Immunolocalization of <Emphasis Type="Italic">Pepper mild mottle virus</Emphasis> in developing seeds and seedlings of <Emphasis Type="Italic">Capsicum annuum</Emphasis>

The location of Pepper mild mottle virus (PMMoV) within seeds as they developed on inoculated seedlings of pepper (Capsicum annuum) was followed over time by detecting the viral coat protein using immunofluorescence microscopy. Seedlings were inoculated with PMMoV when the flower buds on the first and second branching nodes were in bloom. Fluorescence indicating the presence of PMMoV was first observed around immature seeds and placentas in the ovaries on the fourth branching node at 20 days post-anthesis (20 DPA), which corresponded to 39 days post-inoculation (39 DPI). The area with fluorescence gradually expanded from the placenta into the integument and the parenchyma, and finally reached the tip of the immature seeds by 34 DPA (53 DPI). The embryo or endosperm beyond the endothelium never fluoresced during the experiment [i.e., ending at 81 DPA (102 DPI)]. For visualizing viral routes of invasion from seeds into new seedlings, PMMoV-infected C. annuum seeds that were heterozygous for the L ³ tobamovirus-resistance gene were sown in soil at 30°C. After ~2 weeks, the cotyledon developed virally induced necrosis. These findings shed light on the infection cycle of PMMoV through vertical transmission in C. annuum.     

Occurrence of viruses in field-grown pepper crops and some of their reservoir weed hosts in Samsun, Turkey

Pepper production is affected by several viral diseases in Samsun, Turkey. To determine the identity of these viruses, a total of 313 samples from field-grown peppers were collected during surveys in 1998 and 1999, and tested by enzyme linked immunosorbent assay (ELISA). Six viruses,Alfalfa mosaic virus (AMV),Cucumber mosaic virus (CMV),Potato virus Y (PVY),Tomato mosaic virus (ToMV),Tobacco mosaic virus (TMV) andTomato spotted wilt virus (TSWV) were detected in the samples. Of 313 plants tested, 42 were doubly infected, and TMV+PVY (15.4%) was the most common double infection. This is also the first report of AMV in pepper fields in Turkey. The effect of some weed species that may act as reservoir of these viruses was also investigated in the region and of 24 weed species belonging to 15 families tested, 16 were found to be infected with at least one virus.Amaranthus retroflexus (Redroot pigweed) appeared to be a common host of CMV, PVY, ToMV, TMV and TSWV, whereasHibiscus trionum (Venice mallow) was recorded as a new weed host of PVY and TSWV. The majority of weed species found to be virus infected were very common in the pepper growing areas of the region. This indicates that pepper fields contaminated with these weeds are under risk of viral infections. http://www.phytoparasitica.org posting July 21, 2005.     

Survey of viruses infecting open‐field pepper crops in Côte d'Ivoire and diversity of Pepper veinal mottle virus and Cucumber mosaic virus

The prevalence of viruses in pepper crops grown in open fields in the different agro‐ecological zones (AEZs) of Côte d'Ivoire was surveyed. Pepper veinal mottle virus (PVMV; genus Potyvirus) and Cucumber mosaic virus (CMV; genus Cucumovirus) were the most frequent viruses among those surveyed, while tobamoviruses (genus Tobamovirus) were detected at low frequency. PVMV showed a high heterogeneity across AEZs, which may be related to climatic, ecological or agronomical conditions, whereas CMV was more homogeneously distributed. The molecular diversity of CMV and PVMV were analysed from partial genome sequences. Despite the low number of CMV isolates characterized, two molecular groups were revealed, one corresponding to subgroup IA and the other to reassortants between subgroups IA and IB. RNAs 1 and 3 of the reassortants clustered with the IB subgroup of CMV isolates, whereas their RNA 2 clustered with the IA subgroup. Importantly, RNA 1 of CMV isolates of the IB subgroup has been shown to be responsible for adaptation to pepper resistance. The diversity of PVMV in the VPg‐ and coat protein‐coding regions revealed multiple clades. The central part of the VPg showed a high level of amino acid diversity and evidence of positive selection, which may be a signature of adaptation to plant recessive resistance. As a consequence, for efficient deployment of resistant pepper cultivars, it would be desirable to examine the occurrence of virulent isolates in the CMV or PVMV populations in Côte d'Ivoire and to follow their evolution as the resistance becomes more widely deployed.     

