Partial characterization of a bipartite begomovirus infecting yellow passion flower in Brazil

During the spring of 2001, approximately 10 000 yellow passion flower plants, from two orchards in the county of Livramento de Nossa Senhora, Bahia State, Brazil, exhibited intense yellow mosaic symptoms and drastic reduction of the leaf lamina and plant development. A large population of whiteflies ( Bemisia tabaci ) was also found colonizing the plants. All field samples collected tested positive for Passion fruit woodiness virus in DAS-ELISA. Five out of 20 passion flower plants inoculated with adult whiteflies collected from diseased plants in the field developed symptoms 20–30 days after inoculation. Two of these plants gave a positive reaction in TAS-ELISA using antiserum against a begomovirus. Degenerated PCR primers amplified viral DNA fragments from the DNA-A and DNA-B components of a begomovirus infecting these plants. The fragment corresponding to the core region of the coat protein (DNA-A) was cloned and sequenced. A phylogenetic analysis placed this begomovirus isolated from passion flower in the same clade of the New World begomoviruses as several other species from Brazil. Based on the symptoms induced by this virus alone, the disease was tentatively named passion flower little leaf mosaic.     

Effect of Figaren, an Antiviral Protein from Cucumis figarei, on Cucumber mosaic virus Infection

Local lesion formation on cowpea leaves was more than 50% inhibited by treatment with a 23 kDa RNase-like glycoprotein from Cucumis figarei, figaren, from 24 hr before to 1 hr after inoculation with Cucumber mosaic virus (CMV). CMV accumulation detected by ELISA in tobacco leaves treated with figaren 6 or 0 hr before inoculation with CMV was suppressed. When upper leaves of tobacco plants were treated with figaren and inoculated 10 min later with CMV, mosaic symptoms were delayed for 5–7 days on most of the tobacco plants, and some plants remained asymptomatic. From fluorescence in situ hybridization, infection sites were present in figaren-treated cowpea or melon leaves after inoculation with CMV, though the sites were reduced in number and size compared with those in water-treated control leaves. The amount of CMV RNAs and CMV antigen in melon protoplasts inoculated with CMV and subsequently incubated with figaren similarly increased with time as did that in the control. ELISA and local lesion assays indicated that CMV infection on the upper surfaces of the leaves of tobacco, melon, cowpea and C. amaranticolor whose lower surfaces had been treated with figaren 5–10 min before CMV inoculation was almost completely inhibited. Figaren did not inhibit CMV infection on the opposite untreated leaf halves of melon, cowpea and C. amaranticolor, whereas it almost completely inhibited CMV infection on the untreated halves of leaves of tobacco. CMV infection was not inhibited in the untreated upper or lower leaves of the four plants. These data suggest that figaren does not completely prevent CMV invasion but does inhibit the initial infection processes. It may also induce localized acquired resistance in host plants. Received 10 October 2000/ Accepted in revised form 6 February 2001     

Long-lasting systematic roguing for effective management of CABMV in passion flower orchards through maintenance of separated plants

In Brazil, passion flower is grown across almost the entire country. The predominant disease of the passion flower crop is passion fruit woodiness, caused by the potyvirus cowpea aphid-borne mosaic virus (CABMV), and transmitted by aphids in a nonpersistent manner. The disease reduces the useful life of the orchard from 36 months to approximately 18 months. Up to now, there has not been an efficient method for disease management. The aim of this work was to evaluate the efficiency of systematic roguing of diseased plants through weekly inspections, for disease management in the field. The latent and incubation periods of CABMV in passion flower vines were determined in order to optimize roguing efficiency. Passion fruit plants inoculated with CABMV started to act as sources of inoculum from 3 days after inoculation (DAI), and the symptoms were expressed, on average, at 8 DAI. Five field experiments, conducted in the states of São Paulo and Bahia, Brazil, demonstrated that systematic roguing of diseased plants was significantly efficient for managing passion fruit woodiness disease. In order to facilitate identification and subsequent removal of the infected plants, they need to be grown separately. This cultural practice can be recommended for managing passion fruit woodiness disease, provided it is applied on a regional scale by all passion fruit growers. The development of some pilot plantings for the application of roguing in a passion flower-producing region is recommended to validate the use of this technique for managing passion fruit woodiness disease.     

