Diversity and phylogeography of begomoviruses and DNA satellites associated with the leaf curl and mosaic disease complex of eggplant.

Eggplant is one of the important vegetable crops grown across the world, and its production is threatened by both biotic and abiotic stresses. Diseases caused by viruses are becoming major limiting factors for its successful cultivation. A survey for begomovirus-like symptoms in 72 eggplant fields located in six different Indian states revealed a prevalence of disease ranging from 5.2 to 40.2%, and the symptoms recorded were mosaic, mottling, petiole bending, yellowing, and upward curling, vein thickening, and enation of the leaves, and stunting of plants. The causal agent associated with these plants was transmitted from infected leaf samples to healthy eggplant seedlings via grafting and whiteflies (Bemisia tabaci). The presence of begomovirus was confirmed in 72 infected eggplant samples collected from the surveyed fields exhibiting leaf curl and mosaic disease by PCR using begomovirus specifc primers (DNA-A componet), which resulted in an expected amplicon of 1.2 kb. The partial genome sequence obtained from amplified 1.2 kb from all samples indicated that they are closely related begomovirus species, tomato leaf Karnataka virus (ToLCKV, two samples), tomato leaf curl Palampur virus (ToLCPalV, fifty eggplant samples), and chilli leaf curl virus (ChLCuV, twenty samples). Based on the partial genome sequence analysis, fourteen representative samples were selected for full viral genome amplification by the rolling circle DNA amplification (RCA) technique. Analyses of fourteen eggplant isolates genome sequences using the Sequence Demarcation Tool (SDT) indicated that one isolate had the maximum nucleotide (nt) identity with ToLCKV and eight isolates with ToLCPalV. Whereas, four isolates four isolates (BLC1-CH, BLC2-CH, BLC3-CH, BLC4-CH) are showing nucleotide identity of less than 91% with chilli infecting viruses begomoviruses with chilli infecting begomoviruses and as per the guidelines given by the ICTV study group for the classification of begomoviruses these isolates are considered as one novel begomovirus species, for which name, Eggplant leaf curl Chhattisgarh virus (EgLCuChV) is proposed. For DNA-B component, seven eggplant isolates had the highest nt identity with ToLCPalV infecting other crops. Further, DNA satellites sequence analysis indicated that four betasatellites identified shared maximum nucleotide identity with the tomato leaf curl betasatellite and five alphasatellites shared maximum nucleotide identity with the ageratum enation alphasatellite. Recombination and GC plot analyses indicated that the bulk of begomovirus genome and associated satellites presumably originated from of previously known mono and bipartite begomoviruses and DNA satellites. To the best of our knowledge, this is India's first report of ToLCKV and a noval virus, eggplant leaf curl Chhattisgarh virus associated with eggplant leaf curl disease.

Begomovirus Tomato Leaf Curl New Delhi Virus Is Seedborne but Not Seed Transmitted in Melon.

Seed transmission can be of considerable relevance to the dissemination of plant viruses in nature and for their prevalence and perpetuation. Long-distance spread of isolates of the begomovirus species Tomato leaf curl New Delhi virus (genus Begomovirus, family Geminiviridae) has recently occurred from Asia to the Middle East and the Mediterranean Basin. Here, we investigated the possible transmission by melon (Cucumis melo L.) seeds of a tomato leaf curl New Delhi virus (ToLCNDV) isolate of the "Spain" strain widely distributed in the Mediterranean area as an alternative mechanism for long-distance spread. PCR amplification detection of ToLCNDV in floral parts and mature seeds of melon plants reveals that this virus is seedborne. "Seedborne" is defined as the ability of a virus to be carried through seeds, which does not necessarily lead to transmission to the next generation. Treatment with a chemical disinfectant significantly reduced the detectable virus associated with melon seeds, suggesting ToLCNDV contamination of the external portion of the seed coat. Also, when the internal fraction of the mature seed (seed cotyledons + embryo) was analyzed by quantitative PCR amplification, ToLCNDV was detectable at low levels, suggesting the potential for viral contamination or infection of the internal portions of seed. However, grow-out studies conducted with melon progeny plants germinated from mature seeds collected from ToLCNDV-infected plants and evaluated at early (1-leaf) or at late (20-leaf) growth stages did not support the transmission of ToLCNDV from seeds to offspring.