Occurrence and distribution of viruses infecting tomato and pepper in Alibori in northern Benin

In surveys conducted in 2011 and 2012 to identify the viruses causing diseases on pepper and tomato in the department of Alibori in northern Benin, 451 samples of pepper and tomato were analyzed by ELISA using 11 specific antibodies. The highest virus incidence among the surveyed districts was recorded on pepper in Malanville (56.18%), followed by Karimama (39.32%). The most frequently found viruses were Pepper veinal mottle virus (PVMV), Cucumber mosaic virus (CMV), and Potato virus Y-necrotic (PVY-n), accounting respectively for 22.39%, 21.73% and 15.96% of the collected samples. Tomato yellow leaf curl virus (TYLCV) was detected in only 2.43% of the samples, whereas Alfalfa mosaic virus (AMV), Chilli veinal mottle virus (ChiVMV), Tomato mosaic virus (ToMV) and Tomato spotted wilt virus (TSWV) were not detected in any of the samples tested. Double and triple infections involving different virus combinations were found, respectively, in 14.86% and 4% of the samples. Five plant species (Euphorbia hirta Linnaeus, Moringa oleifera Lam, Leucas martinicencis (Jacquin) R. Brown, Combretum micranthum G. Don, Abelmoschus esculentus L. Moench.) out of 30 samples belonging to 13 botanical families, collected within or nearby tomato and pepper fields, were found infected with PVMV, PVY-n, and CMV. Control measures to reduce the impact of viruses on pepper and tomato production are discussed. This is the first report of viruses infecting pepper and tomato in Benin.     

Immunolocalization of Pepper mild mottle virus in Capsicum annuum seeds

To clarify the mechanism of seed transmission of Pepper mild mottle virus (PMMoV), the virus was immunolocalized in Capsicum annuum seeds using fluorescence microscopy. Two distinct patterns were observed: In the first, PMMoV was present in the epidermis and parenchyma but not in the endosperm or embryo; in the second, the virus was restricted to the surface of the epidermis and parenchyma. These findings shed light on the fundamental mechanisms of seed transmission of tobamoviruses and may aid in the design of new methods to prevent the spread of seedborne virus diseases.     

Co‐infection with Cucumber vein yellowing virus and Cucurbit yellow stunting disorder virus leading to synergism in cucumber

Symptom expression and levels of the ipomovirus Cucumber vein yellowing virus (CVYV) and the crinivirus Cucurbit yellow stunting disorder virus (CYSDV) were compared in greenhouse cucumbers in single and mixed infections. Results were contrasted with those obtained for plants infected with the potyvirus Zucchini yellow mosaic virus (ZYMV) in single and in mixed infections with either CVYV or CYSDV. Cucumber showed leaf symptoms of each co‐infecting virus, except for the combination of CYSDV with ZYMV, where the typical CYSDV‐like symptoms of interveinal leaf yellowing were inconspicuous or absent. The progression of CVYV as quantified by real‐time RT‐PCR was similar in plants with single infections and in mixed infection with CYSDV between 15 and 60 days post‐inoculation (dpi). However, CYSDV was detected at significantly enhanced levels in plants when co‐infected with CVYV but not when co‐infected with ZYMV. In the latter case, ZYMV levels were reduced when compared with single infections. During mixed infections of ZYMV and CVYV, the titre levels of the ipomovirus were significantly lower when compared with single infections. Cucumber had reduced plant height, internode length, dry weight and fruit yield, positively correlated with the titre levels of CVYV and not of CYSDV during mixed infections. It is concluded that co‐infections with CVYV enhance the titre of CYSDV, which could have epidemiological significance.     

