First natural co-occurrence of tomato leaf curl New Delhi virus DNA-A and chili leaf curl betasatellite on tomato plants (<Emphasis Type="Italic">Solanum lycopersicum</Emphasis> L.) in India

Tomato leaf curl disease (ToLCD) affected 25% of the tomato crop in Chitrakoot, India and symptomatic leaves were collected for molecular assay. The complete sequences of bipartite begomovirus DNA-A and a betasatellite DNA were amplified. In a sequence analysis, begomovirus DNA-A and betasatellite shared highest sequence identity (91–99%) with Tomato leaf curl New Delhi virus (ToLCNDV) DNA-A and chili leaf curl betasatellite (ChLCB), respectively. The virus was transmitted by whitefly to tomato plants and caused ToLCD symptoms with 70% transmission rate. To our knowledge, this is the first report of the natural occurrence of ToLCNDV and ChLCB in India.     

Role of sponge gourd in apical leaf curl disease of potato in Northern India

Potato (Solanum tuberosum) is one of the important vegetable crops in the world and its production is seriously affected by apical leaf curl disease in northern India. This paper reveals the role of cucurbits in maintaining Tomato leaf curl New Delhi virus (ToLCNDV) and Potato apical leaf curl (PALCD) disease in that region. The affected plants showed severe leaf curling and stunted growth. The begomovirus causing leaf curling and mosaic disease in cucurbits could be easily transmitted by the whitefly to potato crops and develop apical leaf curl disease in northern India. The movement of the virus by whiteflies from cucurbits to potato and tomato is possible because of overlapping of planting and harvesting dates of these crops. The causal virus was identified as a begomovirus on the basis of whitefly transmission, PCR, dot blot hybridization, cloning and sequencing of the coat protein gene. The comparison of full length coat protein gene sequence homology revealed that 90% identity with the coat protein gene of ToLCNDV- [Luffa] isolate and the phylogenetic tree derived from these sequences with other selected begomoviruses formed a close cluster with ToLCNDV isolates. The findings proved that the virus causing disease in cucurbits could easily move to tomato and potato and cause leaf curl disease naturally. This is the first observation on the role of sponge gourd for maintenance of ToLCNDV and serving as a host for PALCD in northern India. The findings indicate that the causal organism is a strain of ToLCNDV.     

Optimized loop-mediated isothermal amplification assay for <Emphasis Type="Italic">Tomato leaf curl New Delhi virus-[potato]</Emphasis> detection in potato leaves and tubers

Apical leaf curl disease of potato is caused by a whitefly transmitted begomovirus, Tomato leaf curl New Delhi virus-[potato] (ToLCNDV-[potato]) in India. Detection of this virus is essential to manage the disease, particularly in healthy potato seed production systems. Large scale testing of micro-plants demands a simple, rapid and sensitive assay. Hence, loop-mediated isothermal amplification (LAMP) method was developed for specific detection of ToLCNDV-[potato]. Six primers that recognize the coat protein gene sequence of ToLCNDV-[potato] were designed and LAMP assay was optimized using different concentrations of magnesium sulphate, betaine, dNTPs, Bst DNA polymerase and temperature. The results were assessed by visual observation of turbidity, colour change using SYBR green dye and also by gel electrophoresis. The assay successfully detected the virus in infected plants collected from potato fields whereas no cross-reactions were observed with healthy plants and other potato viruses. The optimized assay was as sensitive as PCR assay and could detect up to 0.002 pg of total DNA. The assay could detect the virus in infected potato tubers and also in asymptomatic plants. Print-capture LAMP assay was developed and its application could reduce the cost and time of the assay in large scale testing under seed production.     

Biological characterization of Tomato leaf curl New Delhi virus from Spain

Tomato leaf curl New Delhi virus (ToLCNDV; family Geminiviridae, genus Begomovirus) is an emerging virus in horticulture crops in Asia, and has recently been introduced in Spain, Tunisia and Italy. No betasatellite DNA was detected in infected tomato and zucchini squash samples from Spain, and agroinoculated viral DNA‐A and DNA‐B were sufficient to reproduce symptoms in plants of both crop species. Infected tomato and zucchini squash plants also served as inoculum sources for efficient transmission either mechanically or using Bemisia tabaci whiteflies. Cucumber, melon, watermelon, zucchini squash, tomato, eggplant and pepper, but not common bean, were readily infected using viruliferous whiteflies and expressed symptoms 8–15 days post‐inoculation. New full‐length sequences from zucchini squash and tomato indicated a high genetic homogeneity (>99% sequence identity) in the ToLCNDV populations in Spain, pointing to a single recent introduction event.     

