Pathogenicity analysis and seed transmission of watermelon virus A in bottle gourd.

Seed transmission is among the primary strategies utilized by plant viruses for long-distance dissemination, leading to the widespread occurrence of viral diseases globally. Watermelon virus A (WVA) is a novel wamavirus first found in watermelon. However, the pathogenicity and transmission mode of WVA are still unclear. Our previous work found that the incidence of WVA in bottle gourd is very high. Based on that, the pathogenicity and seed transmission mode of WVA in bottle gourd were studied. Compared with healthy plant, bottle gourd infected by WVA showed no visible disease symptom. Moreover, in the seeds of 20 bottle gourd cultivars, the occurrence of WVA varies from 0 to 90%, and one cultivar even reaches 100%. We also found that the transmission rate from seeds to the resulting seedlings was 100%. Furthermore, WVA was present in both the seed coat and embryo, and seed disinfection cannot eliminate WVA. Besides the seed and leaf, WVA can also be detected in stem, flower, and fruit, but not in the root. To our surprise, the level of transmission from WVA-infected plants to seeds was more than 85%. In addition, the viral accumulations of both WVA and CGMMV were increased in plants with co-infection of WVA and CGMMV. Taken together, these findings reveal that WVA is a seed-transmitted virus which causes no disease symptom in bottle gourd, and there may be synergism between WVA and CGMMV.

Improvement of plant resistance to geminiviruses via protein de-S-acylation.

Geminiviruses are an important group of viruses that infect a variety of plants and result in heavy agricultural losses worldwide. The homologs of C4 (or L4) in monopartite geminiviruses and AC4 (or AL4) in bipartite geminiviruses are critical viral proteins. The C4 proteins from several geminiviruses are the substrates of S-acylation, a dynamic post-translational modification, for the maintenance of their membrane localization and function in virus infection. Here we initiated a screening and identified a plant protein ABAPT3 (Alpha/Beta Hydrolase Domain-containing Protein 17-like Acyl Protein Thioesterase 3) as the de-S-acylation enzyme of C4 encoded by BSCTV (Beet severe curly top virus). Overexpression of ABAPT3 reduced the S-acylation of BSCTV C4, disrupted its plasma membrane localization, inhibited its function in pathogenesis, and suppressed BSCTV infection. Because the S-acylation motifs are conserved among C4 from different geminiviruses, we tested the effect of ABAPT3 on the C4 protein of ToLCGdV (Tomato leaf curl Guangdong virus) from another geminivirus genus. Consistently, ABAPT3 overexpression also disrupted the S-acylation, subcellular localization, and function of ToLCGdV C4, and inhibited ToLCGdV infection. In summary, we provided a new approach to globally improve the resistance to different types of geminiviruses in plants via de-S-acylation of the viral C4 proteins and it can be extendedly used for suppression of geminivirus infection in crops.

Comparative genomics and host range analysis of four Ralstonia pseudosolanacearum strains isolated from sunflower reveals genomic and phenotypic differences.

BACKGROUND: Bacterial wilt caused by Ralstonia solanacearum species complex (RSSC) is one of the devastating diseases in crop production, seriously reducing the yield of crops. R. pseudosolanacearum, is known for its broad infrasubspecific diversity and comprises 36 sequevars that are currently known. Previous studies found that R. pseudosolanacearum contained four sequevars (13, 14, 17 and 54) isolated from sunflowers sown in the same field. RESULTS: Here, we provided the complete genomes and the results of genome comparison of the four sequevars strains (RS639, RS642, RS647, and RS650). Four strains showed different pathogenicities to the same cultivars and different host ranges. Their genome sizes were about 5.84 ~ 5.94 Mb, encoding 5002 ~ 5079 genes and the average G + C content of 66.85% ~ 67%. Among the coding genes, 146 ~ 159 specific gene families (contained 150 ~ 160 genes) were found in the chromosomes and 34 ~ 77 specific gene families (contained 34 ~ 78 genes) in the megaplasmids from four strains. The average nucleotide identify (ANI) values between any two strains ranged from 99.05% ~ 99.71%, and the proportion of the total base length of collinear blocks accounts for the total gene length of corresponding genome was all more than 93.82%. Then, we performed a search for genomic islands, prophage sequences, the gene clusters macromolecular secretion systems, type III secreted effectors and other virulence factors in these strains, which provided detailed comparison results of their presence and distinctive features compared to the reference strain GMI1000. Among them, the number and types of T2SS gene clusters were different in the four strains, among which RS650 included all five types. T4SS gene cluster of RS639 and RS647 were missed. In the T6SS gene cluster, several genes were inserted in the RS639, RS647, and RS650, and gene deletion was also detected in the RS642. A total of 78 kinds of type III secreted effectors were found, which included 52 core and 9 specific effectors in four strains. CONCLUSION: This study not only provided the complete genomes of multiple R. pseudosolanacearum strains isolated from a new host, but also revealed the differences in their genomic levels through comparative genomics. Furthermore, these findings expand human knowledge about the range of hosts that Ralstonia can infect, and potentially contribute to exploring rules and factors of the genetic evolution and analyzing its pathogenic mechanism.

