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.     

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.     

Phytosanitary status of grapevine in Montenegro

During 2006 and 2007, a survey on the incidence and distribution of fourteen grapevine viruses was carried out in the Skadar Lake basin, one of the two main grapevine‐growing areas of Montenegro. In total 165 samples were collected from four red (‘Vranac’, ‘Krato?ija’, ‘Merlot’ and ‘Cardinal’), two white (‘Chardonnay’ and ‘Rkaciteli’) and a few unknown grapevine varieties in the vicinity of Podgorica and Bar. The phytosanitary status of the collected samples was analysed by DAS‐ELISA and the presence of Grapevine fanleaf virus (GFLV), Grapevine leafroll‐associated virus 1 (GLRaV‐1), Grapevine leafroll‐associated virus 2 (GLRaV‐2) and Grapevine leafroll‐associated virus 3 (GLRaV‐3) was confirmed in some of them. The most frequently found virus in assayed samples was GLRaV‐3 (54.5%), followed by GFLV (23%), GLRaV‐1 (20%) and GLRaV‐2 (0.6%). These serological analyses showed the absence of Grapevine leafroll‐associated virus 6 (GLRaV‐6), Grapevine leafroll‐associated virus 7 (GLRaV‐7), Raspberry bushy dwarf virus (RBDV), Strawberry latent ringspot virus (SLRSV), Tomato ringspot virus (ToRSV), Raspberry ringspot virus (RpRSV), Arabis mosaic virus (ArMV), Tobacco ringspot virus (TRSV), Tomato black ring virus (TBRV) and Cherry leaf roll virus (CLRV) from all tested samples.     

Leonotis nepetaefolia: An important plant virus reservoir in central Mexico

The presence of viruses in the weedLeonotis nepetaefolia in central México is reported from two field surveys.L. nepetaefolia, with viral-like symptoms such as mosaic, leaf deformation and calico, was observed growing next to cultivated fields in the Valley of Atlixco, Puebla, an important agricultural region in Mexico. The viruses harbored by this plant were characterized biologically, serologically and by molecular methods. The viruses detected wereAlfalfa mosaic virus (AMV),Cucumber mosaic virus (CMV), a satellite RNA of CMV (CMV satRNA) andTobacco mosaic virus (TMV). This last one was detected only during the first survey. CMV was the predominant virus found in both surveys, and was associated mostly to mosaic symptom. Phylogenetic analysis based on the coat protein gene sequence of CMV indicated that this isolate belongs to subgroup IA and confirmed that it is a mosaic-inducing isolate, whereas AMV belongs to subgroup II. Finally, CMV satRNA was found to be a non-necrogenic ameliorative variant, both by symptomatology and by phylogenetic analysis. Our results suggest thatL. nepetaefolia is a reservoir for several viruses in central Mexico, and given its wide distribution in several parts of the world, its role as a virus reservoir could be more general. http://www.phytoparasitica.org posting Aug. 31, 2005.     

Association between genetic variability and titre of Grapevine Pinot gris virus with disease symptoms

The present work was carried out in order to verify the possible association between a new grapevine disease, characterized by leaf mottling and deformation, and the genetic variability and concentration of Grapevine Pinot gris virus (GPGV), a recently identified virus tentatively associated with the pathology. After vineyard surveys and the establishment of real‐time qPCR assays, characterization of GPGV isolates and evaluation of GPGV titre were assessed in more than 100 samples of grapevine Glera, collected from plants regardless of whether or not they showed the symptomatology. Results showed that there was an important association between the GPGV variants and manifestation of the symptoms, and that grapevines with symptoms harboured significantly higher GPGV titre than symptomless vines. Moreover, an interesting relationship among the phylogenetic clustering of the isolates originating from plants with symptoms and some epidemiological characteristics of the disease was found. The current study confirmed the role of GPGV in the emergent disease characterized by grapevine leaf mottling and deformation.     

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.     

<Emphasis Type="Italic">In situ</Emphasis> localization of <Emphasis Type="Italic">Grapevine fanleaf virus</Emphasis> and phloem-restricted viruses in embryogenic callus of <Emphasis Type="Italic">Vitis vinifera</Emphasis>

In grapevine, somatic embryogenesis is particularly effective in eliminating several important virus diseases. However, the mechanism whereby regenerated somatic embryos are freed of the viruses is not clear. The distribution of Grapevine fanleaf virus (GFLV), Grapevine leafroll-associated virus-3 (GLRaV-3) and Grapevine virus A (GVA) in embryogenic callus of grapevine was investigated by in situ hybridization using digoxygenin-labelled oligonucleotide probes. Four months after culture initiation, in callus originated by GFLV-infected explants we observed a mosaic of infected and uninfected cells, with high concentrations of viruses in some cell groups in peripheral zones of the callus. In addition some abnormal somatic embryos showed a high hybridization signal. In callus originated by GVA- and GLRaV-3-infected explants the viruses were concentrated in few cells surrounded by areas of virus-free cells. The two viruses were generally localized in different clusters of cells inside the callus and the levels of infection were lower than those observed in GFLV-infected callus. No virus was detected in callus nor in somatic embryos after 6 months of culture. The results highlight the difficulties of some viruses at stably invading callus tissues and the differential ability of GFLV to spread in the callus cells compared to the phloem-limited viruses.     