Characterization of <Emphasis Type="Italic">Pepper yellow leaf curl virus,</Emphasis> a tentative new <Emphasis Type="Italic">Polerovirus</Emphasis> species causing a yellowing disease of pepper

Pepper (Capsicum annuum) is an important crop worldwide. In Israel, approximately 2,500 ha are grown all year round for the local and export markets. Herein, we report the identification of a viral pathogen causing a new devastating disease in pepper crops. The disease syndrome includes shortening of stem internodes, interveinal yellowing, and upward rolling of the leaf blade, accompanied by fruit discoloration and size reduction. Virus purification from infected plants yielded isometric particles, 25 nm in diameter. The causal agent of the disease was tentatively named Pepper yellow leaf curl virus (PYLCV). The virus cross-reacted in DAS ELISA with antisera against Cucurbit aphid-borne yellows virus and Potato leafroll virus, members of the Polerovirus genus. The partial nucleotide sequence obtained from the cloned viral coat protein and movement protein genes indicated 92% identity at the amino acid level with Tobacco vein distortion virus (TVDV), another member of the Polerovirus genus. However, the host range of PYLCV is significantly different from the host range described for TVDV. Based on our findings, the taxonomic status of PYLCV is discussed.     

Synergistic Disease in Pepper Caused by the Mixed Infection of Cucumber mosaic virus and Pepper mottle virus

ABSTRACT The occurrence of more than one virus species in a single plant is not uncommon in cultivated and native plant species. A mixed virus infection may lead to greater disease severity than individual viral components and this is sometimes referred to as a synergistic disease. Although, in some cases, synergism has been demonstrated for various plant growth parameters such as plant height, weight, and yield, proof of synergy typically has not been demonstrated for symptom severity when the mixed virus infection was not lethal. We demonstrated synergy in bell pepper plants co-infected with Cucumber mosaic virus (CMV) and Pepper mottle virus (PepMoV) relative to each virus alone for stem height (two of three trials) and aboveground fresh weight (one of three trials) using factorial analysis and Abbott's equation for synergy. This approach allowed affirmation of the type of response (i.e., synergistic rather than antagonistic) and statistical proof of synergy. A detailed evaluation of symptom severity for each viral treatment revealed three phases associated with host plant developmental stages. A numerical symptom severity rating scale was developed and used in each of two equations to demonstrate statistical proof for synergy based on symptom severity for co-infected plants. Virus accumulation in noninoculated leaves was determined by enzyme-linked immunosorbent assay. In singly infected plants, CMV titers declined in mildly symptomatic leaves representing later stages of plant development, but titers increased in similar leaves of co-infected plants. In contrast, PepMoV titers did not differ in singly or co-infected plants.     

辣椒轻斑驳病毒凤城分离物的鉴定、全基因测序及系统进化分析

辣椒轻斑驳病毒Pepper mild mottle virus(PMMoV)属于烟草花叶病毒属,是辣椒上重要的病原病毒之一,近年来PMMoV对辽宁部分辣椒产区造成严重危害。本文采用DAS-ELISA检测以及RT-PCR的方法首次从辽宁省凤城市蔬菜产区辣椒病叶中检测出PMMoV,暂命名为PMMoV-FC。设计3对特异性引物对其进行扩增并测序后得到该病毒分离物全基因序列。将PMMoV-FC的全基因序列与已报道的国内外9个PMMoV分离物进行同源性分析,结果表明,其同源性介于94.3%~99.7%之间。基于全基因序列的系统发育进化分析表明,PMMoV-FC与国内分离物、日本分离物以及美洲分离物亲缘关系密切,而与韩国和西班牙分离物亲缘关系稍远。鉴于该病毒在辣椒上造成的严重危害,对于PMMoV-FC在辽宁地区的发生以及防治仍需进行更为详细的研究。     

Pepper 9- and 13-lipoxygenase genes are differentially activated by two tobamoviruses and by hormone treatments

