Cucumber Mosaic Virus Satellite RNA Research Articles

Heterologous replicase driven 3′ end repair of Cucumber mosaic virus satellite RNA

To investigate the extent of the 3′ end repair in a satellite RNA of Cucumber mosaic virus (CMV) strain Q (Qsat) by a heterologous Tomato aspermy virus (TAV), a set of biologically active agrotransformants corresponding to the three genomic RNAs of TAV was developed. Analysis of Nicotiana benthamiana plants agroinfiltrated with TAV and either wild type or each of the six 3′ deletion mutants of Qsat revealed that (i) heterologous replicase failed to generate Qsat multimers, a hallmark feature of homologous replicase dependent replication of Qsat; (ii) manifestation of severe symptom phenotypes and progeny analysis suggested that heterologous replicase was competent to repair Qsat deletion mutants lacking up to 3′13 nucleotides (nt) but not beyond and (iii) comparative in silico analysis indicated that the 3′ secondary structural features of the repaired Qsat progeny from heterologous vs homologous driven replicases are remarkably very similar. The significance of these observations is discussed.

Nicotiana small RNA sequences support a host genome origin of cucumber mosaic virus satellite RNA.

Satellite RNAs (satRNAs) are small noncoding subviral RNA pathogens in plants that depend on helper viruses for replication and spread. Despite many decades of research, the origin of satRNAs remains unknown. In this study we show that a β-glucuronidase (GUS) transgene fused with a Cucumber mosaic virus (CMV) Y satellite RNA (Y-Sat) sequence (35S-GUS:Sat) was transcriptionally repressed in N. tabacum in comparison to a 35S-GUS transgene that did not contain the Y-Sat sequence. This repression was not due to DNA methylation at the 35S promoter, but was associated with specific DNA methylation at the Y-Sat sequence. Both northern blot hybridization and small RNA deep sequencing detected 24-nt siRNAs in wild-type Nicotiana plants with sequence homology to Y-Sat, suggesting that the N. tabacum genome contains Y-Sat-like sequences that give rise to 24-nt sRNAs capable of guiding RNA-directed DNA methylation (RdDM) to the Y-Sat sequence in the 35S-GUS:Sat transgene. Consistent with this, Southern blot hybridization detected multiple DNA bands in Nicotiana plants that had sequence homology to Y-Sat, suggesting that Y-Sat-like sequences exist in the Nicotiana genome as repetitive DNA, a DNA feature associated with 24-nt sRNAs. Our results point to a host genome origin for CMV satRNAs, and suggest novel approach of using small RNA sequences for finding the origin of other satRNAs.

Cloning and profiling of small RNAs from cucumber mosaic virus satellite RNA.

RNA silencing is not only a gene regulation mechanism that is conserved in a broad range of eukaryotes but also an adaptive immune response against foreign nucleic acids including viruses in plants. A major feature of RNA silencing is the production of small RNA (sRNA) of 21-24 nucleotides (nt) in length from double-stranded (ds) or hairpin-like (hp) RNA by Dicer-like (DCL) proteins. These sRNAs guide the binding and cleavage of cognate single-stranded (ss) RNA by an RNA silencing complex. Like all plant viruses and subviral agents, replication of viral satellite RNAs (satRNAs) is associated with the accumulation of 21-24 nt viral small interfering RNA (vsiRNA) derived from the whole region of a satRNA genome in both plus and minus-strand polarities. These satRNA-derived siRNAs (satsiRNAs) have recently been shown to play an important role in the trilateral interactions among host plants, helper viruses and satRNAs. Here, we describe the cloning and profile analysis of satsiRNAs from satRNAs of Cucumber mosaic virus (CMV). We also describe a method to minimize the strand bias that often occurs during vsiRNA cloning and sequencing.