Detection and molecular characterization of viruses associated with tomato yellow leaf curl disease in cucurbit crops in Jordan

In this study, Tomato yellow leaf curl Sardinia virus (TYLCSV) and the strains Israel and Mild of Tomato yellow leaf curl virus (TYLCV-IL, TYLCV-Mld) were detected for the first time in four cucurbit crops in Jordan by nested polymerase chain reaction (nPCR). These viruses cause the tomato yellow leaf curl disease (TYLCD) in tomato. Cucumber, squash, melon and watermelon plants inoculated with TYLCV-IL[JO:Cuc], TYLCV-Mld, TYLCSV-IT[IT:Sar:88] and the Jordanian isolate of TYLCV (TYLCV-JV) did not show disease symptoms. However, virus-specific fragments were detected in uppermost leaves of symptomless plants by nPCR. A whitefly transmission test showed that Bemisia tabaci could transmit TYLCV-Mld from cucumber into tomato and jimsonweed plants. However, all infected tomato plants remained symptomless. In addition, results of semi-quantitative PCR (sqPCR) analysis showed that the relative amount of TYLCV-Mld DNA acquired by B. tabaci from cucumber plants was less than that acquired from tomato plants.     

Recovery from Cucurbit leaf crumple virus (family Geminiviridae, genus Begomovirus) infection is an adaptive antiviral response associated with changes in viral small RNAs

A strong recovery response occurs in cantaloupe (Cucumis melo) and watermelon (Citrullus lanatus) infected with the bipartite begomovirus Cucurbit leaf crumple virus (CuLCrV). This response is characterized by initially severe symptoms, which gradually become attenuated (almost symptomless). An inverse relationship was detected between viral DNA levels and recovery, indicating that recovered tissues had reduced viral titers. Recovered tissues also were resistant to reinfection with CuLCrV; i.e., recovered leaves reinoculated with the virus did not develop symptoms or have an increased level of viral DNA. In contrast, infection of CuLCrV-recovered leaves with the RNA virus, Cucumber mosaic virus (CMV), disrupted recovery, resulting in the development of severe disease symptoms (more severe than those induced by CMV or CuLCrV alone) and increased CuLCrV DNA levels. Small RNAs with homology to CuLCrV DNA were detected in recovered and nonrecovered tissues; as well as in phloem exudates from infected, but not uninfected plants. Levels of these small RNAs were positively correlated with viral titer; thus, recovered tissues had lower levels than symptomatic tissues. In addition, viral DNA from a host that undergoes strong recovery (watermelon) was more highly methylated compared with that from a host that undergoes limited recovery (zucchini). Furthermore, inoculation of CuLCrV-infected zucchini with a construct expressing an inverted repeat of the CuLCrV common region enhanced recovery and reduced viral symptoms and viral DNA levels in newly emerged leaves. Taken together, these results suggest that recovery from CuLCrV infection is an adaptive antiviral defense mechanism, most likely mediated by gene silencing.     

黄瓜花叶病毒M株系引致烟草症状恢复的初步研究

 黄瓜花叶病毒M株系在白肋烟上具有典型的症状恢复现象,本研究用提纯病毒和DAS-ELISA建立了CMV-M病毒定量检测方法。研究发现,症状严重程度与叶片中的病毒浓度呈正相关:最早发病的黄化叶每克病组织中病毒可高达790 μg,而恢复叶片上部再发病的花叶症状病叶中每克病组织中病毒也可高达508 μg,恢复叶片中病毒含量很低,每克叶片中最高也没有超过6 μg,仅为发病叶片病毒浓度的1/85~1/135,远低于根和茎中的病毒浓度。RT-PCR和生物学检测结果表明恢复叶片中确实存在具侵染活性的病毒,而且病毒在长达半个月以上一直保持很低浓度。结果表明恢复叶中可能存在有效的病毒防御机制,其具体机理有待进一步研究。     

Coat protein-mediated transgenic resistance in tomato against a IB subgroup Cucumber mosaic virus strain

Transgenic tomato plants containing the coat protein (CP) gene of Cucumber mosaic virus (CMV) of subgroup IB were developed through Agrobacterium-mediated transformations. The progenies of transgenic plants showed the presence of transgene, its expression and translation of 26 KDa CP. The T₁ and T₂ generation plants were evaluated for resistance against challenge inoculations by a homologous strain of CMV. Visual observations of challenged transgenic plants categorized them into resistant, tolerant and susceptible as compared with untransformed control plants. Out of 33 plants of the T₁ generation, 36.3% showed resistance and remained symptomless throughout their life, 48.4% showed tolerance which developed delayed symptoms of mild mosaic, and 15.1% showed susceptibility to CMV which developed severe systemic mosaic and leaf distortion symptoms after 30?days of virus challenge. Out of 120 plants of the T₂ generation, 60% showed resistance, 26.6% were tolerant and only 13.3% were found susceptible to challenge inoculations of CMV. Resistant transgenic plants also showed less CP accumulation in systemic upper leaves as compared with challenged untransformed plants. In this study, CP of a CMV subgroup IB strain has demonstrated a significant level of resistance in transgenic tomato plants against the CMV strain. The strategy may be applied for better quality and productivity of tomato crops.     