First report of tomato leaf curl New Delhi virus infecting several cucurbit plants in China.

In autumn 2022, a novel and devastating viral disease affecting cucurbits emerged in Ningbo (Zhejiang province), Haimen (Jiangsu province), and Shanghai, China, causing an approximate 650-hectare infestation and resulting in nearly US$15 million in economic losses. The incidence rates of infection reached up to 72.5% on muskmelon (Cucumis melo L. ssp melo), oriental melon (Cucumis melo L. var. agrestis), pumpkin (Cucurbita moschata), luffa (Luffa acutangula), and squash (Cucurbita pepo), and were highly associated with the presence of whitefly (Bemisia tabaci). Infected plants exhibited symptoms such as dwarf stunting, reduced leaf size, leaf chlorotic patches, malformation, fruit deformation, leaf downward rolling, and yellowing (Figure 1). To identify the pathogen, forty cucurbit leaf samples were collected from Haimen (18), Ningbo (19), and Shanghai (3) and tested for cucurbits chlorotic yellows virus (CCYV), cucurbit yellow stunting disorder virus (CYSDV), and Begomovirus using RT-PCR or PCR. All samples tested negative for CCYV and CYSDV using species-specific primers; however, 29 out of 40 samples tested positive (see Supplementary Table 1) for Begomovirus using the degenerate primer pairs PA/PB (Deng et al. 1994). PCR products from seven samples, representing different regions and hosts, underwent Sanger sequencing. The nucleotide sequences of these products showed 98.2-99% identity to tomato leaf curl New Delhi virus (ToLCNDV) by BLASTn. Subsequently, the 29 positive cucurbit samples were confirmed using ToLCNDV-specific primer pairs NDVAF/NDVAR and NDVBF/NDVBR (Jyothsna et al. 2013) for DNA-A and DNA-B, respectively. The DNA-A and DNA-B genome sequences of ToLCNDV isolates from Haimen (Haimen4), Ningbo (Ningbo6), and Shanghai (Shanghai1) were obtained using the primer pairs NDVAF/NDVAR, A1961F/A2645R (covering complete DNA-A sequences), NDVBF/NDVBR, and B1613F/B2579R (covering complete DNA-B sequences，see Supplementary Table 2). No amplicon was produced with primer pairs UNA101/UNA102 and beta01/beta02 (Supplementary Table 2) for detecting Alphasatellite and Betasatellite DNAs, respectively. The complete DNA-A genome sequences (2739 bp) of Haimen 4 (accession no. OP585369), Ningbo 6 (accession no. OP585370), and Shanghai 1 (accession no. OP683993) isolates exhibited 99.5-99.6% nucleotide identity to each other, and their highest nucleotide sequence identity (99.3-99.4%) was shared with the DNA-A of ToLCNDV-Zhejiang isolate (accession no. OP356207) from tomato in Zhejiang Province, China. The complete nucleotide sequences (2693 nt) of DNA-B for Haimen 4 (accession no. OP683995), Ningbo 6 (accession no. OP683996), and Shanghai 1 (accession no. OP683994) isolates showed 99.0-99.1% identity to each other, and their highest nucleotide sequence identity (~99.1%) was shared with the DNA-B of ToLCNDV-Zhejiang isolate (accession no. OP356208).All ToLCNDV isolates from mainland China, including the Zhejiang isolate and the three isolates in this study, shared 98.3-98.7% nucleotide sequence identity and 98.2-98.4% with the DNA-A genome of the severe isolate (accession no. HM159454) from tomato in New Delhi, India, and the DNA-B genome of the India:Delhi:Cucumis:2012 isolate from cucumber in New Delhi, India, respectively. However, the genome sequence identities between mainland and Taiwan isolates (accession nos. GU180095 and GU180096) were below 93%, suggesting that mainland China isolates of ToLCNDV are more closely related to the India isolate than to the Taiwan isolate.To fulfill Koch's postulates, infectious clones of the Haimen 4 isolate were constructed and agroinfiltrated into muskmelon, oriental melon, pumpkin, luffa, and squash plants. In brief, two plasmids, containing 1.56-mer DNA-A and 1.4-mer DNA-B genome sequences, were constructed using enzyme digestion and ligation, transformed into Agrobacterium tumefaciens strain GV3101, respectively, and then co-agroinfiltrated into cucurbit plants. Initial symptoms appeared in the new leaves at 7 days post-inoculation (DPI), followed by severe leaf curling, dwarfing, stunting, reduced leaf size, and chlorotic leaf patches at 18 DPI. The presence of DNA-A and DNA-B of ToLCNDV in inoculated plants was confirmed by PCR using primer pairs A1961F/A2645R and B1613F/B2579R, respectively. Collectively, the pathogen of this emerging disease has been identified as ToLCNDV. ToLCNDV was first reported on tomato in India and is now the most predominant and economically significant disease affecting cucurbit and solanaceous crops in Southeast and East Asia, the Middle East, and the Mediterranean Basin (Moriones et al. 2017). In China, ToLCNDV was initially reported on oriental melon in Taiwan (Chang et al. 2010) and subsequently on tomato (Lycopersicon esculentum) in Zhejiang province (Li et al. 2022). To the best of our knowledge, this is the first report of ToLCNDV infecting muskmelon, pumpkin, luffa, and squash in China. Further investigations on the epidemiology of this viral disease in China are needed.