Two novel pepper 13-lipoxygenase (LOX) genes were cloned and their expressions were compared with those of three 9-LOX genes in pepper leaves inoculated with two different tobamoviruses. Obuda pepper virus (ObPV) inoculation led to a massive induction of pathogenesis-related genes and to the development of hypersensitive reaction (incompatible interaction), while Pepper mild mottle virus (PMMoV) inoculation resulted in a compatible interaction. Both virus infections markedly activated the expression of the two novel 13-LOXs. The magnitudes of induction of 13-LOXs did not differ substantially between the ObPV- and PMMoV-inoculated leaves. The induction of three 9-LOXs was markedly more robust and rapid in ObPV-inoculated leaves than in PMMoV-inoculated ones. LOXs were very differentially activated in pepper leaves treated with defense hormones. A large number of hormone-related cis-regulatory elements were identified in the promoter regions of LOXs. ObPV inoculation resulted also in the substantial up-regulation of an omega-6-fatty acid desaturase gene. Our results suggest that 9-LOX-dependent pathways are more probably involved in the suppression of virus replication than 13-LOX-dependent plant responses.     

Production of discernible disease phenotypes in Canna by five plant viruses belonging to the genera Potyvirus,Cucumovirus and Badnavirus

Cannas are tropical and subtropical flowering perennial plants. The genus contains many species but most commercially grown cultivars are interspecific hybrids selected for their attractive foliage and flowers. Canna production is so lucrative that there are farmers and nurseries dedicated solely to its production. The specific issue that the canna industry faces is virus diseases. In this study, rhizomes of 24 canna cultivars were gathered and diagnostics conducted to detect Bean yellow mosaic virus (BYMV, Potyvirus), Canna yellow mottle virus (CaYMV, Badnavirus), Canna yellow streak virus (CaYSV, Potyvirus), Cucumber mosaic virus (CMV, Cucumovirus) and Tomato aspermy virus (TAV, Cucumovirus). Visual assessment of disease symptoms and diagnostic tests were carried out to identify the prevalent diseases and describe the symptoms that are associated with virus infection. BYMV, CaYMV and CaYSV caused severe mosaic and necrosis either in the leaf lamina or veins of infected leaves. Potyvirus infection suppressed red colouration in the foliage of some varieties. CaYMV and CaYSV often appeared in the same plant, suggesting they might represent a viral complex. CMV and TAV were rarely seen in these populations. Interestingly, CaYMV but not CaYSV could be mechanically inoculated to Phaseolus vulgaris plants.     

实时荧光RT-PCR检测辣椒轻斑驳病毒的初步研究

本研究选取辣椒轻斑驳病毒PMMoV、烟草花叶病毒TMV、黄瓜花叶病毒CMV和马铃薯Y病毒PVY作为试验材料,根据PMMoV衣壳蛋白(CP)RNA的序列特异性位点,设计出Taqman荧光探针及其引物,采用实时荧光RT-PCR技术对PMMoV进行快速检测,同时与ELISA方法进行了灵敏度比较,结果表明：该方法具有较高的灵敏度及较强的特异性,PMMoV检测结果为阳性,TMV、CMV和PVY均无荧光信号,为阴性,灵敏度是传统的ELISA方法的100倍,而且大大缩短了检测时间。该方法快速、准确、灵敏、简便、安全,具有实际应用价值,适用于植物病毒病害的快速检测。     

High incidence of <Emphasis Type="Italic">Pelargonium line pattern virus</Emphasis> infecting asymptomatic <Emphasis Type="Italic">Pelargonium</Emphasis> spp. in Spain

Geraniums (Pelargonium spp.) are traditional ornamental plants largely cultivated in Europe and northern America. Vegetative propagation makes them prone to viral infections, which have detrimental effects on crop production and quality. Asymptomatic samples collected in Spain were tested for a range of viruses using ELISA. The tobamovirus, Tobacco mosaic virus (TMV), the cucumovirus, Cucumber mosaic virus (CMV), and several viruses in the family Tombusviridae, namely, Pelargonium line pattern virus (PLPV), Pelargonium flower break virus (PFBV), and Pelargonium leaf curl virus (PLCV), were detected either singly or in combination in 59.2% of 800 samples. PLPV and PFBV infections were confirmed by dot-blot hybridisation. The most relevant viral infection found on Spanish asymptomatic geraniums was by Pelargonium line pattern virus (PLPV). Symptoms did not develop for 3 years on most of the PLPV infected geranium plants under greenhouse conditions.     