Tomato leaf curl Guangxi virus is a distinct monopartite begomovirus species

Three begomovirus isolates were obtained from tomato plants showing leaf curl symptoms in Guangxi province of China. Typical begomovirus DNA components representing the three isolates (GX-1, GX-2 and GX-3) were cloned and their full-length sequences were determined to be 2752 nucleotides. Nucleotide identities among the three viral sequences were 98.9–99.7%, but all shared <86.7% nucleotide sequence identity with other reported begomoviruses. The sequence data indicated that GX-1, GX-2 and GX-3 are isolates of a distinct begomovirus species for which the name Tomato leaf curl Guangxi virus (ToLCGXV) is proposed. Further analysis indicated that ToLCGXV probably originated through recombination among viruses related to Ageratum yellow vein virus, Tomato leaf curl China virus and Euphorbia leaf curl virus. PCR and Southern blot analyses demonstrated that isolates GX-1 and GX-2 were associated with DNAβ components, but not isolate GX-3. Sequence comparisons revealed that GX-1 and GX-2 DNAβ components shared the highest sequence identity (86.2%) with that of Tomato yellow leaf curl China virus (TYLCCNV). An infectious construct of ToLCGXV isolate GX-1 (ToLCGXV-GX) was produced and determined to be highly infectious in Nicotiana benthamiana, N. glutinosa, tobacco cvs. Samsun and Xanthi, tomato and Petunia hybrida plants inducing leaf curl and stunting symptoms. Co-inoculation of tomato plants with ToLCGXV-GX and TYLCCNV DNAβ resulted in disease symptoms similar to that caused by ToLCGXV-GX alone or that observed in infected field tomato plants.     

Recombinase Polymerase Amplification assay for rapid detection of a geminivirus associated with potato apical leaf curl disease

Journal of Plant Diseases and Protection - Recombinase Polymerase Amplification (RPA) assay was developed for specific detection of Tomato leaf curl New Delhi virus-potato (ToLCNDV [potato]),...     

Tomato leaf curl Gujarat virus, a New Begomovirus Species Causing a Severe Leaf Curl Disease of Tomato in Varanasi, India

ABSTRACT The biological and molecular properties of Tomato leaf curl Gujarat virus from Varanasi, India (ToLCGV-[Var]) were characterized. ToLCGV-[Var] could be transmitted by grafting and through whitefly transmission in a persistent manner. The full-length genome of DNA-A and DNA-B of ToLCGV-[Var] was cloned in pUC18. Sequence analysis revealed that DNA-A (AY190290) is 2,757 bp and DNA-B (AY190291) is 2,688 bp in length. ToLCGV-[Var] could infect and cause symptoms in tomato, pepper, Nicotiana benthamiana, and N. tabacum when partial tandem dimeric constructs of DNA-A and DNA-B were co-inoculated by particle bombardment. DNA-A alone also is infectious, but symptoms were milder and took longer to develop. ToLCGV-Var virus can be transmitted through sap inoculation from infected tomato plants to the above-mentioned hosts causing the same symptoms. Open reading frames (ORFs) in both DNA-A and DNA-B are organized similarly to those in other begomoviruses. DNA-A and DNA-B share a common region of 155 bp with only 60% sequence identity. DNA-B of ToLCGV-[Var] shares overall 80% identity with DNA-B of Tomato leaf curl New Delhi virus-Severe (ToLCNDV-Svr) and 75% with ToLCNDV-[Lucknow] (ToLCNDV-[Luc]). Comparison of DNA-A sequence with different begomoviruses indicates that ToLCGV-[Var] shares 84% identity with Tomato leaf curl Karnataka virus (ToLCKV) and 66% with ToLCNDV-Svr. ToLCGV-[Var] shares a maximum of 98% identity with another isolate of the same region (ToLCGV-[Mir]; AF449999) and 97% identity with one isolate from Gujarat (ToLCGV-[Vad]; AF413671). All three viruses belong to the same species that is distinct from all the other geminivirus species described so far in the genus Begomovirus of the family Geminiviridae. The name Tomato leaf curl Gujarat virus is proposed because the first sequence was taken from an isolate of Gujarat, India.     