Seed and Pollen Transmission of Tomato Leaf Curl New Delhi Virus, Tomato Leaf Curl Taiwan Virus, and Tomato Yellow Leaf Curl Thailand Virus in Cucumbers and Tomatoes.

Understanding the seedborne nature of plant viruses is essential for developing disease control strategies and is impactful to the seed market. Here, we investigated seed transmissibility of tomato leaf curl New Delhi virus-cucumber isolate (ToLCNDV-CB) and -oriental melon isolate (ToLCNDV-OM) in cucumber and seed transmissibility of tomato leaf curl Taiwan virus (ToLCTV) and tomato yellow leaf curl Thailand virus (TYLCTHV) in tomato. Parent plants were inoculated using agroinfiltration with virus infectious clones, and virus infection was confirmed by PCR with virus-specific primers. ToLCNDV-CB and ToLCNDV-OM were detected in different parts of the female and male flowers and the fruits of cucumbers. ToLCNDV-CB and ToLCNDV-OM were also detected in cucumber seed coats and seedlings with an infection rate higher than 79%. Similar results were observed with ToLCTV and TYLCTHV as they were detected in different parts of the female and male flowers and fruits of three tomato cultivars. ToLCTV and TYLCTHV were also detected in tomato seed coats and seedlings with an infection rate higher than 36%. In addition, pollen-mediated transmission assays of these four begomoviruses were conducted with pollen derived from virus-infected plants to healthy plants. Results showed that ToLCNDV-CB and ToLCNDV-OM were detected in cross-pollinated cucumber progenies with an infection rate higher than 70%. ToLCTV and TYLCTHV were also detected in cross-pollinated tomato progenies with an infection rate higher than 77%. Our results indicated that ToLCNDV, ToLCTV, and TYLCTHV can be transmitted via seeds or pollens of cucumber and tomato plants. To our knowledge, this is the first report documenting the pollen-mediated transmission of begomoviruses.

Transcriptional Analysis of the Differences between ToLCNDV-India and ToLCNDV-ES Leading to Contrary Symptom Development in Cucumber.

Tomato leaf curl New Delhi virus-ES (ToLCNDV-ES), a high threat to cucurbits in the Mediterranean Basin, is listed as a different strain from the Asian ToLCNDV isolates. In this study, the infectivity of two clones previously isolated from Italy and Pakistan were compared in cucumbers, which resulted in the opposite symptom appearance. The swapping subgenome was processed; however, the mechanisms related to the disease phenotype remain unclear. To identify the disease-associated genes that could contribute to symptom development under the two ToLCNDV infections, the transcriptomes of ToLCNDV-infected and mock-inoculated cucumber plants were compared 21 days postinoculation. The number of differentially expressed genes in ToLCNDV-India-infected plants was 10 times higher than in ToLCNDV-ES-infected samples. The gene ontology (GO) and pathway enrichment were analyzed using the Cucurbits Genomics Database. The flavonoid pathway-related genes were upregulated in ToLCNDV-ES, but some were downregulated in ToLCNDV-India infection, suggesting their role in resistance to the two ToLCNDV infections. The relative expression levels of the selected candidate genes were validated by qRT-PCR under two ToLCNDV-infected conditions. Our results reveal the different infectivity of the two ToLCNDVs in cucumber and also provide primary information based on RNA-seq for further analysis related to different ToLCNDV infections.