Transcriptome and Metabolome Analyses Reveal That Jasmonic Acids May Facilitate the Infection of Cucumber Green Mottle Mosaic Virus in Bottle Gourd.

Cucumber green mottle mosaic virus (CGMMV) is a typical seed-borne tobamovirus that mainly infects cucurbit crops. Due to the rapid growth of international trade, CGMMV has spread worldwide and become a significant threat to cucurbit industry. Despite various studies focusing on the interaction between CGMMV and host plants, the molecular mechanism of CGMMV infection is still unclear. In this study, we utilized transcriptome and metabolome analyses to investigate the antiviral response of bottle gourd (Lagenaria siceraria) under CGMMV stress. The transcriptome analysis revealed that in comparison to mock-inoculated bottle gourd, 1929 differently expressed genes (DEGs) were identified in CGMMV-inoculated bottle gourd. Among them, 1397 genes were upregulated while 532 genes were downregulated. KEGG pathway enrichment indicated that the DEGs were mainly involved in pathways including the metabolic pathway, the biosynthesis of secondary metabolites, plant hormone signal transduction, plant-pathogen interaction, and starch and sucrose metabolism. The metabolome result showed that there were 76 differentially accumulated metabolites (DAMs), of which 69 metabolites were up-accumulated, and 7 metabolites were down-accumulated. These DAMs were clustered into several pathways, including biosynthesis of secondary metabolites, tyrosine metabolism, flavonoid biosynthesis, carbon metabolism, and plant hormone signal transduction. Combining the transcriptome and metabolome results, the genes and metabolites involved in the jasmonic acid and its derivatives (JAs) synthesis pathway were significantly induced upon CGMMV infection. The silencing of the allene oxide synthase (AOS) gene, which is the key gene involved in JAs synthesis, reduced CGMMV accumulation. These findings suggest that JAs may facilitate CGMMV infection in bottle gourd.

Cotton leaf curl Multan virus differentially regulates innate antiviral immunity of whitefly (Bemisia tabaci) vector to promote cryptic species-dependent virus acquisition.

Begomoviruses represent the largest group of economically important, highly pathogenic, DNA plant viruses that contribute a substantial amount of global crop disease burden. The exclusive transmission of begomoviruses by whiteflies (Bemisia tabaci) requires them to interact and efficiently manipulate host responses at physiological, biological and molecular scales. However, the molecular mechanisms underlying complex begomovirus-whitefly interactions that consequently substantiate efficient virus transmission largely remain unknown. Previously, we found that whitefly Asia II 7 cryptic species can efficiently transmit cotton leaf curl Multan virus (CLCuMuV) while MEAM1 cryptic species is a poor carrier and incompetent vector of CLCuMuV. To investigate the potential mechanism/s that facilitate the higher acquisition of CLCuMuV by its whitefly vector (Asia II 7) and to identify novel whitefly proteins that putatively interact with CLCuMuV-AV1 (coat protein), we employed yeast two-hybrid system, bioinformatics, bimolecular fluorescence complementation, RNA interference, RT-qPCR and bioassays. We identified a total of 21 Asia II 7 proteins putatively interacting with CLCuMuV-AV1. Further analyses by molecular docking, Y2H and BiFC experiments validated the interaction between a whitefly innate immunity-related protein (BTB/POZ) and viral AV1 (coat protein). Gene transcription analysis showed that the viral infection significantly suppressed the transcription of BTB/POZ and enhanced the accumulation of CLCuMuV in Asia II 7, but not in MEAM1 cryptic species. In contrast to MEAM1, the targeted knock-down of BTB/POZ substantially reduced the ability of Asia II 7 to acquire and accumulate CLCuMuV. Additionally, antiviral immune signaling pathways (Toll, Imd, Jnk and Jak/STAT) were significantly suppressed following viral infection of Asia II 7 whiteflies. Taken together, the begomovirus CLCuMuV potentiates efficient virus accumulation in its vector B. tabaci Asia II 7 by targeting and suppressing the transcription of an innate immunity-related BTB/POZ gene and other antiviral immune responses in a cryptic species-specific manner.