Tomato dwarf leaf curl virus, a new bipartite geminivirus associated with tomatoes and peppers in Jamaica and mixed infection with tomato yellow leaf curl virus

Genomic characterization using nonradioactive probes, polymerase chain reaction with degenerate primers for whitefly transmitted geminiviruses and nucleotide sequencing were used to describe a new bipartite geminivirus, associated with dwarfing and leaf curling of tomatoes and peppers in Jamaica. Partial DNA-A and DNA-B clones were obtained. DNA sequence analysis showed that tomato and pepper samples have a similar geminivirus associated with them. Nucleotide sequence identity > 92% between the common regions of DNA-A and DNA-B confirmed the bipartite nature of the Jamaican geminivirus isolates. Nucleotide sequence comparisons of DNA-A and DNA-B with those of geminiviruses representing the major phylogenetic groups of Western Hemisphere geminiviruses showed the greatest similarity to potato yellow mosaic virus and members of the Abutilon mosaic virus cluster of geminiviruses. This new virus is given the name tomato dwarf leaf curl virus (TDLCV) because of the dwarfing and leaf curling symptoms associated with infected tomato plants. Polymerase chain reaction and Southern hybridization showed mixed infections of TDLCV with tomato yellow leaf curl virus from Israel in 16% of the field samples of tomatoes and peppers.     

南疆温室番茄黄化曲叶病病毒种类的分子鉴定

为明确南疆温室番茄黄化曲叶病的病毒种类,利用双生病毒的兼并引物通过PCR扩增,对采集的20个番茄病株进行了分子检测.从20个病株中均扩增到约500 bp的目标片段,对其中4株进行克隆和测序,其相互间序列同源性为97.1％ ～99.3％,与番茄黄化曲叶病毒(Tomato yellow leaf curl virus,TYLCV)的同源性较高,为98.6％ ～ 99.5％.随机选取莎车分离物KS2-5进行全基因组的克隆和测序,KS2-5 DNA全长为2781 nt(序列号:JQ807735),具有典型的双生病毒基因组特征,与TYLCV其它分离物同源性达到98.9％～99.5％,而与其它粉虱传双生病毒的序列同源性较低,为68.3％ ～75.5％,表明危害南疆温室番茄的病毒种类为番茄黄化曲叶病毒TYLCV.     

广东省番茄黄化曲叶病毒的分子鉴定及序列分析

番茄黄化曲叶病毒病是世界番茄生产上一种毁灭性病害,番茄黄化曲叶病毒Tomato yellow leaf curl virus(TYLCV)是引起该病害的主要病原病毒之一。本文采用滚环扩增及基因克隆方法,获得了侵染广东佛山和肇庆番茄的TYLCV 4个分离物全基因组;它们均为2 781 nt,编码6个ORF,其中病毒链上编码AV1和AV2,互补链上编码AC1、AC2、AC3和AC4。同源性比较结果表明,4个广东分离物基因组序列两两间同源性为99%以上;与已报道的TYLCV各分离物同源性在90%以上,而与来自中国不同地区的TYLCV分离物的同源率均在98%以上。系统进化分析显示,广东分离物与来自中国不同地区的TYLCV分离物亲缘关系较近,并聚类在一个分支。因此,侵染引起广东佛山和肇庆番茄黄化曲叶病的病毒应来自国内其他地区。本研究是对TYLCV广东分离物分子特征的首次报道。     

Characterization of Egyptian <Emphasis Type="Italic">Phytophthora infestans</Emphasis> population using simple sequence repeat markers

The plant pathogenic oomycete, Phytophthora infestans, is the causal agent of late blight disease in tomato and potato. For characterizing Egyptian P. infestans isolates by DNA marker analysis, 40 isolates of P. infestans were collected from different locations in Egypt during two growing seasons (2012/2013 and 2013/2014). The 40 isolates were grouped into seven genotypes, in which 24 alleles were detected. The identified genotypes were not completely associated with geographic location and sample collection years. These results provide genetic and geographical information for developing a program to manage late blight disease.     