VIRAL SATELLITE RNA AS A BIOLOGICAL CONTROL AGENT FOR PLANTS

Biological control technology based on viral satellite mediated protection is a novel concept of the viral disease control strategies. Cucumber mosaic virus (CMV) is considered one of the most economically damaging viruses among field grown vegetables such as tomato (Solanum lycopersicum L). CMV can harbor molecular parasites known as satellite RNAs that dramatically alter the symptom phenotype induced by the virus. Two indigenous strains of CMV satellite RNA were isolated and have been sequenced. One of these strains designated as CMV‐KU1 reduced the viral disease to a virtually asymptomatic condition. The other strain was found to aggravate the symptoms and cause severe crop loss and tomato necrosis. Thus, satellites have been referred to as “natural inhibitors of crop damaging viruses’. These satellites RNAs are small nucleic acids whose nucleotide sequences are unrelated to, but are replicatively dependent upon the viral genome; they have a molecular parasitic relationship. Particularly, the KU1 strain may be used to protect plants against a particular viral strain of CMV, referred to as the KU2 strain. The attenuating properties of some satellite RNAs can be exploited to control viral diseases by preinoculating crops with an attenuating satellite RNA variant combined with virus. This technique can effectively protect against CMV disease in the field.

Functional significance of a hepta nucleotide motif present at the junction of Cucumber mosaic virus satellite RNA multimers in helper-virus dependent replication

Satellite RNAs (satRNA) associated with Cucumber mosaic virus (CMV) have been shown to generate multimers during replication. We have discovered that multimers of a CMV satRNA generated in the absence of its helper virus (HV) are characterized by the addition of a hepta nucleotide motif (HNM) at the monomer junctions. Here, we evaluated the functional significance of HNM in HV-dependent replication by ectopically expressing wild type and mutant forms of satRNA multimers in planta either in (+) or (−)-strand polarity. Comparative replication profiles revealed that (−)-strand multimers with complementary HNM (cHNM) are the preferred initial templates for HV-dependent replication than (−)-strand monomers and multimers lacking the cHNM. Further mutational analyses of the HNM accentuate that preservation of the sequence and native length of HNM is obligatory for efficient replication of satRNA. A model implicating the significance of HNM in HV-dependent production of monomeric and multimeric forms of satRNA is presented.

Duplex polymerase chain reaction method for detection of unapproved genetically modified tomato (Solanum lycopersicon L.) with cucumber mosaic virus (CMV) satellite RNA gene

The complete sequence of the cucumber mosaic virus (CMV) satellite RNA (satRNA) gene that was controlled by the cauliflower mosaic virus (CaMV) 35S promoter (P-35S) and the Agrobacterium nopaline synthase terminator (T-nos) was first identified in an unapproved genetically modified (GM) tomato (Solanum lycopersicum L.), and a duplex polymerase chain reaction (PCR) method was developed based on the CMV satRNA nucleotide sequence. To detect the unapproved GM tomato, the metallocarboxypeptidase inhibitor (Mcpi) gene was selected as an endogenous reference gene for tomato and was validated using 13 different crops. The primer pair PTD-F/R was designed to amplify the junction sequences between the 35S promoter and CMV satRNA gene introduced in the unapproved GM tomato, and its specificity was also validated using several different GM events. The detection limit of the duplex PCR method is approximately 1 copy. Using the duplex PCR method, 35 processed tomato foods and 13 tomato seeds were analyzed. Of these samples, 2 GM tomato seeds were identified using the duplex PCR method. The results indicate that this duplex PCR method could be useful for detecting the unapproved GM tomato.

Characterization of potato and tobacco isolates of Cucumber mosaic virus from Syria and the first report on CMV satellite RNA from potato

Cucumber mosaic virus (CMV) has been reported from potato production areas in Europe, USA, Japan and more frequently in regions with warm climates such as Egypt, India, Saudi Arabia and Syria. As it is considered as an uncommon virus in potato, the characterization of potato isolates of CMV is far behind those from other hosts. In addition to potato, CMV is a common virus infecting many crops in Syria, but nothing is known about its molecular characteristics. The present study aimed to characterize Syrian CMV isolates collected from potato and neighboring tobacco fields. All potato isolates of CMV (total of four) co-infected potato plants with Potato virus Y (PVY) which is the most frequent potato virus in Syria. According to the sequence analyses of the coat protein (CP) coding region, three potato and three tobacco CMV isolates were found to be closely related regardless of the host species or geographic origin, and all belonged to the IA strain subgroup of CMV. A potato CMV isolate, PoCMV7-5, readily infected solanaceous plants in which it induced systemic infection, but was less infectious to other hosts including those of Leguminosae and Cucurbitaceae . When inoculated on potato plants, PoCMV7-5 alone or with various PVY strains was able to cause local but not systemic infection in all potato cultivars inoculated. PoCMV7-5 contained heterogeneous variants of satellite RNA which varied in length due to A or/and T deletion/insertion at approximate nucleotide position 225‒240. This is the first report on CMV satellite RNA from potato.