The natural occurrence of bean yellow mosaic, cucumber mosaic and tobacco mosaic viruses in lisianthus in Israel

Mosaic on leaves, necrosis, stunting and flower symptoms were observed in lisianthus ( Eustoma russellianum ) raised from seed. Three viruses, bean yellow mosaic (BYMV), cucumber mosaic (CMV) and tobacco mosaic (TMV), were isolated. Virus identification was based on electron microscopy, host range, aphid transmission and serology.     

Identification and transmission of Piper yellow mottle virus and Cucumber mosaic virus infecting black pepper (Piper nigrum) in Sri Lanka

Sri Lankan black pepper with symptoms of yellow mottle disease contained a mixture of viruses: Piper yellow mottle virus (PYMV) particles (30 × 130 nm), Cucumber mosaic virus (CMV, 30 nm diameter isometric particles), and unidentified, isometric virus-like particles (30 nm diameter). An effective purification procedure is described for PYMV. Immunosorbent and conventional electron microscopy successfully detected badnavirus particles only when at least partially purified extracts were used. PYMV was confirmed as the cause of the disease, with the other two viruses apparently playing no part in producing symptoms. PYMV was transmitted by grafting, by the insect vectors citrus mealy bug ( Planococcus citri ) and black pepper lace bug ( Diconocoris distanti ), but not by mechanical inoculation or through seeds. The CMV isolate was transmitted to indicator plants by mechanical inoculation and by the vector Aphis gossypii , but not by Myzus persicae ; but neither mechanical nor insect transmission of CMV to black pepper was successful. A sensitive polymerase chain reaction assay was developed to detect PYMV in black pepper.     

Characterization of symptom determinants in two mutants of cucumber mosaic virus Y strain,causing distinct mild green mosaic symptoms in tobacco

Two mutants of Cucumber mosaic virus, CMV(Y/GM1) and CMV(Y/GM2), which induced mild green mosaic symptoms in tobacco, were isolated from plants regenerated from tobacco leaves with yellow mosaic symptoms originally infected with the yellow strain of CMV [CMV(Y)]. Although the appearance of mild green mosaic symptoms in tobacco infected with CMV(Y/GM2) was unstable, CMV(Y/GM3) derived from CMV(Y/GM2) reproducibly induced mild green mosaic symptoms in tobacco similar to CMV(Y/GM1). A comparison of the deduced amino acid sequences of the coat proteins of CMV(Y), CMV(Y/GM1) and CMV(Y/GM3), showed single amino acid substitutions from Thr to Ile at position 124 in the CMV(Y/GM1) coat protein and from Val to Ile at position 111 in the CMV(Y/GM3) coat protein. When the amino acid at the 124 or 111 position in the CMV(Y) coat protein was changed to Ile at the cDNA level, CMV RNA3 transcribed in vitro from each cDNA induced mild green mosaic symptoms in tobacco after inoculation with in vitro transcribed CMV(Y) RNA1 and RNA2. The results indicated that amino acids at positions 111 and 124 in the coat protein were responsible for the phenotypic changes caused by the two CMV isolates.     

Latent infection of winter oilseed rape by Leptosphaeria maculans

Plants of oilseed rape, cultivars Primer and Jet Neuf, were grown in a glasshouse and inoculated at G.S. 2.4–2.7 with pycnidiospores or ascospores of Leptosphaeria maculans. The plants were kept for a further 2–4 weeks at 14°C and then transferred, together with uninoculated plants, to a polythene tunnel in winter. The majority of stems of inoculated plants did not have macroscopic symptoms of L. maculans infection 6 weeks after inoculation. Examination of whole mounts of peripheral tissue and transverse sections of fixed and embedded portions of these stems revealed intercellular septate fungal hyphae, often deep in non-necrotic cortical tissue, in symptomless inoculated plants but not in uninoculated plants. L. maculans was recovered following surface sterilization of adjacent portions of the same stems. When symptomless inoculated plants were transferred to a glasshouse at 18–20°C, cankers soon developed. The significance of these latent mycelial infections to canker development in the field is discussed.     

