Evidence from nucleic acid hybridization tests for geminivirus infection of ornamental crotons in India

Reduced growth of plants and excessive generalised and spotty yellowing of leaves occurred in ornamental crotons that were infected with a geminivirus, which was detected by spot hybridization tests using a cloned probe derived from DNA-A of acalypha yellow mosaic geminivirus. The croton virus was transmitted by the whitefly,Bemisia tabaci, toNicotiana tabacum andAcalypha indica, in which it caused leaf curl and yellow mosaic, respectively, but it was not transmitted to five other species. Infected crotons may therefore serve as reservoirs of a geminivirus that is able to infect a crop species.     

Tomato leaf curl Karnataka virus from Bangalore, India, Appears to be a Recombinant Begomovirus

ABSTRACT The genome of Tomato leaf curl virus (ToLCV) from Bangalore, India, a whitefly-transmitted geminivirus, was cloned (pIND9) and sequenced. The circular DNA of 2,759 nucleotides (U38239) is organized similarly to that of other begomoviruses with monopartite genomes. Comparison of the nucleotide sequence of pIND9 with other tomato-associated begomoviruses from India (Tomato leaf curl Bangalore virus [ToLCBV, Z48182]) and Tomato leaf curl New Delhi virus-Severe (ToLCNdV-Svr, U15015) showed moderate DNA sequence identities (82 to 87%) between capsid protein (CP) genes but low identities (66 to 67%) for the intergenic regions and the replication-associated protein (Rep) genes (75 to 81% identity). Phylogenetic trees generated with nucleotide sequences of the Rep and CP genes of 26 begomoviruses indicated that this ToLCV is distinct from other begomoviruses and that it may be a recombinant virus derived from at least three different viral lineages. Tomatoes (Lycopersicon esculentum) inoculated with the cloned DNA monomer of ToLCV (pIND9) via particle bombardment developed leaf curling and yellowing symptoms. The virus was transmitted by Bemisia tabaci biotype B from tomatoes infected via particle bombardment to healthy tomatoes and by sap inoculation from infected tomatoes to tomato, Nicotiana benthamiana and N. tabacum. This ToLCV is a distinct member of the genus Begomovirus from India that differs from the previously characterized Tomato leaf curl Sadasivanagar virus isolate Bangalore 1 (L12739), ToLCBV (Z48182), ToLCBV isolate Bangalore 4 (AF165098), and the bipartite ToLCNdV (U15015, U15016). Thus, this ToLCV is named Tomato leaf curl Karnataka virus (ToLCKV).     

First report of a geminivirus causing leaf curl disease on Trigonella corniculata evidenced by PCR using degenerate primers

A leaf curl and mottle disease was observed naturally affecting plants of Trigonella corniculata. A geminivirus was detected in infected plants by PCR using degenerate geminivirus primers and by Southern hybridization with a probe specific to tomato leaf curl begomovirus. This is the first report of a member of the Geminiviridae naturally occurring in T. corniculata.     

Seed transmission of Sweet potato leaf curl virus in sweet potato (Ipomoea batatas)

Sweet potato leaf curl virus (SPLCV) infects sweet potato and is a member of the family Geminiviridae (genus Begomovirus). SPLCV transmission occurs from plant to plant mostly via vegetative propagation as well as by the insect vector Bemisia tabaci. When sweet potato seeds were planted and cultivated in a whitefly‐free greenhouse, some sweet potato plants started to show SPLCV‐specific symptoms. SPLCV was detected by PCR from all leaves and floral tissues that showed leaf curl disease symptoms. More than 70% of the seeds harvested from SPLCV‐infected sweet potato plants tested positive for SPLCV. SPLCV was also identified from dissected endosperm and embryos. The transmission level of SPLCV from seeds to seedlings was up to 15%. Southern blot hybridization showed SPLCV‐specific single‐ and double‐stranded DNAs in seedlings germinated from SPLCV‐infected seeds. Taken altogether, the results show that SPLCV in plants of the tested sweet potato cultivars can be transmitted via seeds and SPLCV DNA can replicate in developing seedlings. This is the first seed transmission report of SPLCV in sweet potato plants and also, to the authors' knowledge, the first report of seed transmission for any geminivirus.     