Genomic dissection of ROS detoxifying enzyme encoding genes for their role in antioxidative defense mechanism against Tomato leaf curl New Delhi virus infection in tomato.

In the present study, genes encoding for six major classes of enzymatic antioxidants, namely superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR), Peroxidase (Prx) and glutathione S-transferase (GST) are identified in tomato. Their expression was studied in tomato cultivars contrastingly tolerant to ToLCNDV during virus infection and different hormone treatments. Significant upregulation of SlGR3, SlPrx25, SlPrx75, SlPrx95, SlGST44, and SlGST96 was observed in the tolerant cultivar during disease infection. Virus-induced gene silencing of SlGR3 in the tolerant cultivar conferred disease susceptibility to the knock-down line, and higher accumulation (~80%) of viral DNA was observed in the tolerant cultivar. Further, subcellular localization of SlGR3 showed its presence in cytoplasm, and its enzymatic activity was found to be increased (~65%) during ToLCNDV infection. Knock-down lines showed ~3- and 3.5-fold reduction in GR activity, which altogether underlines that SlGR3 is vital component of the defense mechanism against ToLCNDV infection.

Silencing AC1 of Tomato leaf curl virus using artificial microRNA confers resistance to leaf curl disease in transgenic tomato.

KEY MESSAGE: Expression of artificial microRNA targeting ATP binding domain of AC1 in transgenic tomato confers resistance to Tomato leaf curl disease without impacting the yield of tomato. Tomato curl leaf disease caused by Tomato leaf curl virus (ToLCV) is a key constraint to tomato cultivation worldwide. Engineering transgenic plants expressing artificial microRNAs (amiRNAs) against the AC1 gene of Tomato leaf curl New Delhi virus (ToLCNDV), which is important for virus replication and pathogenicity, would consequently confer virus resistance and reduce crop loss in the economically important crops. This study relates to an amiRNA developed on the sequence of Arabidopsis miRNA319a, targeting the ATP/GTP binding domain of AC1 gene of ToLCNDV. The AC1-amiR was found to regulate the abundance of AC1, providing an excellent strategy in providing defense against ToLCNDV. Transgenic lines over-expressing AC1-amiR, when challenged with ToLCNDV, showed reduced disease symptoms and high percentage resistance ranging between ∼ 40 and 80%. The yield of transgenic plants was significantly higher upon ToLCNDV infection as compared to the non-transgenic plants. Although the natural resistance resources against ToLCNDV are not available, this work streamlines a novel amiRNA-based mechanism that may have the potential to develop viral resistance strategies in tomato, apart from its normal symptom development properties as it is targeting the conserved region against which higher accumulation of small interfering RNAs (siRNA) occurred in a naturally tolerant tomato cultivar.

Identification, characterization, expression profiling, and virus-induced gene silencing of armadillo repeat-containing proteins in tomato suggest their involvement in tomato leaf curl New Delhi virus resistance.

Armadillo repeat family is well-characterized in several plant species for their involvement in multiple regulatory processes including growth, development, and stress response. We have previously shown a three-fold higher expression of ARM protein-encoding in tomato cultivar tolerant to tomato leaf curl New Delhi virus (ToLCNDV) compared to susceptible cultivar upon virus infection. This suggests the putative involvement of ARM proteins in defense response against virus infection; however, no comprehensive investigation has been performed to address this inference. In the present study, we have identified a total of 46 ARM-repeat proteins (SlARMs), and 41 U-box-containing proteins (SlPUBs) in tomato. These proteins and their corresponding genes were studied for their physicochemical properties, gene structure, domain architecture, chromosomal localization, phylogeny, and cis-regulatory elements in the upstream promoter region. Expression profiling of candidate genes in response to ToLCNDV infection in contrasting tomato cultivars showed significant upregulation of SlARM18 in the tolerant cultivar. Virus-induced gene silencing of SlARM18 in the tolerant tomato cultivar conferred susceptibility, which suggests the involvement of this gene in resistance mechanism. Further studies are underway to functionally characterize SlARM18 to delineate its precise role in defense mechanism.

Identification and comparative analysis of microRNAs from tomato varieties showing contrasting response to ToLCV infections.

