Transmissibility of Cucumber mosaic virus by Aphis gossypii Correlates with Viral Accumulation and Is Affected by the Presence of Its Satellite RNA

ABSTRACT Satellite RNAs (satRNAs) are associated with Cucumber mosaic virus (CMV) in tomato, most often causing severe epidemics of necrotic plants, and not associated with specific host symptoms. Laboratory studies on virus transmission by the aphid vector Aphis gossypii were performed to better understand the dynamics of field populations of CMV. The presence of satRNAs correlated with lower concentrations of virus in infected plants and with a decrease in the efficiency of transmission from satRNA-infected plants. Both the concentration of virus in CMV-infected tomato and the efficiency of transmission varied more extensively with nonnecrogenic satRNAs than with necrogenic satRNAs. A negative effect of satRNAs on virus accumulation can account, in part, for a decrease in the field transmission and recovery of CMV + satRNAs. Aphids behaved differently and probed less readily on plants infected with CMV + necrogenic satRNAs compared with plants containing non-necrogenic satRNAs. Aphid-mediated satRNA-free CMV infections were observed in test plants when aphids were fed on source plants containing CMV + nonnecrogenic satRNA; no comparable satRNA-free test plants occurred when aphids were fed on source plants containing necrogenic satRNAs. These results indicate that factors associated with transmission can be a determinant in the evolution of natural populations of CMV and its satRNA.     

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.     

A study of viral coat protein accumulation in lily chloroplasts from mixed virus infections of Lily mottle virus and Cucumber mosaic virus

Two viruses that frequently occur in many Lilium species are Lily mottle virus (LMoV) and Cucumber mosaic virus (CMV), which usually co-infect lilies causing severe disease symptoms. Recent reports have revealed that the viral coat protein (CP) affects chloroplast ultrastructure and symptom development. This study used western blot analysis to confirm that in leaves infected by mixed virus infections of LMoV and CMV, CPs of both viruses were accumulated in lily chloroplasts. Immunogold labelling further demonstrated that both the LMoV CP and CMV CP were localized in the stroma and the thylakoid membranes of the chloroplasts. In addition, it was found that CPs of both viruses were rapidly transported into isolated, intact chloroplasts (in vitro), and their transport efficiencies were positively related to CP concentrations. The lowest transmembrane concentration of CMV CP decreased from 38 μg mL⁻¹ recorded in the single CMV CP import system to 10 μg mL⁻¹ in the mixed import system of LMoV CP and CMV CP. CPs of both viruses exhibited species selection in their transmembrane transport into chloroplasts. This is the first report that the CPs from two viruses (LMoV and CMV) are simultaneously present in lily chloroplasts. Accumulation of high levels of LMoV CP and CMV CP inside the chloroplast appears to contribute to a synergistic interaction inducing the development of mosaic symptoms.     

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.     

Identification and characterization of Pestalotiopsis spp. causing twig blight disease of bayberry (Myrica rubra Sieb. & Zucc) in China

Bayberry (Myrica rubra) is a fruit tree native to the subtropical regions of China. It produces fruit with a unique taste and pharmacological characteristics, thus making it a widely cultivated tree commercially in many regions of China, including Zhejiang, Fujian, Guizhou, Sichuan and Yunnan Provinces. Recently, a twig blight disease occurred on the fruit tree and caused destructive damage of plantings in the Zhejiang Province. However, the etiology of the disease was unclear. This study was carried out to identify the causal agent(s) of the blight disease on bayberry. Fungal isolates were obtained from blighted twig samples collected from bayberry fields in Xianju, Rui’an, and Huangyan of Zhejiang Province. The majority (87.9 %) of the 257 fungal isolates were identified as Pestalotiopsis spp. based on their conidial morphology. DNA sequences of the internal transcribed spacer (ITS) region of the ribosomal RNA gene and the β-tubulin gene were obtained from six representative strains (XJ27, XJ42, RA2-1, YS26, YS44 and RA1-2) of the Pestalotiopsis spp. Phylogenetic analysis showed that three of the strains (XJ27, XJ42, and RA2-1) grouped with P. versicolor (Speg.) Steyaert while the other three strains (YS26, YS44 and RA1-2) grouped with P. microspora (Speg.) Batista & Peres. Pathogenicity tests in the greenhouse showed that all these six isolates of Pestalotiopsis spp. caused twig blight disease symptoms on bayberry plants, which were the same as observed in naturally infected plants in the field. Our results clearly indicated that P. versicolor and P. microspora were the major pathogens causing the twig blight disease on bayberry in southern China.     