湖南省番茄和牵牛花上双生病毒的分子鉴定

双生病毒是一类在全世界范围内广泛发生的单链环状DNA病毒。本文对从湖南采集到的6例(洋姜、番茄、萝卜、赛葵、甘薯、牵牛花)疑似双生病毒侵染的植物叶片进行了分子鉴定。利用滚环扩增技术(RCA)对样品DNA进行扩增,分别对其RCA产物进行酶切,并将酶切得到的片段测序后进行BLAST比对,结果显示番茄样品中的病毒分离物与番茄黄化曲叶病毒相似性最高(99%),牵牛花样品中的病毒分离物与甘薯卷叶病毒相似性最高(99%),证明这两个分离物是单组分DNA-A双生病毒。这是在湖南省首次发现并报道双生病毒的全核酸序列。     

侵染一品红的一品红曲叶病毒山东分离物的全基因组克隆和序列分析

2012年,在山东青州花卉市场采集一品红样品,利用双生病毒通用引物PA/PB对样品进行扩增并测序。所得序列经NCBI BLAST比对,发现是一种新的双生病毒,为一品红曲叶病毒（Euphorbia leaf curl virus, ELCV）。随后对该分离物（ELCV-Shandong）进行全基因组序列扩增和测序（GenBank登录号：KC852148）,经分子比对和进化分析,发现其与广西报道的一品红曲叶病毒G35 (AJ558121) 的相似性最高为99%,同处于一个小分支,并且中国报道的所有一品红曲叶病毒分离物都处于一个分支内,从而进一步确定了山东青州一品红病毒分离物为一品红曲叶病毒。这是一品红曲叶病毒在北方地区发生的首次报道。     

Spread of Tomato yellow leaf curl virus in Sicily: Partial Displacement of Another Geminivirus Originally Present

The geminivirus Tomato yellow leaf curl virus (TYLCV) was reported for the first time in Italy in 2002. We have followed its spread in Sicily, where Tomato yellow leaf curl Sardinia virus (TYLCSV), another tomato-infecting geminivirus, is endemic and has been causing severe crop losses since 1989. The presence of the two viruses was monitored in the main tomato growing area, the Ragusa province, analyzing samples with yellow leaf curling symptoms. At first (spring–summer 2002) both viruses were always found in mixed infections, but in 2003 and 2004 18–35% of plants were found infected by TYLCV alone and 8–28% by TYLCSV alone, with 41–69% carrying both viruses. TYLCV has spread quickly in the area, demonstrating, as in other parts of the world, its high virulence and invasiveness; however it has not, so far, completely displaced TYLCSV. An infectious clone of TYLCV from Sicily (TYLCV-IT) was sequenced. The nucleotide sequence was 97% identical to other TYLCV strains of the ‘severe’ type, found in many countries worldwide.     

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.     

Possible recombination of tomato-infecting begomoviruses in Thailand

Nucleotide sequences of the three distinct Tomato yellow leaf curl Thailand virus (TYLCTHV) strains (CM, NK, SK) were analyzed for recombination events. Recombination detection program analyses and a sequence alignment survey provided evidence of recombination between AC1 sequences of TYLCV, TYLCTHV-[MM], and TYLCTHV-[NK] as major parents and of ToLCLV, ToLCTWV, and TYLCTHV-[SK] as minor parents of TYLCTHV-[NK], -[SK], -[CM], respectively. The results further support the notion that interspecies recombination may play a significant role in geminivirus diversity and their emergence as important pathogens.     

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.     

Incidence of root rot of muskmelon in different soil management practices

The objective of this study was to estimate the effects of tillage systems and cover crops on the incidence of root rot in melon and to identify the fungal pathogens associated with the disease. Two consecutive trials were carried out in a randomized complete block design with four replications in each trial. The treatments were arranged in split-plots. Two tillage systems (no-tillage (NT) and conventional tillage (CT)) were assigned in the main plots and in the subplot the six types of ground cover crops were tested (sunn hemp, pearl millet, sunn hemp + pearl millet, corn + brachiaria, spontaneous vegetation, spontaneous vegetation + polyethylene film) or bare soil. At the end of the trials all melon plants were collected and assessed for disease incidence, isolations from symptomatic plants were made for fungal identification. Root rot incidence was lower in the NT treatments with sunn hemp, pearl millet, and spontaneous vegetation. The main fungi isolated from symptomatic roots were Fusarium solani, Macrophomina phaseolina, Monosporascus cannonballus and Rhizoctonia solani, but F. solani was the most frequently isolated fungus in both tillage systems. The results suggest that the NT system has the potential to control incidence of root rot of muskmelon, but is necessary to realize crop rotation between the planting cycles.     