Increasing incidence of viral infections in crop plants adversely affects their growth and yield. Tomato (Solanum lycopersicum) is considered to be a favorite host for viruses with over 50 species of begomoviruses naturally infecting this crop. Tomato leaf curl virus (ToLCV) is among the most widespread and devastating begomoviruses affecting tomato production. microRNAs (miRs) have been established as key regulators of gene expression and plant development. The miR pathways are disturbed during infection by viruses. Thus, comprehension of regulatory miR networks is crucial in understanding the effect of viral pathogenicity. To identify key miRs involved in ToLCV infection, a high throughput approach involving next generation sequencing was employed. Healthy and infected leaf tissues of two tomato varieties, differing in their susceptibility to ToLCV infection were analyzed. NGS data analysis followed by computational predictions, led to identification of 91 known miRs, 15 novel homologs and 53 novel miRs covering two different varieties of tomato, susceptible (Pusa Ruby) and tolerant (LA1777) to ToLCV infection. The cleaved targets of these miRs were identified using online available degradome libraries from leaf, flower and fruit of tomato and showed their involvement in various biological pathways through KEGG Orthology. With detailed comparative profiling of expression pattern of these miRs, we could associate the specific miRs with the resistant and infected genotypes. This study depicted that in depth analysis of miR expression patterns and their functions will help in identification of molecules that can be used for manipulation of gene expression to increase crop production and developing resistance against diseases.

Molecular characterization of a first begomovirus associated with lentil (Lens culinaris) from India.

A disease of lentil with symptoms of distortion, mottling and chlorosis in the leaves, shortening of internodes and excessive branching was noticed in lentil at Kanpur, India, during 2012-2014. Results of polymerase chain reaction and reverse transcriptase polymerase chain reaction employed to detect suspected RNA and DNA viruses indicated involvement of a geminivirus, which was further characterized by sequencing of full genome amplified by rolling circle amplification. Analysis of full length DNA-A revealed 96.4-96.7% nucleotide similarity with bitter gourd yellow vein virus (BGYVV) isolates and tomato leaf curl New Delhi virus (ToLCNDV) isolate. As per the recent revision of begomovirus species demarcation criteria, if a new virus isolate shares ≥91% nt sequence identity with any other isolate of an existing species, it should be treated as an isolate of that species, even if it is <91% identical to all other isolates from that species. This made BGYVV an isolate of ToLCNDV and resulted in the de-recognizing of the BGGYV. Hence, the present virus has been named as a strain of ToLCNDV and designated as Tomato leaf curl New Delhi virus-Lentil-[India:Kanpur:Lentil:2014] with the acronym as ToLCNDV-Lentil-[IN:Knp:Len:14]. This is the first report of a begomovirus found associated with a disease in lentil from India.

Identification of new stable resistant sources and assessing agro-morphological performance of sponge gourd germplasm against Tomato Leaf curl New Delhi Virus incidence.

Tomato leaf curl New Delhi virus (TolCNDV) causes yellow mosaic disease, which poses a significant biotic constraint for sponge gourd cultivation, potentially resulting in crop loss of up to 100%. In the present investigation, 50 diverse genotypes were screened for 3 years under natural epiphytotic conditions. A subset of 20 genotypes was further evaluated across four different environments. The combined analysis of variance revealed a significant genotype × environment interaction. Eight genotypes consistently exhibited high and stable resistance in the preliminary screening and multi-environment testing. Furthermore, genotype plus genotype × environment interaction biplot analysis identified DSG-29 (G-3), DSG-7 (G-2), DSG-6 (G-1), and DSGVRL-18 (G-6) as the desirable genotypes, which have stable resistance and better yield potential even under diseased conditions. The genotype by yield × trait biplot analysis and multi-trait genotype-ideotype distance index analysis further validated the potential of these genotypes for combining higher yield and other desirable traits with higher resistance levels. Additionally, resistant genotypes exhibited higher activities of defense-related enzymes as compared to susceptible genotypes. Thus, genotypes identified in our study will serve as a valuable genetic resource for carrying out future resistance breeding programs in sponge gourd against ToLCNDV.

Molecular characterization and pathogenicity of an infectious clone of tomato leaf curl New Delhi virus isolate infecting Cucumis melo.