Cucumber mosaic virus satellite RNAs that induce similar symptoms in melon plants show large differences in fitness

Two groups of Cucumber mosaic virus (CMV) satellite RNAs (satRNAs), necrogenic and non-necrogenic, can be differentiated according to the symptoms they cause in tomato plants, a host in which they also differ in fitness. In most other CMV hosts these CMV-satRNA cause similar symptoms. Here, we analyse whether they differ in traits determining their relative fitness in melon plants, in which the two groups of CMV-satRNAs cause similar symptoms. For this, ten necrogenic and ten non-necrogenic field satRNA genotypes were assayed with Fny-CMV as a helper virus. Neither type of CMV-satRNA modified Fny-CMV symptoms, and both types increased Fny-CMV virulence similarly, as measured by decreases in plant biomass and lifespan. Necrogenic and non-necrogenic satRNAs differed in their ability to multiply in melon tissues; necrogenic satRNAs accumulated to higher levels both in single infection and in competition with non-necrogenic satRNAs. Indeed, multiplication of some non-necrogenic satRNAs was undetectable. Transmission between hosts by aphids was less efficient for necrogenic satRNAs as a consequence of a more severe reduction of CMV accumulation in leaves. The effect of CMV accumulation on aphid transmission was not compensated for by differences in satRNA encapsidation efficiency or transmissibility to CMV progeny. Thus, necrogenic and non-necrogenic satRNAs differ in their relative fitness in melon, and trade-offs are apparent between the within-host and between-host components of satRNA fitness. Hence, CMV-satRNAs could have different evolutionary dynamics in CMV host-plant species in which they do not differ in pathogenicity.

Multiplex RT-PCR detection of <italic>Cucumber mosaic virus</italic> subgroups and <italic>Tobamoviruses</italic> infecting Tomato using 18S rRNA as an internal control

A multiplex reverse-transcription polymerase chain reaction (RT-PCR) protocol was developed for simultaneous detection and discrimination of subgroups of Cucumber mosaic virus (CMV), including its satellite RNA, Tomato mosaic virus (ToMV) and Tobacco mosaic virus (TMV), using 18S rRNA as an internal control. Species- and subgroups-specific primers designed to differentiate CMV subgroups I and II, ToMV and TMV, were assessed using the cDNA clones of viral genomes, CMV satellite RNA and 18S rRNA gene from tomato (Solanum lycopersicum L.) or tobacco (Nicotiana tobacum). Using total RNA extracted from artificial mixture of tomato leaf tissues infected by each virus, the reaction components and cycling parameters were optimized and a multiplex RT-PCR procedure was established. Six fragments of 704, 593, 512, 421, 385, 255 bp, specific to CMV subgroup II, CMV subgroup I, ToMV, TMV, satellite RNA and 18S rRNA, respectively, were simultaneously amplified. The sensitivity of the multiplex RT-PCR method for detecting CMV was 100 times higher than that of double-antibody sandwich-enzyme-linked immunosorbent assay (DAS-ELISA). This method was successfully used for field detection. Among 141 samples collected from East China through tomato growth seasons, 106 single infections with one of the above isolates were detected and 13 mixed infections were found. The results showed the potential use of this method for investigating the epidemiology of viral diseases infecting tomato.