Seasonal dynamics and virus translocation of Grapevine leafroll‐associated virus 3 in grapevine cultivars

Grapevine leafroll‐associated virus 3 (GLRaV‐3) is associated with grapevine leafroll disease, one of the most economically important viral diseases of grapevines. This disease impacts on both vine health and grape quality; reduction in yield, brix and wine colour are among its detrimental effects. Many methods, including serological and molecular procedures, have been developed for the detection of GLRaV‐3; however, there is no PCR‐based assay available to quantify virus populations within plant tissues. A real‐time RT‐PCR assay with TaqMan probe was developed for specific and reliable quantitative detection of GLRaV‐3 in infected tissues. The designed primers and probes target the conserved sequence in the RNA‐dependent RNA polymerase (RdRp) domain of the viral genome to prevent amplification of most subgenomic and defective RNAs. This protocol was used to examine the seasonal dynamics and translocation of GLRaV‐3 in field‐grown grapevines. The results showed that the virus spread quickly from trunks to new growing shoots and leaves early in the growing season, and most samples still harboured detectable virus during late summer and autumn. The seasonal progress of one GLRaV‐3 isolate was compared in four grapevine cultivars (Chardonnay, Cabernet Sauvignon, Italia and Thompson Seedless). Within cultivars there was little variability in the distribution and translocation of GLRaV‐3, except for in Thompson Seedless. This quantitative detection assay will be a valuable tool for GLRaV‐3 diagnosis, disease monitoring and population ecology studies.     

Effect of two satellite RNAs on Nicotiana glutinosa infected with Cucumber mosaic virus (CMV)

Two types of satellite RNAs (XJs1 and XJs2) were found to be associated with the two Cucumber mosaic virus (CMV) beet isolates XJ1 and XJ2 consisting of 384 nucleotides and 336 nucleotides, respectively. Our work showed that XJs1 and XJs2 could attenuate symptoms on Nicotiana glutinosa induced by CMV and the effect of XJs1 on symptoms was stronger than XJs2. Co-inoculation of CMV with XJs1 produced lighter oxidative stress than that with XJs2. Oxygen uptake rates through the alternative pathway in leaves infected with CMV and XJs2 were always higher than that with CMV and XJs1 and lower than infection with CMV alone. The non-photochemical fluorescence quenching (qN) increased more in N. glutinosa infected with CMV and XJs2 than that with CMV and XJs1. The relationship among helper virus, satRNAs and the host plants is discussed. Two closely-related satRNAs from the same plant has not been reported so far. The effects of these two satRNAs on virus-infected N. glutinosa were significantly different. The difference may be caused by deletion and mutation of the satRNAs.     

Insights into the genetic diversity and evolution of Little cherry virus 1

Little cherry virus 1 (LChV‐1), a member of the recently proposed genus Velarivirus, is a sweet cherry pathogen that has been recently reported to infect other Prunus species and is associated with various plant disorders. In this work the incidence of the virus on its putative hosts and possible mechanisms driving its evolution were investigated. Due to problems encountered with LChV‐1 detection, a new nested RT‐PCR assay was developed and applied. The virus was found to be prevalent in cherry plantations in Greece and only occasionally detected in other Prunus species. Sequences corresponding to the partial RNA‐dependent RNA polymerase (RdRp), heat‐shock protein homologue (HSP70h) and coat protein (CP) genes were determined from Greek LChV‐1 isolates originating from different hosts; these were analysed, along with published homologous genomic regions from other isolates. Phylogenetic analysis of the three genes revealed the segregation of four evolutionary distinct groups showing no host or geography‐based clustering. Mean genetic distances among the four groups were high with the CP region showing the highest divergence, although intragroup variability levels were low. Nevertheless, estimations of the mean ratio of nonsynonymous substitutions per synonymous site to synonymous substitutions per synonymous site (dN/dS) for the partial RdRp, HSP70h and CP indicated that these genomic regions are under negative selection pressure. Interestingly, a recombination event was identified at the 3′ end of RdRp on a Greek virus isolate, thus highlighting the role of this mechanism in the evolutionary history of LChV‐1.     

