Sweepoviruses cause disease in sweet potato and related Ipomoea spp.: fulfilling Koch's postulates for a divergent group in the genus begomovirus

Sweet potato (Ipomoea batatas) and related Ipomoea species are frequently infected by monopartite begomoviruses (genus Begomovirus, family Geminiviridae), known as sweepoviruses. Unlike other geminiviruses, the genomes of sweepoviruses have been recalcitrant to rendering infectious clones to date. Thus, Koch's postulates have not been fullfilled for any of the viruses in this group. Three novel species of sweepoviruses have recently been described in Spain: Sweet potato leaf curl Lanzarote virus (SPLCLaV), Sweet potato leaf curl Spain virus (SPLCSV) and Sweet potato leaf curl Canary virus (SPLCCaV). Here we describe the generation of the first infectious clone of an isolate (ES:MAL:BG30:06) of SPLCLaV. The clone consisted of a complete tandem dimeric viral genome in a binary vector. Successful infection by agroinoculation of several species of Ipomoea (including sweet potato) and Nicotiana benthamiana was confirmed by PCR, dot blot and Southern blot hybridization. Symptoms observed in infected plants consisted of leaf curl, yellowing, growth reduction and vein yellowing. Two varieties of sweet potato, 'Beauregard' and 'Promesa', were infected by agroinoculation, and symptoms of leaf curl and interveinal loss of purple colouration were observed, respectively. The virus present in agroinfected plants was readily transmitted by the whitefly Bemisia tabaci to I. setosa plants. The progeny virus population present in agroinfected I. setosa and sweet potato plants was isolated and identity to the original isolate was confirmed by sequencing. Therefore, Koch's postulates were fulfilled for the first time for a sweepovirus.     

Infectibility and sensitivity of UK raspberry, blackberry and hybrid berry cultivars to Rubus viruses

Six blackberry or hybrid berry cultivars and 19 raspberry cultivars were assessed for their infectibility with, and sensitivity to, graft inoculation with 10 distinct viruses found infecting Rubus in the UK. Cultivars were grafted with each of, two isolates of the pollen borne raspberry bushy dwarf virus (RBDV), five aphid borne viruses: black raspberry necrosis, raspberry leaf mottle (RLMV), raspberry leaf spot (RLSV), rubus yellow net and raspberry vein chlorosis (RVCV); and isolates of the nematode transmitted nepoviruses, arabis mosaic, raspberry ringspot, strawberry latent ringspot and tomato black ring. All tested cultivars were infectible with a resistance breaking isolate of RBDV but only about half of that number with the Scottish type isolate of the virus. The raspberry cvs Autumn Bliss, and occasionally Glen Garry and Glen Prosen, developed leaf yellowing symptoms following infection with RBDV, but none of the other infected cultivars showed obvious leaf symptoms when kept in a heated glasshouse during the growing season. All tested cultivars were infectible with each of the four viruses transmitted in nature by the aphid, Amphorophora idaei. Most were infected symptomlessly, but seven cultivars developed severe leaf spotting symptoms due to infection with RLMV or RLSV. All but one of the raspberry cultivars were infectible with RVCV, which is transmitted in nature by the aphid Aphis idaei, and almost all infected plants developed leaf symptoms; only one of the hybrid berry or blackberry cultivars tested was infected with RVCV. In tests with the four nepoviruses, all tested cultivars, except Tummelberry, were infectible with at least one or more of these viruses. However, cultivars responded differently to challenge inoculation with different isolates of individual nepoviruses. Several cultivars developed chlorotic leaf mottling following infection with some nepovirus isolates. The implications of these results for virus control are discussed in the light of the changing pattern of virus and virus vector incidence in the UK.     

Host range, vector relations and serological relationships of cotton leaf curl virus from southern India