侵染四川辣椒的番茄斑萎病毒和辣椒轻斑驳病毒的小RNA测序检测及鉴定

 调查发现四川省汶川县当地辣椒的病毒病严重且症状多样,病样粗提液摩擦接种辣椒、矮牵牛和三生烟,出现辣椒系统性花叶焦枯和茎尖坏死,指示植物表现局部枯斑。对3个不同症状的病果进行sRNA深度测序鉴定,发现均含有番茄斑萎病毒(Tomato spotted wilt virus,TSWV)和辣椒轻斑驳病毒(Pepper mild mottle virus,PMMoV)。通过RT-PCR技术进行验证,结果显示所有病样的果皮和部分新鲜种子以及回接寄主的病叶均检测到TSWV和PMMoV,表明该地辣椒病毒病是由TSWV和PMMoV复合侵染引起。这是TSWV侵染四川辣椒的首次报道。分别基于TSWV N基因序列和PMMoV CP基因序列构建系统发育树,汶川分离物TSWV-WC(MK468469)与贵州分离物(KP684518)亲缘关系最近,PMMoV-WC(MK408614)与北京分离物(AY859497)亲缘关系最近。推测该地辣椒病毒病可能与品种引进有关。     

Genetic diversity and mixed infections of begomoviruses infecting tomato, pepper and cucurbit crops in Nicaragua

Begomoviruses were detected in Nicaraguan fields of tomato ( Lycopersicon esculentum ) and adjacently growing plants of pepper ( Capsicum annuum ), chilli pepper ( C . baccatum ), cushaw ( Cucurbita argyrosperma ) and Mexican fireplant ( Euphorbia heterophylla ) using polymerase chain reaction (PCR) and universal begomovirus primers. All tomato and Mexican fireplant plants showing symptoms were infected with begomoviruses, while only 30–46% of the pepper, chilli pepper and cushaw plants showing symptoms tested virus-positive. No begomoviruses were found in potato. The virus species were provisionally identified by sequencing 533 bp of the viral coat protein gene ( AV1 ). Tomato severe leaf curl virus (ToSLCV), Tomato leaf curl Sinaloa virus (ToLCSinV) and Pepper golden mosaic virus (PepGMV) were found to infect both tomato and pepper. A new provisional species designated Tomato leaf curl Las Playitas virus (ToLCLPV) was detected in a tomato plant. Squash yellow mottle virus (SYMoV) and PepGMV were found in cucurbits, the latter for the first time in this host. Euphorbia mosaic virus (EuMV) was detected in Mexican fireplant. Sequencing of a larger number of PCR-amplified clones from selected plants revealed intraspecific viral sequence variability, and also multiple begomovirus infections which could represent up to three species in a single tomato or cushaw plant. Phylogenetic grouping of virus sequences did not correlate with the host of origin.     

Detection of Tobamoviruses from Soils by Non-precoated Indirect ELISA

A method for detecting tobamoviruses from field soils was developed using non-precoated indirect enzyme-linked immunosorbent assay (Id-ELISA). Absorbance values in Id-ELISA were relatively low after directly applying Pepper mild mottle virus (PMMoV)-infested soil extract. However, heat treating the soil extract before application greatly enhanced the absorbance values. The heat treatment was essential for the Id-ELISA detection of tobamoviruses from infested soil, although the efficiency of virus recovery varied depending on the properties of soil. The number of local lesions in the infectivity assay was consistent with the absorbance values in Id-ELISA. Moreover, the absorbance values in Id-ELISA were correlated with the incidence of soil transmission of PMMoV. Thus, Id-ELISA combined with heat treatment is a practical technique for the diagnosis of infestation with Tobamovirus in field soils, Gray Lowland soil and Sand-dune Regosol. Received 4 October 1999/ Accepted in revised form 9 December 1999