我国部分地区常见农作物上黄瓜花叶病毒分离物核酸多样性分析

为明确我国黄瓜花叶病毒株系分化及系统进化基本情况,从湖南、新疆、青海和海南4省区采集1 367个样品对其进行酶联免疫和RT-PCR检测,并对分离获得的15个黄瓜花叶病毒(Cucumber mosaic virus,CMV)纯化分离物CP、MP、2b核苷酸序列进行相似性和进化树分析及生物学性状比较。结果表明,辣椒、龙葵和黄瓜的CMV阳性检出率较高,分别为54.13%、29.19%和18.46%。进化树分析显示CMV-Q5与CMV亚组II的亲缘性较高;CMV-N7为新发现的重组株系,其CP、2b基因属于CMV亚组IB,MP基因却属于CMV亚组II;其余13个分离物均属于CMV亚组IB。CMV-N7和CMV-Q5在系统寄主心叶烟和枯斑寄主苋色藜上引发的症状相似,但比对照株系CMV-P3613(IB)的发病时间要晚1~2 d,系统花叶较温和,枯斑较小。表明在以上4省区常见农作物上广泛流行的CMV存在分子变异。     

Variability of some plant species from different origins and their suitability for virus work

Plants of four species obtained from various sources were raised and compared for their suitability as test plants for a number of viruses. The species comprisedChenopodium amaranticolor (local lesion host of red clover mottle virus),Nicotiana glutinosa (local lesion host of tobacco mosaic virus (TMV)),N. rustica (systemic host of TMV, cucumber mosaic virus and tomato spotted wilt virus) andN. tabacum ‘White Burley’ (systemic host of TMV). Some sources proved to be more attractive as test plants or experimental hosts because of, e.g., production of more local lesions per unit of leaf area (C. amaranticolor andN. glutinosa), distinctness of lesions (C. amaranticolor), development of more pronounced systemic symptoms (N. rustica andN. tabacum ‘White Burley’), better growth characteristics (particularlyC. amaranticolor andN. rustica), smoother leaves being easier to rub (N. glutinosa) or more and bigger leaf formation (particularlyN. rustica). The implications for the interpretation of host range studies and the quest for propagation and assays hosts are discussed.     

Induction of systemic resistance against Cucumber mosaic virus by Penicillium simplicissimum GP17‐2 in Arabidopsis and tobacco

The plant growth‐promoting fungus, Penicillium simplicissimum GP17‐2, was evaluated for its ability to induce resistance against Cucumber mosaic virus (CMV) in Arabidopsis thaliana and tobacco plants. Treatment with barley grain inoculum (BGI) of GP17‐2 significantly enhanced fresh weight, dry weight and leaf number of A. thaliana and tobacco plants 6 weeks after planting. Two weeks after CMV inoculation, all plants treated with BGI of GP17‐2 or its culture filtrate (CF) showed a significant reduction in disease severity compared with non‐treated control plants, which exhibited severe mosaic symptoms by the end of the experiment. The enzyme‐linked immunosorbent assay (ELISA) demonstrated that CMV accumulation was significantly reduced in plants treated with GP17‐2 or its CF relative to control plants. Based on RT‐PCR, plants treated with GP17‐2 (BGI or CF) also exhibited increased expression of regulatory and defence genes involved in the SA and JA/ET signalling pathways. These results suggested that multiple defence pathways in A. thaliana and tobacco were involved in GP17‐2‐mediated resistance to CMV, although neither the transgenic NahG line, nor the npr1, jar1 or ein3 mutants disrupted the response in A. thaliana. This is the first report to demonstrate the induction of systemic resistance against CMV by GP17‐2 or its CF.     