Molecular epidemiology of cassava mosaic disease in Madagascar

Cassava is the staple food for hundreds of millions of people in Africa but its cultivation is seriously constrained by cassava mosaic disease (CMD) in Madagascar, and in Africa in general. This study identified the cassava mosaic geminiviruses (CMGs) involved in CMD in Madagascar and their associated epidemiological characteristics from countrywide surveys. Molecular characterization of CMGs in Madagascar revealed an unprecedented diversity and co‐occurrence of six viruses: African cassava mosaic virus (ACMV), East African cassava mosaic Cameroon virus (EACMCV), East African cassava mosaic Kenya virus (EACMKV), East African cassava mosaic virus (EACMV), South African cassava mosaic virus (SACMV) and the recently described Cassava mosaic Madagascar virus (CMMGV). Distinct geographical distributions were observed for the six viruses. While ACMV was more prevalent in the central highlands, EACMV and EACMKV were prevalent in lowlands and coastal regions. Both EACMCV and SACMV occurred in almost all the localities visited. PCR diagnosis revealed that mixed infection (up to four co‐infected viruses) occurred in 21% of the samples and were associated with higher symptom severity scores. Pairwise comparisons of virus associations showed that EACMCV was found in mixed infections more often than expected while ACMV and SACMV were mostly found in single infections. A greater abundance of whiteflies was observed in lowland and coastal areas. Nevertheless, infected cuttings remain the primary source of CMD propagation (95%) in Madagascar.     

Characterization of Tomato curly stunt virus: a new tomato‐infecting begomovirus from South Africa

The biological and molecular characterization of a virus recognized as a distinct begomovirus species, Tomato curly stunt virus (ToCSV), first observed in South Africa in 1997, is reported here. Whitefly‐transmission and host‐range studies were carried out using a Bemisia tabaci colony identified as the B‐biotype. The experimental host range of ToCSV spanned primarily species in the Solanaceae and Fabaceae. The complete ToCSV genome (2·766 kb) was amplified by PCR, cloned, and the DNA sequence determined. Phylogenetic analysis revealed that ToCSV was most closely related to Tobacco leaf curl Zimbabwe virus (TbLCZV), at 84% nucleotide identity, indicating that ToCSV is a new species in the genus Begomovirus that is probably endemic to southern Africa. The ToCSV genome sequence contained all of the hallmark coding and non‐coding features characteristic of other previously recognized monopartite begomoviruses. ToCSV is only the second begomovirus described from southern Africa that infects solanaceous species. Neither a begomoviral DNA‐B component nor a satellite‐like DNA molecule was detected by PCR in extracts of ToCSV‐infected plants.     

Distribution and accumulation of cassava brown streak viruses within infected cassava (Manihot esculenta) plants

Cassava brown streak disease (CBSD), caused by Cassava brown streak virus (CBSV) and Ugandan cassava brown streak virus (UCBSV), ranks among the top seven biological threats to global food security. The disease poses a significant threat to cassava production in East and Central Africa (ECA). In Uganda, overall CBSD incidence increased by c. 20% since it re‐emerged in 2004, and the disease persistently reduces cassava yields and storage root qualities. The spread of CBSD has been studied spatially in fields in different agroecologies. However, within‐host distribution and accumulation of CBSV and UCBSV in naturally infected cassava plants is unknown. Therefore, within‐host CBSV and UCBSV distribution was studied to correlate CBSD symptoms with virus titre in organs of infected cassava. Leaf, stem and storage root samples, with and without symptoms, were collected from 10 genotypes of field‐grown cassava. Presence of CBSV and UCBSV was detected by RT‐PCR and virus levels determined by qRT‐PCR. CBSV was present in 100% of CBSD samples with symptoms, with 45·3% positive for presence of both CBSV and UCBSV. Tolerant cassava genotypes were infected with CBSV alone and accumulated higher titre in roots than in aerial organs. Susceptible genotypes were co‐infected with CBSV and UCBSV and exhibited variation in virus titre in each organ. Across genotypes, virus titre was lowest in the youngest leaves and highest in mature non‐senescing leaves. This information provides insight into the relationship between CBSV, UCBSV and their cassava host, and is valuable for CBSD resistance breeding, epidemiology studies and CBSD control.     

Occurrence of aphidborne viruses in southernmost South American populations of Fragaria chiloensis ssp. chiloensis

Wild and cultivated Fragaria chiloensis ssp. chiloensis (Fcc) plants were collected at different locations in southern Chile in order to determine the current viral status of this native strawberry. The following aphidborne viruses (ABVs): Strawberry mild yellow edge virus (SMYEV), Strawberry mottle virus (SMoV), Strawberry crinkle virus (SCV) and Strawberry vein banding virus (SVBV), were found in wild and cultivated Fcc plants, but severe symptoms were not associated with viral infection. Furthermore, partial gene sequences of these ABV isolates were determined and displayed a high degree of conservation with virus isolates reported previously. In addition, partial gene sequences of SCV and SVBV from southernmost South American populations of Fcc are described for the first time. High‐throughput parallel sequencing (Illumina) of double‐stranded RNA was used to provide viral profiles of Fcc from different locations. Although strong evidence of novel viruses affecting Fcc was not found, it was confirmed that ABVs are the most frequent viruses affecting this subspecies. The information provided will help in the development of high‐quality molecular tools for virus detection and control in Fcc.