sequence comparisons of four AMV isolates (AM1, AM2, AM3 and AM4) showed the highest nucleotide identity of 99.7% with the Gomchi isolate from South Korea infecting Gomchi (Ligularia fischeri) plants. Phylogenetic analysis showed that the present isolates were grouped together into a distinct separate clade (GPI) along with the Gomchi isolate from South Korea. Similarly, the deduced amino acid sequence comparisons of Egyptian AMV isolates revealed that amino acids Q29, S30, T34, V92 and V175 were conserved among the Egyptian isolates in GPI.ConclusionThe present study found strong evolutionary evidence for the genetic diversity of AMV isolates by the identification of potential recombination events involving parents from GPI and GPII lineages. Additionally, the study found that Egyptian AMV isolates are genetically stable with low nucleotide diversity. Genetic analysis of the AMV population suggested that the AMV populations differ geographically, and AMV CP gene is under mild purifying selection. Furthermore, the study proposed that the Egyptian AMV population had common evolutionary ancestors with the Asian AMV population. Antioxidant enzymes activity was assessed on N. glutinosa plants in response to infection with each AMV isolate studied, and the results revealed that the enzyme activity varied.

Mixed viral infection constrains the genome formula of multipartite cucumber mosaic virus

Many plant viruses have a multipartite organization, with multiple genome segments packaged into separate virus particles. The genome formula describes the relative frequencies of all viral genome segments, and previous work suggests rapid changes in these frequencies facilitate virus adaptation. Many studies have reported mixed viral infections in plants, often resulting in strong virus–virus interactions. Here, we tested whether mixed infections with tripartite alfalfa mosaic virus (AMV) and monopartite potato virus Y (PVY) affected the genome formula of the tripartite cucumber mosaic virus (CMV), our experimental model. We found that the CMV titer was reduced in mixed infections with its tripartite Bromoviridae relative AMV and in triple infections with both AMV and PVY, indicating notable virus–virus interactions. The variability of the CMV genome formula was significantly lower in mixed infections (CMV and AMV, CMV and PVY, and CMV and AMV and PVY) than in single infections (CMV only). These observations led to the surprising conclusion that mixed infections with two distinct viruses constrain the CMV genome formula. It remains unclear how common these effects are for different combinations of virus species and strains and what the underlying mechanisms are. We, therefore, extended a simulation model to consider three putative scenarios in which a second virus affected the genome formula. The simulation results also suggested that shifts in the genome formula occur, but may not be widespread due to the required conditions. One scenario modeled—co-infection exclusion through niche differentiation—was congruent with the experimental data, as this scenario led to reductions in genome formula variability and titer of the multipartite virus. Whereas previous studies highlighted host–species effects, our results indicate that the genome formula is also affected by mixed infections, suggesting that there is a broader set of environmental cues that affect the genome formula.

First report of Sw-5 resistance-breaking strain of tomato spotted wilt orthotospovirus infecting tomato in Texas.