新疆番茄病毒病检测及南方番茄病毒全基因组序列测定与传播特性分析

为了解新疆番茄上病毒病的发生情况,利用一步法RT-PCR技术检测了南北疆番茄上南方番茄病毒(Southern tomato virus,STV)、黄瓜花叶病毒(Cucumber mosaic virus,CMV)、番茄花叶病毒(Tomato mosaic virus,ToMV)以及马铃薯Y病毒(Potato virus Y,PVY)的感染情况,并利用分段克隆的方法进行全基因组测序,通过RT-PCR方法检测健康植株与携带病毒植株杂交育种的F1代植株带毒率以分析STV的种子传播特性。结果显示,新疆番茄上CMV、STV、ToMV和PVY在北疆的检出率分别为52%、37%、27%和14%;在南疆的检出率分别为79%、60%、69%和0;且以STV、CMV及ToMV的复合侵染为主。从我国加工番茄上首次获得了长3 437 nt的STV SHZ-1核苷酸序列,序列比对分析发现其与已报道STV只有1~9个核苷酸的变异,且序列变异与地域无相关性。分析杂交F1代加工番茄植株上STV的传播特性,发现其除可由种子传播外,也可通过花粉传播。表明STV是侵染新疆番茄的主要病毒之一,且该病毒可通过种子或杂交育种途径进行传播。     

Exogenous application of melatonin improves plant resistance to virus infection

In this study, melatonin (MEL)-mediated plant resistance to tobacco mosaic virus (TMV) was examined to study local infection in Nicotiana glutinosa and systemic infection in Solanum lycopersicum. Exogenous application of 100 µm MEL increased anti-virus infection activity to 37.4% in virus-infected N. glutinosa plants. The same treatment significantly reduced relative levels of virus RNA analysed by qRT-PCR and virus titres measured by dot-ELISA, and increased the relative expression levels of the PR1 and PR5 genes analysed by qRT-PCR, in virus-infected S. lycopersicum. MEL treatment induced considerable accumulations of salicylic acid (SA) and nitric oxide (NO) but did not significantly affect production of hydrogen peroxide (H₂O₂) in the virus-infected S. lycopersicum plants. Transgenic nahG N. tabacum was used to determine whether MEL-induced TMV resistance was dependent on the SA pathway. The results showed that the relative RNA level of the TMV analysed by qRT-PCR and virus titres analysed by dot-ELISA were not reduced by the MEL treatment in the nahG transgenic N. tabacum seedlings treated twice with 100 µm MEL. The increased relative expression levels of PR1 and PR5 were greatly reduced when cPTIO, an NO scavenger, was included in the MEL treatment. A working model of MEL-mediated plant resistance to TMV is proposed. MEL-mediated plant resistance to viruses provides a new avenue to control plant viral diseases.     

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.     

Genetic Diversity of Panicum mosaic virus Satellite RNAs in St. Augustinegrass

ABSTRACT St. Augustine decline is a viral disease caused by Panicum mosaic virus (PMV) alone or in combination with a satellite virus (SPMV) and/or satellite RNAs (satRNAs). A ribonuclease protection assay (RPA) was used to evaluate the genetic diversity of PMV satRNAs isolated from 100 naturally infected St. Augustinegrass plants (Stenotaphrum secundatum). Distinctive satRNA RPA profiles were observed for 40 of 52 samples from College Station (CS) and 37 of 48 samples from Corpus Christi (CC), Texas. A dendrogram constructed from the RPA data revealed that satRNAs were grouped in two distinct clusters based on their place of origin. From 100 samples, only 4 satRNAs from CS were placed in the CC group, and only 2 satRNAs from CC were placed in the CS group. The data show that there is genetic variability in PMV satRNAs in naturally occurring infections, and distinct geographically separate populations can be identified from CC and CS.     