In 1989 to 1991, leaf curl disease was observed in cotton (Gossypium bar-badense cv. Local) grown in kitchen gardens in five districts in Karnataka State, India, and in 1994 it was recorded in G. hirsutum cv. Sharada in two districts. Symptoms consist of leaf curling, vein thickening, leaf enations, and stunting and distortion of plants. The disease is caused by cotton leaf curl virus (CLCuV-K), which was transmitted by the whitefly Bemisia tabaci to 24 plant species in six families. Hosts include bean (Phaseolus vulgaris), pepper, tobacco, tomato and several weeds, almost all of which developed leaf curl, with or without vein thickening. CLCuV-K was transmitted from cotton to cotton by adult B. tabaci after an acquisition access period of 1 h, could be inoculated in 5 min, had a minimum latent period of 8 h and was retained by viruliferous insects for up to 9 days. Female B. tabaci transmitted more frequently than males. CLCuV-K is a whitefly-transmitted geminivirus. It reacted with two out of 17 monoclonal antibodies (MAbs) raised to African cassava mosaic virus and five out of 10 MAbs raised to Indian cassava mosaic virus. CLCuV-K isolates from different locations in Karnataka had similar epitope profiles. As judged by these profiles, CLCuV-K is closely related to Indian tomato leaf curl virus from Karnataka, is distinguishable from several other whitefly-transmitted geminiviruses found in India and is still more distantly related to those, including cotton leaf crumple virus from the USA, found in other continents. CLCuV-K infected all cultivars tested of G. barbadense and one of six cultivars of G. hirsutum but none of G. arboreum or G. herbaceum.     

Recognition and differentiation of seven whitefly-transmitted geminiviruses from India, and their relationships to African cassava mosaic and Thailand mung bean yellow mosaic viruses

Particles resembling those of geminiviruses were found by immunosorbent electron microscopy in extracts of plants infected in India with bhendi yellow vein mosaic, croton yellow vein mosaic, dolichos yellow mosaic, horsegram yellow mosaic, Indian cassava mosaic and tomato leaf curl viruses. All these viruses were transmitted by Bemisia tabaci whiteflies, all reacted with at least one out of ten monoclonal antibodies to African cassava mosaic virus (ACMV), and all reacted with a probe for ACMV DNA-1, but scarcely or not at all with a full-length probe for ACMV DNA-2. Most of the viruses were distinguished by their host ranges when transmitted by whiteflies, and the rest could be distinguished by their pattern of reactions with the panel of monoclonal antibodies. Horsegram yellow mosaic virus was distinguished from Thailand mung bean yellow mosaic virus by its lack of sap transmissibility, ability to infect Arachis hypogaea, failure to react strongly with the probe for ACMV DNA-2 and its pattern of reactions with the monoclonal antibodies. Structures resembling a ‘string of pearls’, but not geminate particles, were found in leaf extracts containing malvastrum yellow vein mosaic virus. Such extracts reacted with two of the monoclonal antibodies, suggesting that this whitefly-transmitted virus too is a geminivirus. All seven viruses from India can therefore be considered whitefly-transmitted geminiviruses.     

Short Communication: Ageratum,Croton and Malvastrum harbour geminiviruses: evidence through PCR amplification

Ageratum conyzoides, Croton bonpladianum and Malvastrum coromandelianum are common weeds found around agricultural fields. In several cases these were found to exhibit vein yellowing and yellow mosaic symptoms. Using degenerate primers specific for whitefly-transmitted geminiviruses (WTGs), and total DNA isolated from such infected plants (exhibiting the above symptoms) as a template, 1.2kbp fragments were amplified and were shown to have homology to DNA-A of Indian tomato leaf curl virus (ITLCV) by Southern hybridization. In control experiments the same primers failed to amplify any DNA fragments from the total DNA isolated from healthy plants (no symptoms as above). These results show that Ageratum, Croton and Malvastrum harbour geminivirus(es).     

番茄曲叶病及其血清学和PCR测定

我国曾报道的番茄病毒病有多种,其中最常见的是黄瓜花叶病毒(CMV)和烟草花叶病毒(TMV)引起的花叶病。柯冲等(1964)在大陆首次报道烟粉虱(Bemisia tabaci)传播的番茄病毒病——番茄黄顶病,此病在50～60年代曾在广州市郊流行,造成大面积减产。Green等(1984)报道台湾发生番茄黄曲叶病,此病与日本的番茄黄矮病(Tomato yellow dwarf)相似,并且与烟草曲叶病毒(TLCV)有血清学关系。印度、委内瑞拉等国也曾报道发生由烟粉虱传播的番茄曲叶病和番茄黄曲叶病。1991和1992年秋,在广西南宁市郊发现一种症状表现为植株矮缩,叶片向上向内卷曲,叶背面产生耳状或杯状增生物,对光看有时可见叶脉呈墨绿色,不结果或少结果的番茄病害。1992年秋广西农业科学院的番茄试验地发病率高达6.8％,对当地秋番茄生产构成了威胁。作者对病害症状、传播、血清学反应及PCR分析等方面与烟草曲叶病毒进行了比较研究,证实了该病的病原与烟草曲叶病毒有很高的同源性。现将研究结果简报如下。     