Differential effects of Cucumber mosaic virus satellite RNAs in the perturbation of microRNA-regulated gene expression in tomato

Viral infections generally cause disease symptoms by interfering with the microRNA (miRNA)-mediated regulation of gene expression of host plants. In tomato leaves, the accumulation levels of eleven miRNAs and ten target mRNAs were enhanced by different degrees upon Cucumber mosaic virus (CMV)-Fny and Tomato aspermy virus (TAV)-Bj infections. The ability of CMV-Fny to interfere with miRNA pathway was dramatically suppressed in the addition of the benign satellite (sat) RNA variant (satYn12), but was slightly affected when CMV-Fny was co-inoculated with the aggressive satRNA variant (satT1). In plants harboring the infection of CMV-FnyΔ2b (a CMV-Fny 2b-deletion mutant), the unaltered miRNAs and target mRNAs levels compared with mock inoculated plants indicated that 2b ORF was essential for perturbation of miRNA metabolism. When the amounts of viral open reading frames (ORFs) in these infections were quantified, we found satYn12 caused a higher reduction of CMV-Fny accumulation levels than satT1. These results indicate the complex mechanism by which satRNAs participate in CMV-tomato interaction, and suggest that the severity of disease symptoms positively correlates to some extent with the perturbation of miRNA pathway in tomato.

Characterisation of a satellite RNA of Cucumber mosaic virus that induces chlorosis in Capsicum annuum

The presence of Cucumber mosaic virus (CMV) satellite RNA dramatically changes symptoms on some hosts. A satellite RNA present in a strain of CMV (PepY-CMV) that induced chlorosis in pepper (Capsicum annuum) was shown to induce chlorosis in pepper in combination with another strain (Fny-CMV) that by itself induced a green mosaic symptom. The location of sequences within the PepY satellite RNA (PepY-satRNA) of CMV that conferred the ability to induce chlorosis on pepper plants were analyzed by exchanging sequence domains between cDNA clones of PepY-satRNA and an attenuated mosaic satellite RNA (Paf-satRNA), as well as site-directed mutagenesis of various clusters of the 22-nt sequence differences between the two satellite RNAs in the delimited central domain. The symptoms induced by site-directed mutants of PepY-satRNA and Paf-satRNA in the presence of Fny-CMV demonstrated an insertion within PepY-satRNA of 11 nt at positions 86-96 relative to Paf-satRNA determined the chlorosis-inducing phenotype. Within the chlorosis-inducing domain, deletion of nucleotides did not affect the satRNA replication but abolished the ability of PepY-satRNA to elicit chlorosis symptom. Conversely, a mutant satellite RNA derived from Paf-satRNA in which eleven nucleotides were inserted indicated that sequences of 11 nucleotides were found to be sufficient for chlorosis induction in pepper.

Oligonucleotide microarray-based detection and identification of 10 major tomato viruses

A DNA microarray chip was developed for screening 10 major economically important tomato viruses from infected plants using “Combimatrix” platform 40-mer oligonucleotide probes. A total of 279 oligonucleotide virus probes were specific for simultaneous multiple detection, identification, differentiation and/or genotyping of each of the following tomato RNA viruses and/or strains and a virus satellite: Cucumber mosaic virus, Cucumber mosaic virus satellite RNA, Tomato infectious chlorosis virus, Tomato chlorosis virus, Tomato spotted wilt virus, Pepino mosaic virus, Potato virus Y, Tobacco mosaic virus and Tomato mosaic virus. This selection included both positive and negative single-stranded RNA viruses. The single-stranded DNA viruses, Tomato yellow leaf curl virus and Tomato yellow leaf curl Sardinia virus were detected but were not differentiated using probes designed from their coat protein genes. A sectored oligonucleotide microarray chip containing four sets of 2000 features (4 × 2K) was designed. In this way, four samples were tested simultaneously in a hybridization event and 16 samples were analyzed by re-using the chip four times. The hybrids had low background signals. Many of the 40-mer oligonucleotide probes were specific for the detection and identification of each RNA viral species, RNA viral satellite and genotyping strains of Cucumber mosaic virus, Pepino mosaic virus and Potato virus Y. Universal probes were developed for strains of the last three viruses and also for the genus Tobamovirus which includes both Tobacco mosaic virus and Tomato mosaic virus.