Comparative transcriptome profiling reveals a network of differentially expressed genes in Asia II 7 and MEAM1 whitefly cryptic species in response to early infection of Cotton leaf curl Multan virus.

Cotton leaf curl Multan virus (CLCuMuV) is a whitefly-vectored begomovirus that poses ramping threat to several economically important crops worldwide. The differential transmission of CLCuMuV by its vector Bemisia tabaci mainly relies on the type of whitefly cryptic species. However, the molecular responses among different whitefly cryptic species in response to early CLCuMuV infection remain elusive. Here, we compared early-stage transcriptomic profiles of Asia II 7 and MEAM1 cryptic species infected by CLCuMuV. Results of Illumina sequencing revealed that after 6 and 12 h of CLCuMuV acquisition, 153 and 141 genes among viruliferous (VF) Asia II 7, while 445 and 347 genes among VF MEAM 1 whiteflies were differentially expressed compared with aviruliferous (AVF) whiteflies. The most abundant groups of differentially expressed genes (DEGs) among Asia II 7 and MEAM1 were associated with HTH-1 and zf-C2H2 classes of transcription factors (TFs), respectively. Notably, in contrast to Asia II 7, MEAM1 cryptic species displayed higher transcriptional variations with elevated immune-related responses following CLCuMuV infection. Among both cryptic species, we identified several highly responsive candidate DEGs associated with antiviral innate immunity (alpha glucosidase, LSM14-like protein B and phosphoenolpyruvate carboxykinase), lysosome (GPI-anchored protein 58) and autophagy/phagosome pathways (sequestosome-1, cathepsin F-like protease), spliceosome (heat shock protein 70), detoxification (cytochrome P450 4C1), cGMP-PKG signaling pathway (myosin heavy chain), carbohydrate metabolism (alpha-glucosidase), biological transport (mitochondrial phosphate carrier) and protein absorption and digestion (cuticle protein 8). Further validation of RNA-seq results showed that 23 of 28 selected genes exhibited concordant expression both in RT-qPCR and RNA-seq. Our findings provide vital mechanistic insights into begomovirus-whitefly interactions to understand the dynamics of differential begomovirus transmission by different whitefly cryptic species and reveal novel molecular targets for sustainable management of insect-transmitted plant viruses.

First report of tomato yellow mottle-associated virus infecting Solanum nigrum in China.