Characterisation of datura yellow vein virus, a newly described rhabdovirus from Australia

A previously undescribed sub-group 2 rhabdovirus was isolated in Queensland from Datura stramonium with symptoms of vein yellowing, leaf distortion and reduced leaf size. Particles accumulated in the perinuclear space of infected cells of D. stramonium and measured 77 × 166 nm in preparations from sap. The virus was named datura yellow vein virus (DYVV) and was graft-transmitted to several hosts in the Solanaceae including Lycopersicon esculentum, Nicotiana tabacum and Solanum melongena, but not to Capsicum annuum or Solanum tuberosum. DYVV was not transmitted by mechanical inoculation and no insect vector was found. Purified particles of DYVV contained four structural proteins with molecular weights of about 78, 47, 41 and 36 kd. The 78 kd protein bound the lectin concanavalin A, thus identifying it as the viral glycoprotein. DYVV was serologically distinct from 11 other rhabdoviruses belonging to both subgroups, including potato chlorotic stunt, potato yellow dwarf (2 isolates) and tomato vein yellowing viruses. The glycoprotein only of DYVV cross-reacted with a polyclonal antiserum to sonchus yellow net virus.     

Involvement of Volatile Substances in Systemic Resistance of Barley Against Erysiphe graminis f. sp. hordei Induced by Pruning of Leaves

Horsegram yellow mosaic disease was shown to be caused by a geminivirus; horsegram yellow mosaic virus (HYMV). The virus could not be transmitted by mechanical sap inoculation. Leaf dip and purified virus preparations showed geminate virus particles, measuring 15-18 * 30 nm. An antiserum for HYMV was produced and in enzyme-linked immunosorbent assay (ELISA) and immunosorbent electron microscopy (ISEM) tests HYMV was detected in leaf extracts of fieldinfected bambara groundnut, french bean, groundnut, limabean, mungbean, pigeonpea and soybean showing yellow mosaic symptoms. Bemisia tabaci fed on purified HYMV through a parafilm membrane transmitted the virus to all the hosts listed above but not to Ageratum conyzoides, okra, cassava, cowpea, Croton bonplandianus, Lab-lab purpureus, Malvastrum coromandalianum and tomato. No reaction was obtained in ELISA and ISEM tests between HYMV antibodies and extracts of plants diseased by whitefly-transmitted agents in India such as A. conyzoides yellow mosaic, okra yellow vein mosaic, C. bonplandianus, yellow vein mosaic, M. coromandalianum yellow vein mosaic, tomato leaf curl and cassava mosaic. HYMV was also not found to be related serologically to bean golden mosaic, virus.     

Transmission of tomato leaf curl geminiviruses by Bemisia tabaci: effects of virus isolate and vector biotype

Cultures of Bemisia tabaci from Ivory Coast (IC), Pakistan (PK) and USA (US B-type) were compared for the frequency with which they transmitted three tomato geminivirus isolates: Indian tomato leaf curl virus from Bangalore (ITmLCV), and tomato yellow leaf curl viruses from Nigeria (TYLCV-Nig) and Senegal (TYLCV-Sen). Frequency of transmission from tomato to tomato depended both on the whitefly culture and the virus isolate. US B-type and IC whiteflies transmitted TYLCV-Sen more frequently than ITmLCV whereas PK whiteflies transmitted ITmLCV more frequently than TYLCV-Sen. US B-type whiteflies transmitted both viruses four to nine times more frequently than IC whiteflies. TYLCV-Nig was transmitted rarely by US B-type and not at all by IC whiteflies. Previous work indicates that the geminivirus coat protein controls vector transmissibility. The differential adaptation of TYLCV-Sen to transmission by US B-type whiteflies and of ITmLCV to PK whiteflies was associated with a large difference in epitope profile of the coat proteins of the two viruses. Also, the readily transmissible TYLCV-Sen differed appreciably in epitope profile from the poorly transmissible TYLCV-Nig, which reached a consistently greater concentration in source tissues but lacked epitope 18. However, the lack of epitope 18 in ITmLCV did not prevent its transmission by US B-type whiteflies. Differences in frequency and specificity of geminivirus transmission by whitefly cultures from different countries therefore were associated with differences among epitope profiles of the coat proteins of the viruses, but the structural features of the proteins that control transmission remain to be determined.     