Races of <Emphasis Type="Italic">Phytophthora infestans</Emphasis> isolated from potato in Hokkaido,Japan

The virulence of 29 isolates of Phytophthora infestans collected in potato fields in Hokkaido, Japan, in 2013 and 2014, was tested for race identification. Thirteen different races were identified, each of which had five to eight virulence factors. All of the isolates caused a virulent reaction against plants with R1 and R7, and most of the isolates caused a virulent reaction against plants with R3, R4, R10, and R11. On the other hand, no isolate was virulent against plants with R9. These results demonstrate that the current Japanese P. infestans population is more complex than the population in the 1990s from the viewpoint of race.     

Genotypic and phenotypic variability of <Emphasis Type="Italic">Pectobacterium</Emphasis> strains causing blackleg and soft rot on potato in Turkey

Pectinolytic bacteria were isolated from 48 potato plants showing the symptoms of blackleg and collected in different fields of commercial potato production areas at Samsun, Amasya, Corum and Yozgat provinces in Turkey in 2015. The survey resulted in the isolation of 26 pectinolytic strains that belonged to P. atrosepticum, P. carotovorum subsp. brasiliense, P. carotovorum subsp. carotovorum and P. parmentieri species based on molecular identification with species-specific PCR and phenotypic characterization. The identified strains indicated typical biochemical characteristics of the assigned species. For 16 representative Pectobacterium isolates 10 unique rep-PCR band patterns were obtained. The 16S rRNA and recA and gapA gene fragment sequencing confirmed the species identity of the isolates. The phenotypic characterization of the strains revealed that for all assays but one (cellulase, protease activity, swimming but not swarming), the tested Pectobacterium species were significantly different from each other proving the diversity of the strains belonging to these genera. Recent outbreaks of blackleg and/or soft rot in potato production areas in Turkey may pose a threat on other crops, as tomato, pepper, cucumber, onion, cabbage, broccoli and sugar beet are cultivated in the same provinces.     

Assessing the Belgian potato <Emphasis Type="Italic">Alternaria</Emphasis> population for sensitivity to fungicides with diverse modes of action

Early blight and brown spot, caused by respectively Alternaria solani and Alternaria alternata, can lead to severe yield losses in potato-growing areas. To date, fungicide application is the most effective measure to control the disease. However, in recent years, a reduced sensitivity towards several active ingredients has been reported. To shed light on this issue, Alternaria isolates were collected from different potato fields in Belgium during two growing seasons. Subsequently, the sensitivity of these isolates was assessed using four widely used fungicides with different modes of action. Demethylation inhibitors, quinone outside inhibitors, a dithiocarbamate and a carboxylic acid amide were included in this study. Although all fungicides reduced spore germination and vegetative growth of Alternaria species to some extent, the interspecies sensitivity was very variable. In general, A. solani was more suppressed by the fungicides compared to A. alternata. The effectiveness of the dithiocarbamate mancozeb was high, whereas the quinone outside inhibitor azoxystrobin showed a limited activity, especially towards A. alternata. Therefore, a subset of the A. alternata and A. solani isolates was tested for the presence of, respectively, the G143A substitution and the F129L substitution in the cytochrome b. The frequency of A. alternata isolates bearing the resistant G143A allele (approximately 65%) was comparable in both sampling years, although sensitivity of isolates decreased during the growing season. This finding points to a shift of the population towards resistant isolates. Both the European genotype I and American genotype II were present in the A. solani population, with genotype I being the most prevalent. None of the genotype I isolates carried the F129L substitution, whereas in 83% of the genotype II isolates this substitution was present. Our results demonstrate for the first time that the Belgian Alternaria population on potato comprises a considerable broad spectrum of isolates with different sensitivity to fungicides.     