First report of tomato yellow mottle-associated virus infecting Solanum nigrum in China Zhenggang Li, Yafei Tang, Xiaoman She, Guobing Lan, Lin Yu, and Zifu He† Guangdong Provincial Key Laboratory of High Technology for Plant Protection, Plant Protection Research Institute, Guangdong Academy of Agricultural Sciences, Guangzhou, 510640, P. R. China. Tomato yellow mottle-associated virus (TYMaV) is a newly found cytorhabdovirus associated with epinasty of leaflet blades, yellow spots, puckering, and mottling symptoms in tomato plants in China (Xu et al., 2017). In May 2020, Solanum nigrum plants exhibiting leaf crinkling and mosaic symptoms (eXtra S1) were found in Shantou city, Guangdong, China. To identify the causal pathogens, the leaves of three symptomatic plants were collected and subjected to total RNA extraction with TRIzol Reagent (Takara, Kusatsu, Japan). About 100 µg RNA mixture, which consisted of an equal amount of total RNA extracted from the three samples, was subjected to small RNA deep sequencing and assembly (sRSA) (Kreuze et al., 2009). Small RNA cDNA library was constructed with the method described previously (Mi et al., 2008). Small RNA deep sequencing was performed with Illumina HiSeq X Ten platform. VirusDetect (Zheng et al., 2017) was used to analyze the sequence data. The result showed that the sequence data includes about 11 million reads and generated 194 unique contigs after removal of host-derived contigs. Subsequently, the unique contigs were screened using BLASTn search against the virus database. One hundred and five unique contigs were mapped to TYMaV genome (reference sequence, KY075646), 21 unique contigs were mapped to RNA1 segment of tomato chlorosis virus (ToCV) genome (reference sequence, KY618796), 67 unique contigs were mapped to RNA2 segment of ToCV genome (reference sequence, KY618797), and one unique contig was mapped to pepper veinal mottle virus (PVMV) genome (reference sequence, FJ617225) (eXtra S1). To verify the sRSA result for TYMaV detection, RT-PCR was performed with two primer pairs TYMaV-F1/R1 (5'-TCATTAGACTCAGGCCTAATCCTCA AAGT-3'/5'-GATATGGAGACGTCCAAGTTCAAAGGGATGGA-3'), and TYMaV-F2/R2 (5'-TATGCGGCAGCTTTCATGTCTATAGACCCT-3'/5'-ATGACCTAGCTTCAATAACAGTCGCG-3'), which are designed according to the sRSA result. All the symptomatic samples tested positive for TYMaV (eXtra S2). Western blot with TYMaV N protein-specific antibody further verified the result (eXtra S2). To obtain the nearly full-length sequence of TYMaV identified in Shantou, 13 primer pairs were designed to amplify the viral fragments. The amplified PCR products were then introduced into pMD19T (Takara, Kusatsu, Japan) and sequenced by Sangon Biotech Co. (Shanghai, China). The nearly full-length sequence of TYMaV Shantou isolate (TYMaV-ST) was assembled from the 13 overlapping sequences (reference sequence, MW527091). TYMaV-ST genome comprises of 13401 nt and shares 84.93% nucleotide sequence identity with the reference genome (KY075646). In addition, 37 S. nigrum samples and 20 tomato samples nearby with viral disease symptoms were collected from different sites of Guangdong province, China. Six S. nigrum samples and five tomato plant samples tested positive for TYMaV by RT-PCR, suggesting a wide spread of the virus in the surveyed region. These results together with those of the sRSA assay also suggest that the disease symptoms shown in the original S. nigrum plants may not necessarily be caused by TYMaV or by TYMaV alone. To our knowledge, this is the first report of TYMaV infecting S. nigrum in China. S. nigrum is a common weed which belongs to the family Solanaceae and may serve as a reservoir for TYMaV in the fields. Further research is needed to verify whether this is indeed the case, and to understand the characteristics of this virus including its transmission, pathogenicity, and economic significance. The authors declare no conflict of interest. Funding This work was supported by the Key Research and Development Program of Guangdong Province (2018B020202006), the Agricultural Competitive Industry Discipline Team Building Project of Guangdong Academy of Agricultural Sciences (202103TD and 202105TD), the Science and Technology Program of Guangzhou (202102020504), and Special Fund for Scientific Innovation Strategy-Construction of High-Level Academy of Agriculture Science (R2019PY-QF003). References: Kreuze, J. F., et al. 2009. Virology. 388: 1. Mi, S., et al. 2008. Cell. 133: 116. Xu, C., et al. 2017. J Virol. 91: 11. Zheng, Y., et al. 2017. Virology. 500: 130.

Anthracnose: A New Leaf Disease on Radermachera sinica (China Doll) in China.

Radermachera sinica (China doll) is a popular evergreen horticultural crop worldwide. However, little information has been provided to describe the anthracnose disease of R. sinica. In 2018, symptoms suspected of leaf anthracnose were observed on R. sinica in gardens and commercial greenhouses in Guangzhou, China. Lesions on diseased leaves showed thinned and grayish white centers, dark-brown to black borders, and raised black spots. Twenty-seven single-conidia isolates were obtained from symptomatic leaf lesions. Based on morphological characteristics and multilocus phylogenetic analysis, 19 isolates were identified as Colletotrichum siamense and six and two isolates were identified as C. fructicola and C. karstii, respectively. An in vivo pathogenicity test was conducted on leaves of R. sinica plants, and it was discovered that C. siamense was more aggressive under wounded conditions than under unwounded conditions, and caused symptomatic necrotic lesions on the leaf. Afterward, the same pathogen was reisolated from lesions of inoculated leaves to fulfill Koch's postulates. However, neither C. fructicola nor C. karstii caused visible lesions on leaves of R. sinica under wounded or unwounded conditions, indicating that they may be asymptomatic endophytes or opportunistic pathogens on R. sinica. To our knowledge, this study is the first report of Colletotrichum spp. associated with anthracnose disease on R. sinica in China.