Differentiation of three whitefly-transmitted geminiviruses from the Republic of Yemen

Three viruses collected in southern Yemen in 1990, infecting watermelon, tobacco and tomato were shown to be transmitted by the whiteflyBemisia tabaci and to have particle morphologies typical of geminiviruses. Colonies ofB. tabaci collected from different locations and from different hosts were used in virus transmission tests with the same host range of plants. Colonies established from both watermelon and cotton in the Yemen were identified as the squash silverleaf-inducing B biotype. The culture host of the colony did not influence virus acquisition and transmission efficiencies to and from other hosts. The tobacco and tomato geminiviruses had a similar host range, but differed in their severity in some hosts. Both these viruses differed from the watermelon geminivirus in host range and symptoms.Datura stramonium, an alternative host for all three viruses, could be co-infected by the watermelon and tobacco viruses.B. tabaci was able to acquire both viruses from the co-infectedD. stramonium and infect seedlings of either original host plant species with their respective viruses orD. stramonium with both. The viruses were identified as watermelon chlorotic stunt virus, tobacco leaf curl virus and tomato yellow leaf curl virus and were distinguished by cross hybridisation.     

北京地区番茄黄化曲叶病毒病的鉴定及防治对策

番茄黄化曲叶病毒病是一种由烟粉虱传播的病毒病,给番茄生产造成严重威胁。2009年在北京郊区调查时发现部分保护地种植的番茄植株表现典型黄化曲叶症状。通过提取典型症状样品总DNA利用粉虱传双生病毒检测简并引物PA/PB,进行PCR扩增到541bp的特异条带。通过测序和核苷酸序列比对表明该序列与番茄黄化曲叶病毒序列相似性最高为99%。分子检测结果表明北京郊区部分保护地种植的番茄已被烟粉虱传播的番茄黄化曲叶病毒侵染危害。     

Typing of Tomato Yellow Leaf Curl Viruses in Europe

Tomato yellow leaf curl disease is spreading in southern Europe, where it has quickly become a serious problem. In recent years, several virus isolates have been characterised. Although with some genetic variability, all isolates found in Europe belong to one of two species Tomato yellow leaf curl-Sardinia (TYLCV-Sar) or Tomato yellow leaf curl-Israel (TYLCV-Is). Several methods were tested to identify and type TYLCV isolates from field samples: (1) RFLP of a DNA fragment amplified from the coat protein gene; (2) PAGE of a fragment amplified from the C2 gene; (3) dot-blot hybridisation. All methods enabled the detection of the TYLCVs and provided good indications for attributing them to one species or the other. However, for typing purposes, the RFLP method was the most reliable, due to the easily recognisable pattern produced by the two virus species present in Europe. Dot-blot hybridisation is less expensive for identifying TYLCVs in large numbers of samples, particularly when a mixture of two probes is used. PAGE of the C2 fragment is the fastest of the methods tested.     

Biomolecular relationships among isolates of<Emphasis Type="Italic">Tomato Yellow Leaf Curl Tanzania Virus</Emphasis>

An investigation of the biological properties of the virus causing tomato yellow leaf curl disease in Tanzania was initiated to compare it with other known tomato yellow leaf curl viruses. Properties relating to acquisition and inoculation feeding time, persistence, mechanical inoculation, seed transmission and host range were studied. Results obtained indicate that the virus was transmitted persistently byBemisia tabaci Genn., but it was not mechanically, sap- or seed-transmissible. Minimum acquisition and inoculation feeding time was 30 min.Capsicum annuum, Datura stramonium, Nicotiana glutinosa, N. tabacum andLycopersicon esculentum were found to be hosts of the virus among the plant species tested, whereasPhaseolus vulgaris was not. It is concluded that the properties of the agent causing yellow leaf curl symptoms in tomato plants from different regions in Tanzania are similar to those ofTomato yellow leaf curl Sardinia virus species studied elsewhere. http://www.phytoparasitica.org posting Feb. 20, 2003.     