Tomato leaf curl New Delhi virus (ToLCNDV) is a member of the genus Begomovirus, and causes devastating disease in the world. In recent years, ToLCNDV was rapidly spreading in China and induces severe economic losses in agriculture. In this study, we sequenced and characterized the complete genome of ToLCNDV isolates from melon plants showing leaf curling and stunting symptoms in Jiangsu Province of China. We constructed a full-length infectious cDNA clone of ToLCNDV, which could induce systemic infection with typical symptoms in Nicotiana benthamiana, Citrullus melo, and Citrullus lanatus plants through agrobacterium-mediated inoculation. Further experimental evidence demonstrated that the virions produced in plants infected with the infectious clone of ToLCNDV are biologically active and sap-transmissible. We also evaluated the resistance of commercial melon cultivars to ToLCNDV and found all testing melon cultivars were susceptible to ToLCNDV. Collectively, the reverse genetic system developed herein will facilitate further research on biological functions of proteins encoded by ToLCNDV and plant-ToLCNDV interactions, which might provide new insights into breeding resistance germplasm in crops.

Molecular Characterization of a Recombinant Isolate of Tomato Leaf Curl New Delhi Virus Associated with Severe Outbreaks in Zucchini Squash in Southern Italy.

The molecular characterization of a tomato leaf curl New Delhi virus (ToLCNDV) isolate, denoted ToLCNDV-Le, is reported. The virus was associated with severe and recurrent outbreaks in protected crops of zucchini squash grown in the Province of Lecce (Apulia, southern Italy). The fully sequenced genome of ToLCNDV-Le consists of two genomic components named DNA-A and DNA-B of 2738 and 2683 nt in size, respectively. Like other ToLCNDV isolates, ToLCNDV-Le DNA-A contains the AV2 and AV1 open reading frames (ORFs) in the virion-sense orientation and five additional ORFs named AC1, AC2, AC3, AC4 and AC5 in the complementary-sense orientation. The DNA-B contains BV1 ORF in the virion-sense orientation and BC1 ORF in the complementary-sense orientation. No DNA betasatellites were found associated with ToLCNDV-Le in naturally infected samples. Phylogenetic analysis clustered ToLCNDV-Le with the ToLCNDV-ES strain of western Mediterranean Basin isolates. Consequently, the ToLCNDV-ES-[IT-Zu-Le18] name is proposed as the descriptor for ToLCNDV-Le. Using recombination detection program RDP4, one putative recombination breakpoint (Rbp) was identified close to nucleotide positions 2197-2727, covering approximately half of the AC1 region, including the AC4 ORF and the 3' UTR. RDP4 indicated the event represents an Rbp of an isolate similar to ToLCNDV [Pk-06] (Acc. No. EF620534) found in Luffa acutangula in Pakistan and identified as putative minor parent into the background of ToLCNDV [BG-Jes-Svr-05] (Acc. No. AJ875157), found in tomato in Bangladesh, and identified as putative major parent. To the best of our knowledge, this is the first report of a ToLCNDV-ES recombinant isolate in the AC1-AC4 region in Italy.

Tomato leaf curl New Delhi virus: an emerging plant begomovirus threatening cucurbit production.

Tomato leaf curl New Delhi virus (ToLCNDV), a bipartite begomovirus, was first reported to infect tomato and has recently spread rapidly as an emerging disease to Cucurbitaceae crops. To date, the virus has been reported to infect more than 11 cucurbit crops, in 16 countries and regions, causing severe yield losses. In autumn 2022, ToLCNDV was first isolated from cucurbit plants in Southeastern coastal areas of China. Phylogenetic analysis established that these isolates belong to the Asian ToLCNDV clade, and shared high nucleotide identity and closest genetic relationship with the DNA-A sequence from the Chinese tomato-infecting ToLCNDV isolate (Accession no. OP356207) and the tomato New Delhi ToLCNDV-Severe isolate (Accession no. HM159454). In this review, we summarize the occurrence and distribution, host range, detection and diagnosis, control strategies, and genetic resistance of ToLCNDV in the Cucurbitaceae. We then summarize pathways that could be undertaken to improve our understanding of this emerging disease, with the objective to develop ToLCNDV-resistant cucurbit cultivars. Supplementary Information: The online version contains supplementary material available at 10.1007/s42994-023-00118-4.

Detecting Tomato Leaf Curl New Delhi Virus Causing Ridge Gourd Yellow Mosaic Disease, and Other Begomoviruses by Antibody-Based Methods.