Sequence variation in ribosomal DNA and in the nuclear <Emphasis Type="Italic">hsp</Emphasis>90 gene of <Emphasis Type="Italic">Pratylenchus penetrans</Emphasis> (Nematoda: Pratylenchidae) populations and phylogenetic analysis

Tomato bacterial canker and wilt disease caused by Clavibacter michiganensis subsp. michiganensis (Cmm) is among one of the major bacterial diseases associated with tomato (Solanum lycopersicum L.) in the western Mediterranean region of Turkey. A total of 118 Cmm isolates were obtained from the petiole and the main vein of leaves of different cultivars of diseased tomato plants, and these isolates were cultured in semiselective medium (mSCM). The identity of Cmm isolates was confirmed through gas chromatography-fatty acid methyl-esters (GC-FAME) analysis and polymerase chain reaction (PCR) using the primers, CMM5 and CMM6. The fatty acid analysis of all the Turkish isolates yielded major components that included anteisoheptadeconic acid (a15:0), palmitic acid (i16:0) and anteisoheptadeconic acid (a17:0); the analysis detected and categorized all the isolates into 10 different FAME groups. Among repetitive element sequence PCR (rep-PCR) analysis, Box primer yielded the most reproducible genomic profiles with band sizes that ranged from ~200 bp to 2 kb. The isolates were also separated into 12 groups by pulsed-field gel electrophoresis (PFGE) after digesting the total genomic DNA with SpeI, a rare cutting enzyme. The genome sizes of the different strains of Cmm were also determined after running unrestricted total genomic DNA, which yielded average values between 3.0 and 3.5 MB. All the Cmm isolates had pCM1 and pCM2 plasmids. This is the first report on the detailed characterization of the Cmm population in Turkey.     

Association of Pedilanthus leaf curl virus with yellow mottling and leaf curl symptoms in two jasmine species grown in India

Association of Pedilanthus leaf curl virus (PeLCV) with yellow mottling of crape jasmine (Tabernaemontana coronaria) and leaf curl of night blooming jasmine (Cestrum nocturnum) was investigated using a sequence analysis of the cloned full-length genome amplified by Rolling circle amplification using Φ-29 DNA polymerase. In the sequence analysis, virus isolates from T. coronaria (JN807764) and C. nocturnum (JQ012790) shared 95 % sequence identity with each other and the highest identity (93 %) and the closest phylogenetic relationships with PeLCV (AM712436); therefore, the virus isolates were identified as PeLCV strains. This report from India is the first on the occurrence of PeLCV on T. coronaria and C. nocturnum anywhere in the world.     

Nucleotide sequencing, whitefly transmission, and screening tomato for resistance against two newly described begomoviruses in bangladesh

ABSTRACT The molecular diversity of Tomato leaf curl viruses (ToLCVs), from the two main tomato growing areas of Jessore and Joydebpur, Bangladesh, was investigated. The viral DNA was amplified from tomato plants exhibiting mild and severe symptoms by polymerase chain reaction, and the complete genomes of the ToLCVs were sequenced. An isolate of the bipartite Tomato leaf curl New Delhi virus-Severe (ToLCNDV-Svr) was associated with the severe symptom phenotype from Jessore (ToLCNDV-Svr[Jes]). A previously undescribed monopartite virus, designated Tomato leaf curl Joydebpur virus-Mild (ToLCJV-Mld), was sequenced from plants showing mild symptoms. ToLCNDV-Svr[Jes] was most closely related to ToLCNDV-[Lucknow] at 95.7% nucleotide (nt) identity and Tomato leaf curl Gujarat virus-[Varanasi] at 90.6% nt identity, based on DNA-A and -B component sequences. ToLCJV-Mld was similar to Pepper leaf curl Bangladesh virus at 87.1% DNA-A nt identity. Identification of ToLCNDV-Svr[Jes] and ToLCJV-Mld was in addition to the previously described Tomato leaf curl Bangladesh virus, with which they shared 73.2 and 86.0% DNA-A nt identities, thus demonstrating the existence of at least three distinct viruses infecting tomato in Bangladesh. Nucleotide identities and placement in phylogenetic trees suggested that the three ToLCVs may have had different evolutionary pathways. The whitefly, Bemisia tabaci, transmitted the viruses of this study equally efficiently. Four tomato cultivars (TLB111, TLB130, TLB133, and TLB182) resistant/ tolerant to South Indian ToLCV were screened against the Bangladesh ToLCVs in 2003-04. Although challenged by diverse viruses and potentially mixed infections, disease incidence remained low (6 to 45%) in the resistant cultivars compared with local cultivars (68 to 100%).     