<Emphasis Type="Italic">Cucumber mosaic virus</Emphasis> isolated from Amazon lily (<Emphasis Type="Italic">Eucharis grandiflora</Emphasis>)

Cucumber mosaic virus (CMV) was isolated from a mosaic diseased plant of Eucharis grandiflora. The virus caused mosaic symptoms on leaves and slight distortion of flower petals in E. grandiflora by either mechanical or aphid inoculation. The virus was identified as a strain of CMV subgroup I from its biological and serological characteristics.     

2b Protein is essential to induce a novel gradual cell death in Zucchini yellow mosaic virus-inoculated cucumber cotyledon co-infected with Cucumber mosaic virus

Cucumber cotyledons inoculated with Cucumber mosaic virus (CMV, Pepo strain) or Zucchini yellow mosaic virus (ZYMV, Z5-1 isolate) developed either mild chlorotic spots or no symptoms. Cotyledons treated with CMV plus ZYMV also developed mild chlorotic spots. However, plants ZYMV-inoculated cotyledons had veinal yellowing and gradual cell death by 20 days postinoculation (dpi) when co-inoculated with CMV on the other cotyledon. When analyzing this synergism, an enzyme-linked immunosorbent assay showed that CMV gradually increased in CMV-inoculated cotyledons of plants, with the other cotyledon mock- or ZYMV-inoculated. However, CMV significantly increased at 9 to 14 dpi in the ZYMV-inoculated cotyledons of plants co-infected with CMV. ZYMV similarly increased in cotyledon pairs of both co-infected and singly infected plants. Inoculation with PepoΔ2b, a modified Pepo-CMV that lacks translation of the 2b protein, revealed that PepoΔ2b without the 2b protein systemically infected cucumber but induced no symptoms on cotyledons or true leaves. Plants with a ZYMV-inoculated cotyledon and co-infected with PepoΔ2b did not undergo cell death; nevertheless, PepoΔ2b was at high levels comparable to levels of CMV in the ZYMV-inoculated cotyledon. The 2b protein thus seems essential for induction of the novel gradual cell death in ZYMV-inoculated cotyledons of cucumbers co-infected with CMV.     

福建西番莲病毒病的发生及其病原黄瓜花叶病毒亚组鉴定

1988～1995年对福建西番莲病毒病的发生为害进行调查,发现在西番莲栽培区病毒病普遍发生,发病率通常在30％～40％,严重的达90％以上。田间症状主要表现为叶片环斑、皱缩、花叶、环斑花叶,死顶和果实木质化等。从田间病株采集40份样本,经电镜观察和采用A蛋白夹心ELISA(PAS-ELISA),用西番莲木质化病毒(PWV)、西番莲黄花叶病毒(PFYMV)、紫果西番莲花叶病毒(GMV)、烟草花叶病毒(TMV)及黄瓜花叶病毒(CMV)的抗血清进行测定,其中36个样本检测出CMV,表明福建西番莲病毒病的主要病原为CMV。与此同时,对西番莲上CMV进行亚组鉴定,采用鉴别寄主和单、多克隆抗体双夹心ELISA(DAS-ELISA)对36个CMV阳性样本进行测定,结果35个属CMV亚组Ⅰ,1个属CMV亚组Ⅱ,表明田间以CMV亚组Ⅰ分离物占绝对优势。     

云南美人蕉病毒病病原鉴定

In this study,the causal agents were identified from Canna indica viral diseased plants in Yunnan Province.The diseased C.indica plants mainly exhibited the symptoms like veinal chlorosis and yellowing,streak mosaic or interveinal chlorosis,while older leaves always showed veinal necrosis as well as chlorosis.Viral pathogens were detected by RT-PCR/PCR in 24 diseased C.indica samples collected from Kunming and Yuxi City in Yunnan Province.The results indicated that the main C.indica-infecting viruses were canna yellow mottle virus (CaYMV),bean yellow mosaic virus (BYMV),sugarcane mosaic virus (SCMV).CaYMV showed the highest detection rate of 87.5 %,whereas,the BYMV had the lowest rate of 16.7% in the 24 samples.Co-infections of CaYMV+SCMV,CaYMV+BYMV and CaYMV+SCMV+BYMV were also detected in the diseased samples.However,cucumber mosaic virus (CMV),tobamovirus,luteovirus,orthotospovirus,begomovirus and umbravirus were not detected in these samples.This is the first report of CaYMV and SCMV infecting C.indica in Yunnan province.     