Deploying Viruses against Phytobacteria: Potential Use of Phage Cocktails as a Multifaceted Approach to Combat Resistant Bacterial Plant Pathogens.

Plants in nature are under the persistent intimidation of severe microbial diseases, threatening a sustainable food production system. Plant-bacterial pathogens are a major concern in the contemporary era, resulting in reduced plant growth and productivity. Plant antibiotics and chemical-based bactericides have been extensively used to evade plant bacterial diseases. To counteract this pressure, bacteria have evolved an array of resistance mechanisms, including innate and adaptive immune systems. The emergence of resistant bacteria and detrimental consequences of antimicrobial compounds on the environment and human health, accentuates the development of an alternative disease evacuation strategy. The phage cocktail therapy is a multidimensional approach effectively employed for the biocontrol of diverse resistant bacterial infections without affecting the fauna and flora. Phages engage a diverse set of counter defense strategies to undermine wide-ranging anti-phage defense mechanisms of bacterial pathogens. Microbial ecology, evolution, and dynamics of the interactions between phage and plant-bacterial pathogens lead to the engineering of robust phage cocktail therapeutics for the mitigation of devastating phytobacterial diseases. In this review, we highlight the concrete and fundamental determinants in the development and application of phage cocktails and their underlying mechanism, combating resistant plant-bacterial pathogens. Additionally, we provide recent advances in the use of phage cocktail therapy against phytobacteria for the biocontrol of devastating plant diseases.

A group I WRKY transcription factor regulates mulberry mosaic dwarf-associated virus-triggered cell death in Nicotiana benthamiana.

Geminiviruses constitute the largest group of known plant viruses and cause devastating losses to a wide range of crops and woody plants globally. Mulberry mosaic dwarf-associated virus (MMDaV), identified from Chinese mulberry trees via small RNA-based deep sequencing, is a divergent monopartite geminivirus belonging to the genus Mulcrilevirus of the family Geminiviridae. Previous studies have shown that plants employ multiple layers of defence to protect themselves from geminivirus infection. The interplay between plant and MMDaV is nevertheless less studied. This study presents evidence that MMDaV triggers hypersensitive response (HR)-mediated antiviral defence in Nicotiana benthamiana plants. We show that the RepA protein of MMDaV is engaged in HR-type cell death induction. We find that the RepA mutants with compromised nuclear localization ability impair their capabilities of cell death induction. Virus-induced gene silencing of the key components of the R protein-mediated signalling pathway reveals that down-regulation of the nucleus-targeting NbWRKY1 alleviates the cell death induction activity of RepA. We further demonstrate that RepA up-regulates the transcript level of NbWRKY1. Furthermore, expression of RepA in N. benthamiana confers plant resistance against two begomoviruses. We propose that plant resistance against RepA can be potentially used to improve plant defence against geminiviruses in crops.

Global genetic diversity and evolutionary patterns among Potato leafroll virus populations.

Potato leafroll virus (PLRV) is a widespread and one of the most damaging viral pathogens causing significant quantitative and qualitative losses in potato worldwide. The current knowledge of the geographical distribution, standing genetic diversity and the evolutionary patterns existing among global PLRV populations is limited. Here, we employed several bioinformatics tools and comprehensively analyzed the diversity, genomic variability, and the dynamics of key evolutionary factors governing the global spread of this viral pathogen. To date, a total of 84 full-genomic sequences of PLRV isolates have been reported from 22 countries with most genomes documented from Kenya. Among all PLRV-encoded major proteins, RTD and P0 displayed the highest level of nucleotide variability. The highest percentage of mutations were associated with RTD (38.81%) and P1 (31.66%) in the coding sequences. We detected a total of 10 significantly supported recombination events while the most frequently detected ones were associated with PLRV genome sequences reported from Kenya. Notably, the distribution patterns of recombination breakpoints across different genomic regions of PLRV isolates remained variable. Further analysis revealed that with exception of a few positively selected codons, a major part of the PLRV genome is evolving under strong purifying selection. Protein disorder prediction analysis revealed that CP-RTD had the highest percentage (48%) of disordered amino acids and the majority (27%) of disordered residues were positioned at the C-terminus. These findings will extend our current knowledge of the PLRV geographical prevalence, genetic diversity, and evolutionary factors that are presumably shaping the global spread and successful adaptation of PLRV as a destructive potato pathogen to geographically isolated regions of the world.