RNAi-mediated viral immunity requires amplification of virus-derived siRNAs in Arabidopsis thaliana

In diverse eukaryotic organisms, Dicer-processed, virus-derived small interfering RNAs direct antiviral immunity by RNA silencing or RNA interference. Here we show that in addition to core dicing and slicing components of RNAi, the RNAi-mediated viral immunity in Arabidopsis thaliana requires host RNA-directed RNA polymerase (RDR) 1 or RDR6 to produce viral secondary siRNAs following viral RNA replication-triggered biogenesis of primary siRNAs. We found that the two antiviral RDRs exhibited specificity in targeting the tripartite positive-strand RNA genome of cucumber mosaic virus (CMV). RDR1 preferentially amplified the 5'-terminal siRNAs of each of the three viral genomic RNAs, whereas an increased production of siRNAs targeting the 3' half of RNA3 detected in rdr1 mutant plants appeared to be RDR6-dependent. However, siRNAs derived from a single-stranded 336-nucleotide satellite RNA of CMV were not amplified by either antiviral RDR, suggesting avoidance of the potent RDR-dependent silencing as a strategy for the molecular parasite of CMV to achieve preferential replication. Our work thus identifies a distinct mechanism for the amplification of immunity effectors, which together with the requirement for the biogenesis of endogenous siRNAs, may play a role in the emergence and expansion of eukaryotic RDRs.

Biological and Molecular Characterization of Taiwanese Isolates ofCucumber mosaic virusAssociated with Banana Mosaic Disease

Banana mosaic disease (BMD) caused by Cucumber mosaic virus (CMV) has become an important threat to the banana industry. We collected and characterized 10 CMV isolates associated with BMD in Taiwan and compared their biological characteristics and coat protein sequences. The isolates fell into four pathotypes on the basis of the symptoms they induce on banana, Nicotiana glutinosa and Vigna unguiculata (cowpea). Double-stranded RNA analysis revealed that the different pathotypes are not related to the presence of CMV satellite RNA. Phylogenetic analysis of worldwide CMV coat protein sequences revealed that among the currently known CMV subgroups IA, IB and II, subgroup IB is phylogenetically unresolved. Our CMV isolates form a new subgroup, IT, within subgroup I. In addition, we resolved another new CMV subgroup, IS, within subgroup I. The analysis also revealed that isolates within different subgroups can infect the banana.

Leonotis nepetaefolia: An important plant virus reservoir in central Mexico

The presence of viruses in the weedLeonotis nepetaefolia in central Mexico 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.

Analysis of mechanisms involved in the Cucumber mosaic virus satellite RNA-mediated transgenic resistance in tomato plants.

Transgenic tomato (Lycopersicon esculentum Mill. cv. UC82) plants expressing a benign variant of Cucumber mosaic virus satellite RNA (CMV Tfn-satRNA) were generated. The transformed plants did not produce symptoms when challenged with a satRNA-free strain of CMV (CMV-FL). The same plant lines initially were susceptible to necrosis elicited by a CMV strain supporting a necrogenic variant of satRNA (CMV-77), but a phenotype of total recovery from the necrosis was observed in the newly developing leaves. The features of the observed resistance were analyzed and are consistent with two different mechanisms of resistance. In transgenic plants inoculated with CMV-FL strain, the symptomless phenotype was correlated to the down-regulation of CMV by Tfn-satRNA, amplified from the transgene transcripts, as the first resistance mechanism. On the other hand, the delayed resistance to CMV-77 in transgenic tomato lines was mediated by a degradation process that targets satRNAs in a sequence-specific manner. Evidence is provided for a correlation between a reduced accumulation level of transgenic messenger Tfn-satRNA, the accumulation of small (approximately 23 nucleotides) RNAs with sequence homology to satRNAs, the progressively reduced accumulation of 77-satRNA in infected tissues, and the transition in infected plants from diseased to healthy. Thus, events leading to the degradation of satRNA sequences indicate a role for RNA silencing as the second mechanism determining resistance of transgenic tomato lines.

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.