Patterns in disease progress and the influence of single and multiple viral infections on pepper (<Emphasis Type="Italic">Capsicum annuum</Emphasis> L.) growth

The patterns and progress of disease caused by multiple infections of Cucumber mosaic virus (CMV), Pepper mild mottle virus (PMMoV) and Pepper mottle virus (PepMoV) and their effects on growth of pepper plants (Capsicum annuum L.) were investigated in this study. Each virus induced distinct symptoms, but more severe symptoms, including reduced growth rates, were observed when pepper plants were simultaneously infected by more than one virus. When CMV was included in multiple viral inoculations, co-inoculations and sequential inoculations, PepMoV and PMMoV symptoms were observed but the symptoms characteristic of CMV were not masked, even though CMV titre did not increase greatly. In multiple viral infections, PepMoV titre and CMV did not increase significantly, but PMMoV titre gradually increased in most cases. Growth rates of pepper plants were greatly reduced during the 30 to 40-day post-inoculation period under both single-infection and multiple-infection conditions, but multiple viral infections of CMV pre-inoculated peppers were affected to a greater extent. A significant reduction in fruit size and fruit number was observed in single and multiple viral inoculations, and fruit malformation rates were high in CMV single-infection and multiple viral infections with CMV.     

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.     

Evolution of Virulence in Natural Populations of the Satellite RNA of Cucumber mosaic virus

From 1986 to 1992, an epidemic of tomato necrosis caused by Cucumber mosaic virus (CMV) plus CMV satellite RNAs (satRNAs) occurred in eastern Spain. From 1989 onward, the frequency of tomato necrosis di-minshed, and it almost completely disappeared after 1991. Analyses of plants infected with CMV and with CMV satRNA and of the phenotype (necrogenic or nonnecrogenic for tomato) induced by some CMV satRNA variants, showed that the disappearance of tomato necrosis was due to changes in the genetic composition of the satRNA population (i.e., to its evolution toward decreased virulence). Analysis of components of the fitness of satRNA variants, necrogenic or nonnecrogenic for tomato, showed that necrogenic and nonnecrogenic variants did not differ in infectivity or in their accumulation level in tomato and that they represented the same fraction of encapsidated RNA. Other fitness components were positively correlated with the greater virulence of necrogenic variants, in that they were favored in mixed infections with nonnecrogenic variants and were more effectively passed into CMV progeny than were nonnecrogenic variants. On the other hand, necrogenic CMV satRNA variants caused a more pronounced depression in the accumulation of CMV than did nonnecro-genic variants, which could affect the efficiency of aphid transmission. Thus, the evolution of virulence in the CMV satRNA population can be explained by trade-offs between factors that determine virulence and factors that affect transmission, as predicted by theoretical models on the evolution of virulence in parasites.     

北京瓜类花叶病毒的调查研究

北京郊区南瓜、西葫芦花叶病是由甜瓜花叶病毒类型所致。黄瓜、参瓜、丝瓜的花叶病都是由黄瓜花叶病毒类型所引起;黄瓜花叶病毒除为害瓜类作物外,还广泛分布在郊区其他栽培作物和野生植物上。根据 CMV 各分离物在冬瓜、南瓜、蔓陀罗,心叶菸,菸上的病状反应,建议将 CMV 分离物区分为八个毒株。来自南瓜并侵染西瓜的一个甜瓜花叶病毒分离物在侵染丝瓜后不能用汁接侵染荀瓜。     

Systemic infection of some N-gene-carrying Nicotiana species and cultivars after inoculation with tobacco mosaic virus

Since July 1974Nicotiana tabacum ‘Samsun NN’ plants, inoculated with the common strain of tobacco mosaic virus (TMV), have occasionally been found to develop necrosis on non-inoculated upper leaves 2–7 days after the local necrotic lesions had appeared on the lower leaves. All these plants had been kept in a growth chamber at 17–20°C. Other tobacco species and cultivars carrying theN gene, such asN. glutinosa andN. tabacum ‘Xanthi-nc’, showed the same phenomenon. Substantial amounts of TMV could be recovered from leaves with systemic symptoms. The systemic necrosis somewhat resembled that caused by tobacco rattle virus (TRV). A number of possible causes, such as high concentration of the inoculum, contamination with another strain of TMV or with TRV, change in the genetic composition of the host plants and certain growing conditions (soil, water, pesticides) were investigated. None of these factors could be held fully responsible for the abnormal systemic reaction, although there was evidence that the soil could sometimes play an important rôle.