Molecular phylogenetics and evolutionary analysis of a highly recombinant begomovirus, Cotton leaf curl Multan virus, and associated satellites.

Cotton leaf curl Multan virus (CLCuMuV) and its associated satellites are a major part of the cotton leaf curl disease (CLCuD) caused by the begomovirus species complex. Despite the implementation of potential disease management strategies, the incessant resurgence of resistance-breaking variants of CLCuMuV imposes a continuous threat to cotton production. Here, we present a focused effort to map the geographical prevalence, genomic diversity, and molecular evolutionary endpoints that enhance disease complexity by facilitating the successful adaptation of CLCuMuV populations to the diversified ecosystems. Our results demonstrate that CLCuMuV populations are predominantly distributed in China, while the majority of alphasatellites and betasatellites exist in Pakistan. We demonstrate that together with frequent recombination, an uneven genetic variation mainly drives CLCuMuV and its satellite's virulence and evolvability. However, the pattern and distribution of recombination breakpoints greatly vary among viral and satellite sequences. The CLCuMuV, Cotton leaf curl Multan alphasatellite, and Cotton leaf curl Multan betasatellite populations arising from distinct regions exhibit high mutation rates. Although evolutionarily linked, these populations are independently evolving under strong purifying selection. These findings will facilitate to comprehensively understand the standing genetic variability and evolutionary patterns existing among CLCuMuV populations across major cotton-producing regions of the world.

The C4 protein encoded by Tomato leaf curl Yunnan virus interferes with mitogen-activated protein kinase cascade-related defense responses through inhibiting the dissociation of the ERECTA/BKI1 complex.

Mitogen-activated protein kinase (MAPK) cascades are involved in host defense against pathogens and are often activated by upstream plasma membrane leucine-rich repeat receptor-like kinases (LRR-RLKs). ERECTA (ER) is an LRR-RLK that regulates plant developmental processes through activating MAPK cascades. Tomato leaf curl Yunnan virus (TLCYnV) C4 protein interacts with BKI1, stabilizes it at the plasma membrane and impairs ER autophosphorylation through suppressing the dissociation of the BKI1/ER complex, and then inhibits the activation of downstream MAPK cascades, which ultimately creates a favorable environment for TLCYnV infection. This study provides a novel viral strategy to impair MAPK activation.

Complete genome sequence of a previously undescribed monopartite begomovirus and betasatellite infecting Malvastrum coromandelianum in Cambodia.

A previously undescribed monopartite begomovirus was identified in Kampot province, Cambodia, in Malvastrum coromandelianum plants exhibiting yellow vein symptoms characteristic of begomovirus infections. The apparently full-length viral component was cloned and sequenced following enrichment of circular DNA by rolling-circle amplification and restriction enzyme digestion. The genome of the virus was 2737 nucleotides in length (KP188831) and exhibited an organization like that of other monopartite begomoviruses, sharing the highest nucleotide sequence similarity (87.7% identity) with ageratum yellow vein virus (AM940137). A satellite molecule was amplified from total DNA by PCR amplification, using the betasatellite-specific primer pair ß01/ß02. The satellite molecule (1346 nt, KP188832) had structural characteristics like those of other betasatellites associated with begomoviruses and shared the highest nucleotide sequence similarity (84.8% identity) with malvastrum yellow vein betasatellite (MN205547). According to the criteria established for species demarcation for classification of begomoviruses (family Geminiviridae) and betasatellites (family Tolecusatellitidae), respectively, the virus isolate from M. coromandelianum in Cambodia is a previously undescribed novel monopartite begomovirus, for which the name "malvastrum yellow vein Cambodia virus" (MaYVCV) is proposed, and the betasatellite is a previously undescribed novel betasatellite, for which the name "malvastrum yellow vein Cambodia betasatellite" (MaYVKHB) is proposed.

Nanotechnology and Plant Viruses: An Emerging Disease Management Approach for Resistant Pathogens.