Tomato spotted wilt orthotospovirus (TSWV) is one of the most devastating plant viruses causing crop disease epidemics of global economic significance. A single dominant resistant gene 'Sw-5' offering a broad-spectrum resistance to multiple orthotospoviruses was introduced in tomato cultivars. However, multiple resistance-breaking strains of TSWV were reported worldwide (Ciuffo 2005; Zaccardelli et al. 2008; Batuman et al. 2017; di Rienzo et al. 2018). Symptoms suggestive of orthotospoviral infection including stunting, bronzing, and inward rolling of leaves, and concentric necrotic spots on leaves, petioles, and fruits were observed in two TSWV-resistant tomato cultivars ('BL163' and 'HT 2') planted in a tomato variety trial in Bushland, TX in 2022. Leaf tissues from 45 resistant tomato plants (symptomatic or asymptomatic) from both resistant cultivars were tested using a TaqMan probe-based qPCR assay targeting a 200bp region in nucleoprotein (N) of the TSWV (Gautam et al. 2022). While 25 of those samples tested positive for TSWV, only ten expressed characteristic disease symptoms described above. The possibility of mixed infection in those samples with other endemic viruses in the region viz., alfalfa mosaic virus, groundnut ringspot orthotospovirus, tobacco mosaic virus, tomato chlorotic spot orthotospovirus, tomato mosaic virus, tomato necrotic streak virus, tomato ringspot virus, and tomato torrado virus was discounted through RT-PCR analysis (Kumar et al. 2011; Verbeek et al. 2012; Bratsch et al. 2018). To test the RB phenotype of the observed putative TSWV-RB strains, three-week-old tomato plants from eight commercially available TSWV resistant cultivars and one non-resistant cultivar (n=10 each) were mechanically inoculated with leaf tissues collected from a single symptomatic plant from one of the field-grown resistant cultivars. The experiment was replicated twice. Hypersensitive response was observed on all inoculated leaves of resistant plants one week post inoculation. Furthermore, all eight resistant cultivars started expressing local and systemic TSW symptoms 12 to 16 days post inoculation (dpi), while non-resistant cultivar started expressing symptoms at 9 dpi. TSW incidence across all resistant cultivars was 30-70%, while in susceptible cultivar it was 90%. Symptoms exhibited by all resistant cultivars resembled those of symptoms observed in field collected plants. The expression of Sw-5 gene in all eight resistant cultivars and the lack thereof in a susceptible cultivar was confirmed using Sw-5b specific primers and using Actin as a housekeeping gene in qRT-PCR (Islam et al. 2022). The RB strains in Sw-5 resistant tomato in California (Batuman et al. 2017) had the C118Y mutation in the TSWV NSm protein, consistent with the original reporting of C118Y or T120N RB mutations in 11 TSWV isolates from Spain (NCBI accession # HM015517 & HM015518) (Lopez et al. 2011). The nucleotide and amino acid sequence analysis of NSm gene from Bushland RB isolates from four resistant cultivars (NCBI accessions # OP810513-14 [field], OQ247901-05 [mechanically inoculated]) shared 98.9 and 99.4% homology with the Californian NSm sequences of TSWV RB tomato isolate (KX898453 and ASO67371), respectively. While the Nsm C118Y or T120N RB mutations were absent in all Bushland TSWV RB isolates, they had six additional unique point mutations across the NSm (I163V, P227Q, V290I, N293S, V294I, K296Q), which could potentially be responsible for resistance breaking. Despite the lack of C118Y or T120N RB mutations, Bushland isolates were capable of disrupting Sw-5-mediated TSWV resistance in all eight commercial resistant tomato cultivars. This study suggests a new or a different class of fundamental mechanisms are likely to be responsible for resistance breaking in Sw-5b resistant tomatoes. The new RB strain/s of TSWV therefore pose a substantial threat to tomato production in TX and other tomato-growing regions of the US.

Protective Activity of Rhizobium leguminosarum bv. viciae Strain 33504-Mat209 against Alfalfa Mosaic Virus Infection in Faba Bean Plants.