The incidence and severity of begomovirus diseases have been increasing around the world recently, and the ridge gourd [Luffa acutangula (Roxb.) L.] is the latest example of a crop that has become highly susceptible to the outbreak of the tomato leaf curl New Delhi virus (ToLCNDV, genus Begomovirus) in India. Accurate diagnosis of causal agents is important in designing disease management strategies. In this study the coat protein (CP) gene from a ToLCNDV-Rg ridge gourd isolate was used to produce polyclonal antibodies (ToLCNDV-Rg-CP-PAb) in a rabbit. The antibodies successfully detected a 30.5 kDa ToLCNDV-Rg-CP in extracts of symptomatic ridge gourd leaf samples by several assays, such as Western Blotting (WB), Dot Immuno Binding Assay (DIBA), Direct Antigen Coating Enzyme Linked Immuno Sorbent Assay (DAC-ELISA), Immuno Capture Polymerase Chain Reaction (IC-PCR), and Immuno Capture Loop-Mediated Isothermal Amplification (IC-LAMP) assays. However, none of the negative samples tested positive in either of the detection methods. Among all the methods tested, the immunocapture assay, IC-LAMP, was the most sensitive in detecting ToLCNDV-Rg. Furthermore, antibodies generated in this study also detected other commonly occurring begomoviruses in South India, such as tomato leaf curl Palampur virus and squash leaf curl China virus in cucurbits. Together, ToLCNDV-Rg-CP-PAb can be used for detecting at least three species of begomoviruses infecting cucurbits. The obtained antibodies will contribute to monitoring disease outbreaks in multiple crops.

Tomato Leaf Curl New Delhi Virus Spain Strain Is Not Transmitted by Trialeurodes vaporariorum and Is Inefficiently Transmitted by Bemisia tabaci Mediterranean between Zucchini and the Wild Cucurbit Ecballium elaterium.

Tomato leaf curl New Delhi virus (ToLCNDV) is a bipartite begomovirus (genus Begomovirus, family Geminiviridae) persistently transmitted, as with all other begomoviruses, by whiteflies (Hemiptera: Aleyrodidae) of the Bemisia tabaci cryptic species complex. The virus, originally from the Indian subcontinent, was recently introduced in the Mediterranean basin, where it is currently a major concern for protected and open-field horticulture. The Mediterranean ToLCNDV isolates belong to a novel strain named "Spain strain" (ToLCNDV-ES), which infects zucchini and other cucurbit crops but is poorly adapted to tomato. Recently, it has been reported that another whitefly, Trialeurodes vaporariorum, is able to transmit an isolate of ToLCNDV from India which infects the chayote plant, a cucurbit. The present work aimed to clarify some aspects of whitefly transmission of ToLCNDV-ES. It was shown that T. vaporariorum is not able to transmit ToLCNDV-ES between zucchini plants. In addition, Ecballium elaterium may not act as a relevant reservoir for this virus strain in the Mediterranean basin, as B. tabaci Mediterranean (MED), the most prevalent species of the complex in the region, is not an efficient vector of this begomovirus between cultivated zucchini and wild E. elaterium plants.

Genetic Diversity and Geographic Distribution of Cucurbit-Infecting Begomoviruses in the Philippines.

Cucurbits are important economic crops worldwide. However, the cucurbit leaf curl disease (CuLCD), caused by whitefly-transmitted begomoviruses constrains their production. In Southeast Asia, three major begomoviruses, Tomato leaf curl New Delhi virus (ToLCNDV), Squash leaf curl China virus (SLCCNV) and Squash leaf curl Philippines virus (SLCuPV) are associated with CuLCD. SLCuPV and SLCCNV were identified in Luzon, the Philippines. Here, the genetic diversity and geographic distribution of CuLCD-associated begomoviruses in the Philippines were studied based on 103 begomovirus detected out of 249 cucurbit samples collected from 60 locations throughout the country in 2018 and 2019. The presence of SLCCNV and SLCuPV throughout the Philippines were confirmed by begomovirus PCR detection and viral DNA sequence analysis. SLCuPV was determined as a predominant CuLCD-associated begomovirus and grouped into two strains. Interestingly, SLCCNV was detected in pumpkin and bottle gourd without associated viral DNA-B and mixed-infected with SLCuPV. Furthermore, the pathogenicity of selected isolates of SLCCNV and SLCuPV was confirmed. The results provide virus genetic diversity associated with CuLCD for further disease management, especially in developing the disease-resistant cultivars in the Philippines as well as Southeast Asia.