Symptom development,in planta distribution,and transmission of <Emphasis Type="Italic">Impatiens necrotic spot virus</Emphasis> in gentian: evidence for survival in roots and winter buds

Impatiens necrotic spot virus (INSV) causes serious damage to gentian (Gentiana spp.). Symptom development, in planta distribution, and transmission of INSV were studied after mechanical inoculation of gentian plants and propagation of shoot cuttings from infected stock plants. When young gentian plants at the 2nd-leaf stage were inoculated with INSV, plants developed systemic symptoms that were restricted to a few upper leaves. However, older plants at the 6th-leaf stage did not develop systemic symptoms. After plant inoculation, INSV was detected using DAS-ELISA in symptomatic upper leaves, and rootlets and winter buds, but not in asymptomatic leaves. When asymptomatic shoot cuttings from infected stock plants were vegetatively propagated in a thrips-free glasshouse, 44.4% of those obtained from the apical shoot and 20.6% of those obtained from the middle section of the plant developed systemic symptoms. These results indicated that when gentian plants were infected with INSV, the virus was preferentially transported from the infected leaves to the root and winter buds. However, even asymptomatic shoot cuttings may develop systemic symptoms when obtained from infected stock plants. Therefore, vegetative propagation from infected stock plants can be a source of INSV infection.     

Occurrence and development of Colletotrichum acutatum on symptomless blueberry bushes

The anthracnose fungus Colletotrichum acutatum was detected in symptomless blueberry bushes ( Vaccinium spp.) in a Japanese blueberry field. Naturally diseased bushes and their apparently healthy neighbours were selected, and C. acutatum was isolated from the symptomless tissues of each bush from February 2000 to January 2002. Analysis of the diseased bushes during the dormant period revealed that the fungus was able to survive on symptomless tissues, such as shoot bark and bud scales. Furthermore, C. acutatum was consistently isolated from symptomless leaves and shoots of several surrounding symptomless bushes. Arbitrarily primed PCR (ap-PCR) analyses of the fungal isolates obtained from the diseased and symptomless bushes revealed that most C. acutatum isolates were genotypically identical, regardless of their origins. Inoculation tests using leaves of various blueberry cultivars suggested that the presence or absence of symptoms on each bush can not always be explained by differences in cultivar susceptibility, and other factors may be associated with the appearance of symptoms.     

Breeding tomatoes for resistance to tomato yellow leaf curl begomovirus

Tomato yellow leaf curl begomovirus (TYLCV) can be devastating to tomato crops in tropical and subtropical regions. The development of resistant cultivars is the best option for the control of TYLCV. The TYLCV-resistance level of a new breeding line, TY172, alongside that of commercial cultivars known to be resistant to the virus, was evaluated in a field test by comparing the yield performance of inoculated plants with that of uninoculated plants of the same line or cultivar. There were substantial differences among the different entries tested in the extent of yield loss relative to the corresponding uninoculated control plants. This comparison between inoculated and uninoculated plants of the same entry provides a quantitative assay for resistance level. All resistant commercial cultivars tested developed different levels of disease symptoms. Only line TY172 showed no symptoms of the disease. A low level of viral DNA was detected in infected TY172, showing that it is a symptomless carrier of TYLCV. When TY172 was crossed with susceptible lines, the hybrids exhibited milder symptoms than the susceptible parent, yet higher than that of TY172, suggesting a partial dominance for TY172 resistance. Upon inoculation of F₂ populations, the amount of symptomless individuals appeared in a ratio approximating 7:64. This suggests that at least three genes appear to account for the resistance.     

北京地区花生病毒病防治研究

应用无毒花生种子防治黄瓜花叶病毒(CMV)和花生轻斑驳病毒(PMMV)效果显著,在与大田毒源花生隔离500米条件下基本控制住两种病毒病发生。覆膜栽培有减轻两种病毒病特别是CMV引起的黄花叶病毒病的效果。在病区大面积隔离繁殖无CMV病毒种子获得成功,应用无CMV病毒种子和覆膜栽培示范获得显著防病效果。34个花生栽培品种和资源材料田间抗性鉴定,未发现有抗性品种和材料。