Poinsettia Leaf Curl, a New Disease Caused by a Geminivirus

A serious disorder of unknown aetiology was found on the poinsettia cv. Angelica, a recent introduction to Taiwan. The symptoms of downward leaf curling and vein enation were similar to those of leaf curl disease on tobacco. The causal agent of the disease was transmitted to healthy plants of'Angelica'poinsettia, common poinsettia and tobacco by whiteflies. Twinned particles measuring 16-18 × 30-32 nm were detected in a purified preparation obtained from diseased leaf tissues of'Angelica'poinsettia, indicating that the disease is caused by a geminivirus. An earlier introduction of common poinsettias also showed mild leaf curl symptoms under natural condition. When the causal agent of this disease was transmitted to healthy'Angelica'poinsettia plants by whiteflies or bud grafting, these plants also developed severe leaf curl symptoms, indicating that the disease is not a new introduction to Taiwan.     

Detection and relationships of cotton leaf curl virus and allied whitefly-transmitted geminiviruses occurring in Pakistan

A stock culture of cotton leaf curl virus from Pakistan (CLCuV-PK), was transmitted by whiteflies (Bemisia tabaci) to seven plant species, including French bean, okra, tobacco and tomato, and caused vein thickening and leaf curl symptoms. It was readily detected in triple antibody sandwich ELISA (TAS-ELIS A) by 11 out of 31 monoclonal antibodies raised against the particles of three other geminiviruses: African cassava mosaic, Indian cassava mosaic and okra leaf curl viruses. Reaction strength was enhanced when the tissue extraction fluid contained sodium sulphite. Minor variations in epitope profile were found among virus isolates from cotton (Gossypium hirsutum) collected from different districts in Pakistan over a 5-year period. These epitope profiles were distinguishable from that of cotton leaf curl virus from G. barbadense in southern India but indistinguishable from the profiles of viruses causing yellow vein disease of okra in India or Pakistan, or leaf curl of okra {Abelmoschus esculentus), Hibiscus tiliaceus, radish or sunflower in Pakistan, suggesting that these plants are putative natural hosts of CLCuV-PK. The viruses in cotton, and in okra with leaf curl or yellow vein symptoms, were also detected by PCR with three pairs of CLCuV-PK-specific primers. Five additional whitefly-transmitted geminiviruses were found among isolates from 11 other naturally-infected species in Pakistan, and were distinguished by their epitope profiles. These viruses were associated, respectively, with tobacco leaf curl, squash yellow blotch, tomato yellow leaf curl, watermelon leaf crinkle and soybean yellow mosaic diseases. The first four of these viruses were detected readily by PCR with geminivirus general primers but only weakly, if at all, with two pairs of CLCuV-PK-specific primers. Pakistani crops are infected with a range of distinguishable but relatively closely related whitefly-transmitted geminiviruses, some of which resemble those found in India.     

Complete nucleotide sequence and the genome organization of tobacco leaf curl geminivirus from Japan

The genomic DNA of tobacco leaf curl geminivirus (TLCV) from tomato plants with leaf curl disease in Japan has been sequenced. The single circular DNA molecule comprises 2,761 nucleotides. TLCV DNA contains six open reading frames (ORFs) capable of encoding proteins with a molecular weight greater than 10 K. In total nucleotide sequence comparisons with other geminiviruses, TLCV was most closely related to tomato leaf curl virus from Taiwan (TwToLCV) (76% identity), tomato leaf curl virus from Bangalore (ToLCV-Ba) (74%) and agerantum yellow vein virus (AYVV) (74%), all possessing a monopartite genome. The significant but relatively low sequence similarity in the genomic DNA between TLCV and other geminiviruses suggests it is a distinct geminivirus in genus Begomovirus.     

Chinese tomato yellow leaf curl virus--a new species of geminivirus

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Engineering resistance against tomato yellow leaf curl virus (TYLCV) using antisense RNA

One of the most severe diseases of cultivated tomato worldwide is caused by tomato yellow leaf curl virus (TYLCV), a geminivirus transmitted by the whitefly Bemisia tabaci. Here we describe the application of antisense RNAs to interfere with the disease caused by TYLCV. The target of the antisense RNA is the rare messenger RNA of the Rep protein, encoded by the C1 gene. Transgenic Nicotiana benthamiana plants expressing C1 antisense RNA were obtained and shown to resist infection by TYLCV. Some of the resistant lines are symptomless, and the replication of challenge TYLCV almost completely suppressed. The transgenes mediating resistance were shown to be effective through at least two generations of progeny.     