Phytoviruses are highly destructive plant pathogens, causing significant agricultural losses due to their genomic diversity, rapid, and dynamic evolution, and the general inadequacy of management options. Although an increasing number of studies are being published demonstrating the efficacy of engineered nanomaterials to treat a range of plant pathogens, very little work has been done with phytoviruses. Herein, we describe the emerging field of "Nanophytovirology" as a potential management approach to combat plant viral diseases. Because of their special physiochemical properties, nanoparticles (NPs) can interact with viruses, their vectors, and the host plants in a variety of specific and useful ways. We specifically describe the potential mechanisms underlying NPs-plant-virus interactions and explore the antiviral role of NPs. We discuss the limited literature, as well as the challenges and research gaps that are instrumental to the successful development of a nanotechnology-based, multidisciplinary approach for timely detection, treatment, and prevention of viral diseases.

Carbon-based nanomaterials suppress tobacco mosaic virus (TMV) infection and induce resistance in Nicotiana benthamiana.

Although nanomaterials (NMs) may inhibit viral pathogens, the mechanisms governing plant-virus-nanomaterial interactions remain unknown. Nicotiana benthamiana plants were treated with nanoscale titanium dioxide (TiO2) and silver (Ag), C60 fullerenes, and carbon nanotubes (CNTs) at 100, 200 and 500 mg L-1 for a 21-day foliar exposure before inoculation with GFP-tagged tobacco mosaic virus (TMV). Plants treated with CNTs and C60 (200 mg L-1) exhibited normal phenotype and viral symptomology was not evident at 5 days post-infection. TiO2 and Ag failed to suppress viral infection. RT-qPCR analysis revealed that viral coat protein transcript abundance and GFP mRNA expression were reduced 74-81% upon CNTs and C60 treatment. TEM revealed that the chloroplast ultrastructure in carbon NM-treated plants was unaffected by TMV infection. Fluorescence measurement of CNTs and C60 (200 mg L-1) treated plants indicated photosynthesis equivalent to healthy controls. CNTs and C60 induced upregulation of the defense-related phytohormones abscisic acid and salicylic acid by 33-52%; the transcription of genes responsible for phytohormone biosynthesis was elevated by 94-104% in treated plants. Our findings demonstrate the protective role of carbon-based NMs, with suppression of TMV symptoms via hindered physical movement and viral replication. Given the lack of viral phytopathogen treatment options, this work represents a novel area of nano-enabled agriculture.

C4, the Pathogenic Determinant of Tomato Leaf Curl Guangdong Virus, May Suppress Post-transcriptional Gene Silencing by Interacting With BAM1 Protein.

Tomato leaf curl Guangdong virus (ToLCGdV) is a begomovirus associated with a Tomato yellow leaf curl disease (TYLCD) epidemic in Guangdong province, China. Being the least conserved protein among geminivirus proteins, the function of C4 during ToLCGdV infection has not been elucidated. In this study, the infectious clones of ToLCGdV and a ToLCGdV mutant (ToLCGdVmC4) with disrupted C4 ORF were constructed. Although ToLCGdV and ToLCGdVmC4 could infect Nicotiana benthamiana and tomato plants, ToLCGdVmC4 elicited much milder symptoms compared with ToLCGdV. To further verify the role of C4 in viral pathogenesis, C4 was expressed in N. benthamiana from Potato virus X (PVX) vector. The results showed that ToLCGdV C4 enhanced the pathogenicity of PVX and induced more severe developmental abnormalities in plants compared with PVX alone or PVX-mC4. In addition, ToLCGdV C4 suppresses systemic gene silencing in the transgenic N. benthamiana line 16c, but not local gene silencing induced by sense GFP in wild-type N. benthamiana plants. Moreover, C4 suppresses transcriptional gene silencing (TGS) by reducing the DNA methylation level of 35S promoter in 16c-TGS N. benthamiana plants. Furthermore, C4 could also interact with the receptor-like kinase (RLK) BARELY ANY MERISTEM 1 (BAM1), suggesting that C4 may suppress gene silencing by interfering with the function of BAM1 in the cell-to-cell spread of RNAi. All these results suggest that C4 is a pathogenic determinant of ToLCGdV, and C4 may suppress post-transcriptional gene silencing (PTGS) by interacting with BAM1.

Transmission efficiency of Cotton leaf curl Multan virus by three cryptic species of Bemisia tabaci complex in cotton cultivars.