Different Infectivity of Mediterranean and Southern Asian Tomato Leaf Curl New Delhi Virus Isolates in Cucurbit Crops.

Tomato leaf curl New Delhi virus (ToLCNDV) became an alerting virus in Europe from 2017 to 2020 because of its significant damage to Cucurbitaceae cultivation. Until now, just some cucurbit crops including sponge gourd, melon, pumpkin, and cucumber were reported to be resistant to ToLCNDV, but no commercial cultivars are available. In this study, a new isolate of ToLCNDV was identified in Pakistan and analyzed together with ToLCNDV-ES which was previously isolated in Italy. Furthermore, infectious clones of two ToLCNDV isolates were constructed and agroinoculated into different cucurbit crops to verify their infectivity. Results showed that both isolates exhibited severe infection on all tested cucurbit (>70%) except watermelon. Thus, those cultivars may be good candidates in the first step of screening genetic resources for resistance on both Southeast Asian and Mediterranean ToLCNDV isolates. Additional, comparison pathogenicity of different geographical ToLCNDV isolates will be aided to understand viral characterization as such knowledge could facilitate breeding resistance to this virus.

FKBP-type peptidyl-prolyl cis-trans isomerase interacts with the movement protein of tomato leaf curl New Delhi virus and impacts viral replication in Nicotiana benthamiana.

Begomoviruses belonging to the family Geminiviridae are plant-infecting DNA viruses. Begomoviral movement protein (MP) has been reported to be required for virus movement, host range determination, and symptom development. In the present study, the FK506-binding protein (FKBP)-type peptidyl-prolyl cis-trans isomerase (NbFKPPIase) of Nicotiana benthamiana was identified by a yeast two-hybrid screening system using the MP of tomato leaf curl New Delhi virus (ToLCNDV) oriental melon (OM) isolate (MPOM ) as bait. Transient silencing of the gene encoding NbFKPPIase increased replication of three test begomoviruses, and transient overexpression decreased viral replication, indicating that NbFKPPIase plays a role in defence against begomoviruses. However, infection of N. benthamiana by ToLCNDV-OM or overexpression of the gene encoding MPOM drastically reduced the expression of the gene encoding NbFKPPIase. Fluorescence resonance energy transfer analysis revealed that MPOM interacted with NbFKPPIase in the periphery of cells. Expression of the gene encoding NbFKPPIase was induced by salicylic acid but not by methyl jasmonate or ethylene. Moreover, the expression of the gene encoding NbFKPPIase was down-regulated in response to 6-benzylaminopurine and up-regulated in response to gibberellin or indole-3-acetic acid, suggesting a role of NbFKPPIase in plant development. Transcriptome analysis and comparison of N. benthamiana transient silencing and overexpression of the gene encoding MPOM led to the identification of several differentially expressed genes whose functions are probably associated with cell cycle regulation. Our results indicate that begomoviruses could suppress NbFKPPIase-mediated defence and biological functions by transcriptional inhibition and physical interaction between MP and NbFKPPIase to facilitate infection.

Functional characterization of a new ORF ßV1 encoded by radish leaf curl betasatellite.

Whitefly-transmitted begomoviruses infect and damage a wide range of food, feed, and fiber crops worldwide. Some of these viruses are associated with betasatellite molecules that are known to enhance viral pathogenesis. In this study, we investigated the function of a novel ßV1 protein encoded by radish leaf curl betasatellite (RaLCB) by overexpressing the protein using potato virus X (PVX)-based virus vector in Nicotiana benthamiana. ßV1 protein induced lesions on leaves, suggestive of hypersensitive response (HR), indicating cell death. The HR reaction induced by ßV1 protein was accompanied by an increased accumulation of reactive oxygen species (ROS), free radicals, and HR-related transcripts. Subcellular localization through confocal microscopy revealed that ßV1 protein localizes to the cellular periphery. ßV1 was also found to interact with replication enhancer protein (AC3) of helper virus in the nucleus. The current findings suggest that ßV1 functions as a protein elicitor and a pathogenicity determinant.