Tomato Cell Death Mediated By Complementary Plant Viral Satellite RNA Sequences

Cell death (necrosis) and severe yellowing (chlorosis) in tomato are induced by cucumber mosaic virus (CMV) supporting particular satellite RNAs. To determine whether CMV RNA sequences also are needed to induce necrosis or chlorosis, tomato seedlings were infected with the potato virus X vector expressing either a necrogenic or chlorosis-inducing satellite RNA of CMV. The infected plants did not develop chlorosis, although they did develop necrosis, but only when all or part of a 335-nucleotide necrogenic satellite RNA was expressed in the (-) polarity; i.e., the strand not packaged in virus particles. Computer-assisted secondary structure analysis suggests that the necrogenicity domain is an octanucleotide loop and adjacent base-paired stem of a thermodynamically stable hairpin structure.

Template-independent repair of the 3' end of cucumber mosaic virus satellite RNA controlled by RNAs 1 and 2 of helper virus.

RNA viruses which do not have a poly(A) tail or a tRNA-like structure for the protection of their vulnerable 3' termini may have developed a different strategy to maintain their genome integrity. We provide evidence that deletions of up to 7 nucleotides from the 3' terminus of cucumber mosaic cucumovirus (CMV) satellite RNA (satRNA) were repaired in planta in the presence of the helper virus (HV) CMV. Sequence comparison of 3'-end-repaired satRNA progenies, and of satRNA and HV RNA, suggested that the repair was not dependent on a viral template. The 3' end of CMV satRNA lacking the last three cytosines was not repaired in planta in the presence of tomato aspermy cucumovirus (TAV), although TAV is an efficient helper for the replication of CMV satRNA. With use of pseudorecombinants constructed by the interchange of RNAs 1 and 2 of TAV and CMV, evidence was provided that the 3'-end repair was controlled by RNAs 1 and 2 of CMV, which encode subunits of the viral RNA replicase. These results, and the observation of short repeated sequences close to the 3' terminus of repaired molecules, suggest that the HV replicase maintains the integrity of the satRNA genome, playing a role analogous to that of cellular telomerases.

Cucumber Mosaic Virus, Tobacco Streak Virus, and Cucumber Mosaic Virus Satellite RNA Associated with Mosaic and Ringspot Symptoms in Ajuga reptans in Ohio.

Creeping bugleweed (Ajuga reptans L.) is a perennial ornamental commonly grown as a ground cover in temperate climates. Commercial samples of the A. reptans cultivars Royalty, var. Atropurpurea Bronze, Bronze Beauty, and Burgundy Glow showing mosaic and ringspot symptoms were tested for the presence of virus infection by direct antibody sandwich enzyme-linked immunosorbent assay (ELISA) and viral-associated double-stranded (ds) RNA analysis. Cucumber mosaic cucumovirus (CMV) was detected by ELISA and dsRNA analysis in symptomatic samples of all cultivars tested. ELISA values were considered positive if the absorbance values were twice the negative control. Negative control values were established with asymptomatic tissue of the cv. Bronze Beauty. Tobacco streak ilarvirus (TSV) was detected only by ELISA in symptomatic samples of all cultivars except Royalty. No dsRNA suggestive of TSV was detected. Alfalfa mosaic virus (AMV) was detected by ELISA and dsRNA analysis in symptomatic samples of all cultivars tested except Royalty and var. Atropurpurea Bronze. dsRNA analysis also indicated the presence of a low molecular weight, possible satellite (sat) RNA associated with all symptomatic and asymptomatic Royalty and var. Atropurpurea Bronze plants tested. Northern (RNA) blot analysis with a digoxigenin-labeled full-length clone of the (S) CARNA-5 (-) CMV satRNA (ATCC no. 45124) confirmed that the low molecular weight RNA associated with the Royalty and var. Atropurpurea Bronze cultivars was indeed CMV satRNA. Only AMV has been previously reported in A. reptans in the United States (1). This is the first report of CMV and its satRNA, as well as TSV, in A. reptans in the United States. Reference: (1) W. T. Schroeder and R. Provvidenti. Plant Dis. Rep. 56:285, 1972.