Cotton leaf curl Multan virus (CLCuMuV) is a serious and economically important viral disease agent in cotton and ornamental plants like Hibiscus in many regions of the world, especially in South Asia. CLCuMuV is transmitted exclusively by Bemisia tabaci cryptic species complex. This virus was recently recorded in southern China, presumably an invasion from South Asia. This study was performed to estimate the efficiency of three species of the B. tabaci whitefly complex (tentatively named as MEAM1, MED and Asia II 7, respectively) to transmit CLCuMuV and Cotton leaf curl multan virus betasatelite (CLCuMuB). Transmission assays and real-time quantitative PCR were conducted using three cultivars of cotton, Gossypium hirsutum, including 112-2, Xinhai-21 and Zhongmian-40. The results indicated that Asia II 7 was able to transmit the virus to two of the cotton cultivars, i.e. 112-2 and Xinhai-21, with the highest transmission efficiencies of 40% and 30%, respectively, but was unable to transmit the virus to the cotton cultivar Zhongmian-40. MEAM1 and MED failed to transmit CLCuMuV and CLCuMuB to any of the three cotton cultivars. After the three cryptic species of whiteflies had fed on virus-infected cotton plants for 48 h, the relative quantity of CLCuMuV in Asia II 7 was detected to be significantly higher than that in both MEAM1 and MED (P < 0.05). These results indicate that among the three species of whiteflies Asia II 7 is likely the most efficient vector for CLCuMuV and CLCuMuB in Malvaceae crops in China. Our findings provide valuable information to the control of viral diseases caused by CLCuMuV in the field.

Identification and Genetic Characterization of Ralstonia solanacearum Species Complex Isolates from Cucurbita maxima in China.

Ralstonia solanacearum species complex is a devastating phytopathogen with an unusually wide host range, and new host plants are continuously being discovered. In June 2016, a new bacterial wilt on Cucurbita maxima was observed in Guangdong province, China. Initially, in the adult plant stage, several leaves of each plant withered suddenly and drooped; the plant then wilted completely, and the color of their vasculature changed to dark brown, ultimately causing the entire plant to die. Creamy-whitish bacterial masses were observed to ooze from crosscut stems of these diseased plants. To develop control strategies for C. maxima bacterial wilt, the causative pathogenic isolates were identified and characterized. Twenty-four bacterial isolates were obtained from diseased C. maxima plants, and 16S rRNA gene sequencing and pathogenicity analysis results indicated that the pathogen of C. maxima bacterial wilt was Ralstonia solanacearum. The results from DNA-based analysis, host range determination and bacteriological identification confirmed that the 24 isolates belonged to R. solanacearum phylotype I, race 1, and eight of these isolates belonged to biovar 3, while 16 belonged to biovar 4. Based on the results of partial egl gene sequence analysis, the 24 isolates clustered into three egl- sequence type groups, sequevars 17, 45, and 56. Sequevar 56 is a new sequevar which is described for the first time in this paper. An assessment of the resistance of 21 pumpkin cultivars revealed that C. moschata cv. Xiangyu1 is resistant to strain RS378, C. moschata cv. Xiangmi is moderately resistant to strain RS378, and 19 other pumpkin cultivars, including four C. maxima cultivars and 15 C. moschata cultivars, are susceptible to strain RS378. To the best of our knowledge, this is the first report of C. maxima bacterial wilt caused by R. solanacearum race 1 in the world. Our results provide valuable information for the further development of control strategies for C. maxima wilt disease.

Genomic Analysis of Phylotype I Strain EP1 Reveals Substantial Divergence from Other Strains in the Ralstonia solanacearum Species Complex.

Ralstonia solanacearum species complex is a devastating group of phytopathogens with an unusually wide host range and broad geographical distribution. R. solanacearum isolates may differ considerably in various properties including host range and pathogenicity, but the underlying genetic bases remain vague. Here, we conducted the genome sequencing of strain EP1 isolated from Guangdong Province of China, which belongs to phylotype I and is highly virulent to a range of solanaceous crops. Its complete genome contains a 3.95-Mb chromosome and a 2.05-Mb mega-plasmid, which is considerably bigger than reported genomes of other R. solanacearum strains. Both the chromosome and the mega-plasmid have essential house-keeping genes and many virulence genes. Comparative analysis of strain EP1 with other 3 phylotype I and 3 phylotype II, III, IV strains unveiled substantial genome rearrangements, insertions and deletions. Genome sequences are relatively conserved among the 4 phylotype I strains, but more divergent among strains of different phylotypes. Moreover, the strains exhibited considerable variations in their key virulence genes, including those encoding secretion systems and type III effectors. Our results provide valuable information for further elucidation of the genetic basis of diversified virulences and host range of R. solanacearum species.