Morphological and molecular variability among Indian isolates of <Emphasis Type="Italic">Rhizoctonia solani</Emphasis> causing banded leaf and sheath blight in maize

Rhizoctonia solani, a devastating soil borne fungus inciting banded leaf and sheath blight (BLSB) disease is a constraint in maize production and improvement program. Rhizoctonia isolates collected from seven diverse maize cropping zones of India were examined for morphological and molecular variability. All the tested isolates caused symptoms of BLSB on maize and were also cross infective on rice and sugarcane hosts, but showed significant variability in hyphal diameter, mean hyphal cell size, weight, size and distribution of scleorotia, culture pigmentation, incubation period, pathogenicity and expression of symptoms. Neighbour joining cluster analysis placed the 62 isolates of R. solani into four major groups, A, B, C and D. Group A was more diverse and included isolates of diverse agro-ecological zones. The cluster analysis corresponded well with principle component analysis. Pathogenicity testing of R. solani isolates on maize genotype (CM 501) revealed highly variable virulence pattern of the pathogen population suggesting its high evolutionary potential, and hence adaptability to diverse geographical regions. The study reveals a strong evidence of inherent potential of the R. solani isolates to survive in diverse ecological zones and its probable spread to other maize cultivars across India. Sequence comparisons of the internal transcribed sequence-ribosomal DNA region of 62 isolates did not reveal much diversity among the isolates. Majority of the isolates (n?=?61) clustered together with anastomosis group (AG) AG1-IA used as reference strain in the phylogram, distinct from AG1-IB, AG2–2IIIB and Waitea circinata used as reference strains. BLSB isolates representing distinct geographical locations shared identical sequences indicating long-distance dispersal of the pathogen. The study confirms that the genetic flexibility of the pathogen allows for its adaptation to variable ecological niches and long-distance introduction of new genotypes into the region. The study emphasizes that epidemiological studies may complement the molecular studies.     

Association of Tomato leaf curl Joydebpur virus and a betasatellite with leaf curl disease of eggplant

Leaf samples (five) from brinjal/eggplant fields showing upward leaf curling symptoms were collected from Varanasi, Uttar Pradesh state, India. The full length genome of begomovirus and associated betasatellite were amplified by PCR, cloned and sequenced. Sequences of homologous DNA-A and its betasatellite in all samples were the same. The samples failed to amplify DNA-B, suggesting that the begomovirus associated with leaf curl disease of eggplant was monopartite. The complete genome (homologous of DNA-A) consists of 2758 nts, whereas the betasatellite has 1352 nts and the genome organization is typical of Old World begomoviruses. The sequence analysis showed high levels of nucleotide sequence identity (79.8–91.7%) of virus with Tomato leaf curl Joydebpur virus (ToLCJoV) infecting chilli in India, suggesting it as a strain of ToLCJoV based on the current ICTV taxonomic criteria for begomovirus strain demarcation. However, the betasatellite associated was identified as a variant of Tomato leaf curl Bangladesh betasatellite (ToLCBDB), with which it shared highest sequence identity of 84.7–94.8%. Phylogenetic analyses of the genome further supported the above results. The recombination analyses of both genome and betasatellite showed that a major part of genome sequences are derived from begomoviruses (ToLCJoV, ChiLCuV, AEV) infecting chilli, tomato, ageratum and betasatellite from PaLCuB as the foremost parents in evolution, suggesting this as a new recombinant virus strain. This is the first report of a monopartite begomovirus and a betasatellite molecule associated with the leaf curl disease of eggplant.     

警惕新德里番茄曲叶病毒在我国的传播和危害

新德里番茄曲叶病毒(tomato leaf curl New Delhi virus, ToLCNDV)是双生病毒科菜豆金色花叶病毒属病毒,由烟粉虱Bemisia tabaci以循环持久性方式传播,能对茄科和葫芦科等多种作物造成毁灭性的危害。近年来,ToLCNDV不断扩散蔓延至新的国家和地区。我国于2021年在浙江的温室番茄上发现了ToLCNDV的危害。本文综述了ToLCNDV的发生分布、基因组结构与进化、传播方式和寄主范围,提出了防范ToLCNDV在我国进一步传播和危害的防控建议。