Characterisation of a Quantitative Resistance to Vector Transmission of Tomato yellow leaf curl virus in Lycopersicon pimpinellifolium

Two wild genotypes from the same species Lycopersicon pimpinellifolium, WVA106 (susceptible) and INRA-Hirsute (so-called ‘resistant’), were compared with respect to their reaction to Tomato yellow leaf curl virus isolate Réunion (TYLCV-Mld[RE]), using both whitefly-mediated inoculation and graft inoculation. Disease incidence and symptom severity were scored. Presence and quantification of viral DNA were assessed by dot blot hybridisation. Upon insect inoculation, accession INRA-Hirsute showed a moderate resistance against TYLCV that was overcome by a high inoculation pressure obtained by increasing the cumulative number of inoculative whiteflies. Temporal analyses of the disease progress in relation to this criterion exhibited that the protection was quantitative, mainly reducing the TYLCV-Mld[RE] incidence by at maximum 50% at low inoculation pressure. When graft inoculated, the final TYLCV-Mld[RE] disease incidence was 100% in both susceptible and resistant genotypes with severe symptoms, suggesting a reduction of virus transmission by a vector resistance as a possible mechanism. Implications of using such type of resistance in breeding programmes are discussed.     

Mercurialis ambigua and Solanum luteum: Two Newly Discovered Natural Hosts of Tomato Yellow Leaf Curl Geminiviruses

The yellow leaf curl disease of tomato is caused by a complex of virus species, two of which, tomato yellow leaf curl virus (TYLCV)-Sar and TYLCV-Is, are involved in epidemics of southern Spain. Plants of Mercurialis ambigua and Solanum luteum showing abnormal upward leaf curling and leaf distortion collected in the vicinity of tomato crops were found to be naturally infected with TYLCV-Is and TYLCV-Sar, respectively. These weed species, as well as Datura stramonium and S. nigrum, which had also been found to be naturally infected by TYLCVs in the same region in previous studies, were tested for susceptibility to TYLCV-Sar or TYLCV-Is by Agrobacterium tumefaciens-mediated and by Bemisia tabaci inoculation. Results indicated that both TYLCV-Sar and TYLCV-Is were able to infect D. stramonium and M. ambigua, whereas only TYLCV-Sar infected S. nigrum and S. luteum. Implications for the epidemiology of TYLCV are discussed. This is the first report of M.ambigua and S. luteum as hosts of TYLCV.     

Characterization of a New Begomovirus from Egypt Infecting Hollyhock (Althea Rosea)

A viral isolate from Egypt associated with symptoms of enations and leaf curling on hollyhock (Althea rosea) was characterized at the cytopathological and molecular levels. Microscopic observations showed that enations resulted from a reorganization of the vascular tissues, including activation of a cambial activity in the phloem, the development of a palisade parenchyma in place of a spongy one and the differentiation of minor vascular tissues. From this isolate, the full-length DNA-A of a begomovirus (family Geminiviridae) was cloned and sequenced. This genome exhibited a genetic organization similar to that of other old-world begomoviruses like Tomato yellow leaf curl virus from Israel and Ageratum yellow vein virus from Singapore. However, its sequence was significantly distinct (similarity < 69%) from any other geminivirus. This begomovirus was thus considered as representative of a new viral species named Althea rosea enation virus (AREV). AREV was agroinfectious on Nicotiana benthamiana, on which it induced a severe leaf-curling and vein distortion, but could not re-establish infection on A. rosea. To determine if AREV was also associated with a similar disease affecting okra in Upper-Egypt, the partial sequence of the coat protein gene of an isolate was determined. It exhibited 90% nt identity with the hollyhock isolate (97% amino acid), suggesting a genetic heterogeneity in the begomovirus population associated with the enation diseases.     

Occurrence of a new recombinant begomovirus species infecting tomato in the Al‐Batinah region of Oman

Whitefly‐transmitted begomoviruses are the most important limiting factor for tomato cultivation in Oman, particularly in the Al‐Batinah region, the major agricultural area of the country. Commercial farms in the Al‐Batinah region were surveyed during January–March 2013. Samples of tomato showing leaf curl disease symptoms typical of begomoviruses were collected and analysed. Full‐length sequences of five clones were shown to have relatively low percentage identity values to known begomoviruses, with the highest (88·6%) to isolates of Tomato leaf curl Oman virus (ToLCOMV), a begomovirus previously reported in Oman, indicating that these represent a newly identified species, for which the name Tomato leaf curl Barka virus (ToLCBrV) is proposed. Four isolates of ToLCBrV were found associated with Tomato leaf curl betasatellite (ToLCB). The five isolates of ToLCBrV characterized in this study were shown to be recombinants, with ToLCOMV as the major parent, and a fragment of Croton yellow vein virus (CrYVV) spanning the 3′ half of the replication‐associated protein. The significance of these findings is discussed.