Purification and Identification of a Rhabdovirus from Thunbergia alata

A subgroup 2 rhabdovirus was isolated in south-east Queensland from black-eyed Susan (Thunbergia alata) with symptoms of vein yellowing, vein clearing and leaf distortion. Bacilliform particles accumulated in the perinuclear space of infected plants and measured 69 ± 7 × 161 ± 8 nm in unfixed preparations. The virus was not transmitted mechanically. Purified preparations of the Thunbergia alata rhabdovirus (TaRV) contained four major proteins with molecular weights of 80 kD, 48 kD, 40 kD and 35 kD, similar to those of datura yellow vein virus (DYW), a newly described rhabdovirus from Australia. The 80 kD protein was identified as the viral glycoprotein. In immunoblots, the two largest proteins of TaRV reacted strongly with antiserum to DYW, but were serologically distinct from sonchus yellow net, cereal chlorotic mottle, potato yellow dwarf and lettuce necrotic yellows viruses. TaRV is considered to be a strain of DYW.     

Complete Nucleotide Sequence and Genome Organization of Soybean Crinkle Leaf Virus

The genomic DNA of soybean crinkle leaf virus (SCLV) from Thailand has been sequenced. The single circular DNA molecule comprises 2737 nucleotides, and contains eight open reading frames each capable of encoding a protein with a molecular weight greater than 10 kDa. A 39‐base potential stem‐loop forming region occurs in the intergenic region (IR) that also includes the conserved nonanucleotide sequence TAATATTAC. The iterative sequence (TCAATCGGTGT), which is specific to SCLV, is also found in the IR. SCLV is most closely related (90% identity) to the monopartite geminivirus ageratum yellow vein virus. As the two viruses differ in host range, and the iterative sequence is specific to SLCV, the virus is a distinct monopartite geminivirus of soybean.     

Characterisation of pumpkin yellow vein mosaic virus from India

Yellow vein mosaic disease symptoms occur frequently in pumpkin in India. Diseased plants show vein yellowing, which sometimes coalesces to form chlorotic patches. Infected plants are stunted and flowers drop prematurely, greatly reducing yields. Diseased plants are infected by a begomovirus, designated pumpkin yellow vein mosaic virus (PYVMV), which is transmitted readily and in a persistent manner by the whitefly, Bemisia tabaci. Transmission of PYVMV requires minimum acquisition and inoculation access periods of 30 min and 10 min, respectively. The minimum latent period in the insect is 6 h and the virus persists in the vector for at least 8 days. PYVMV has a narrow host range consisting of a small number of cucurbit species and some tobacco cultivars. It was detected serologically in diseased plants and in viruliferous B. tabaci using polyclonal antibodies in a double‐antibody sandwich enzyme‐linked immunosorbent assay. Reactions with monoclonal antibodies in a triple‐antibody sandwich ELISA showed that PYVMV has an epitope profile distinct from those of other begomoviruses from the Indian sub‐continent. Polymerase chain reaction amplified fragments from the putative viral coat and movement protein genes. Based on comparative phylogeny of complete coat protein gene sequences, PYVMV was most similar to the bipartite Tomato leaf curl New Delhi virus from India and appears to be a new strain of this virus.     

Photosynthetic properties of leaves of Eupatorium makinoi infected by a geminivirus

We examined photosynthetic properties of Eupatorium makinoi leaves infected by a geminivirus. Since a major symptom of the geminivirus infection is variegation or yellowing of leaves, Chl content was used as an index of disease severity. As leaf Chl content was lowered, leaf absorptance, maximal quantum yield of photosynthesis on an absorbed quantum basis (o_2,max) and light-saturated rate of photosynthesis (P_max) decreased. The share of energy allocated to PS II, which can be estimated from fluorescence parameters and oxygen evolution rate, was about 30% lower in the infected yellow leaves than in uninfected leaves. Analyses of the composition of thylakoid polypeptides by gel electrophoresis showed preferential loss of LHC II. The lower o_2,maxin the infected leaves was, thus, attributed to the decreased energy allocation to PS II. These features were largely consistent with those of b-less mutants, but lowered P_maxhas been never reported for b-less mutants. Possible mechanisms causing these changes in photosynthetic properties to the infected leaves are discussed.     

Effects of mist irrigation on the physiology of sugar beet

Bacilliform particles characteristic of plant rhabdoviruses were found in negatively-stained leaf sap and in thin sections of Laburnum anagyroides in England showing vein yellowing. The particles were detected principally in the perinuclear space of parenchyma cells. They were not transmitted by sap inoculation to twelve herbaceous species. The affected trees also showed mosaic symptoms but there was no evidence of an association between these and the bacilliform particles.