The application of Rhizobium spp., nitrogen-fixing plant growth-promoting rhizobacteria, as biocontrol agents to enhance systemic disease resistance against plant viral infections is a promising approach towards achieving sustainable and eco-friendly agriculture. However, their potential as antivirals and biocontrol agents is less studied. Herein, the capability of Rhizobium leguminosarum bv. viciae strain 33504-Mat209 was evaluated to promote plant growth and enhance faba bean systemic resistance against alfalfa mosaic virus (AMV) infection. Under greenhouse conditions, the soil inoculation with 3504-Mat209 resulted in notable improvements in growth and an increase in chlorophyll content. This led to a marked decrease in the disease incidence, severity, and viral accumulation level by 48, 74, and 87%, respectively. The protective effect of 33504-Mat209 was linked to significant decreases in non-enzymatic oxidative stress indicators, specifically H2O2 and MDA. Additionally, there were significant increases in the activity of reactive oxygen species scavenging enzymes, such as peroxidase (POX) and polyphenol oxidase (PPO), compared to the virus treatment. The elevated transcript levels of polyphenolic pathway genes (C4H, HCT, C3H, and CHS) and pathogenesis-related protein-1 were also observed. Out of 18 detected compounds, HPLC analysis revealed that 33504-Mat209-treated plants increased the accumulation of several compounds, such as gallic acid, chlorogenic acid, catechin, pyrocatechol, daidzein, quercetin, and cinnamic acid. Therefore, the ability of 33504-Mat209 to promote plant growth and induce systemic resistance against AMV infection has implications for utilizing 33504-Mat209 as a fertilizer and biocontrol agent. This could potentially introduce a new strategy for safeguarding crops, promoting sustainability, and ensuring environmental safety in the agricultural sector. As far as we know, this is the first study of biological control of AMV mediated by Rhizobium spp. in faba bean plants.

Plant YTHDF proteins are direct effectors of antiviral immunity against an N6‐methyladenosine‐containing RNA virus

In virus–host interactions, nucleic acid‐directed first lines of defense that allow viral clearance without compromising growth are of paramount importance. Plants use the RNA interference pathway as a basal antiviral immune system, but additional RNA‐based mechanisms of defense also exist. The infectivity of a plant positive‐strand RNA virus, alfalfa mosaic virus (AMV), relies on the demethylation of viral RNA by the recruitment of the cellular N6‐methyladenosine (m6A) demethylase ALKBH9B, but how demethylation of viral RNA promotes AMV infection remains unknown. Here, we show that inactivation of the Arabidopsis cytoplasmic YT521‐B homology domain (YTH)‐containing m6A‐binding proteins ECT2, ECT3, and ECT5 is sufficient to restore AMV infectivity in partially resistant alkbh9b mutants. We further show that the antiviral function of ECT2 is distinct from its previously demonstrated function in the promotion of primordial cell proliferation: an ect2 mutant carrying a small deletion in its intrinsically disordered region is partially compromised for antiviral defense but not for developmental functions. These results indicate that the m6A‐YTHDF axis constitutes a novel branch of basal antiviral immunity in plants.

Antiviral Activity of Chitosan Nanoparticles and Chitosan Silver Nanocomposites against Alfalfa Mosaic Virus.

Plant viruses are a global concern for sustainable crop production. Among the currently available antiviral approaches, nanotechnology has been overwhelmingly playing an effective role in circumventing plant viruses. Alfalfa mosaic virus (AMV) was isolated and identified from symptomatic pepper plants in Egypt using symptomatology, serological tests using the direct ELISA technique, differential hosts and electron microscopy. The virus was biologically purified from a single local lesion that developed on Chenopodium amaranticolor. The AMV infection was further confirmed using an AMV coat protein-specific primer RT-PCR. We further evaluated the antiviral potential of chitosan nanoparticles (CS-NPs) and chitosan silver nanocomposites (CS-Ag NC) in different concentrations against AMV infections in pepper plants. All tested concentrations of CS-NPs and CS-Ag NC induced the inhibition of AMV systemically infected pepper plants when applied 24 h after virus inoculation. The foliar application of 400 ppm CS-NPs or 200 ppm CS-Ag NC produced the highest AMV inhibitory effect (90 and 91%) when applied 24 h after virus inoculation. Treatment with CS-NPs and CS-Ag NC considerably increased the phenol, proline and capsaicin contents compared to the infected plants. Moreover, the agronomic metrics (plant height, fresh and dry pod weights and number of pods per plant) were also significantly improved. According to our results, the potential applications of CS-NPs and CS-Ag NC may provide an effective therapeutic measure for better AMV and other related plant virus management.