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International audience; Alphasatellites (family Alphasatellitidae) are circular, single-stranded DNA molecules (similar to 1-1.4 kb) that encode a replication-associated protein and have commonly been associated with some members of the families Geminiviridae, Nanoviridae, and Metaxyviridae (recently established). Here, we provide a taxonomy update for the family Alphasatellitidae following the International Committee on Taxonomy of Viruses (ICTV) Ratification Vote held in March 2021. The taxonomic update includes the establishment of the new subfamily Petromoalphasatellitinae. This new subfamily includes three new genera as well as the genus Babusatellite, which previously belonged to the subfamily Nanoalphasatellitinae. Additionally, three new genera and 14 new species have been established in the subfamily Geminialphasatellitinae, as well as five new species in the subfamily Nanoalphasatellitinae.

Nanoviridae is a family of plant viruses (nanovirids) whose members have small isometric virions and multipartite, circular, single-stranded (css) DNA genomes. Each of the six (genus Babuvirus) or eight (genus Nanovirus) genomic DNAs is 0.9–1.1 kb and is separately encapsidated. Many isolates are associated with satellite-like cssDNAs (alphasatellites) of 1.0–1.1 kb. Hosts are eudicots, predominantly legumes (genus Nanovirus), and monocotyledons, predominantly in the order Zingiberales (genus Babuvirus). Nanovirids require a virus-encoded helper factor for transmission by aphids in a circulative, non-propagative manner. This is a summary of the ICTV Report on the family Nanoviridae, which is available at ictv.global/report/nanoviridae.

[EN] Members of the family Pospiviroidae have single-stranded circular RNA genomes that adopt a rod-like or a quasi-rod-like conformation. These genomes contain a central conserved region that is involved in replication in the nucleus through an asymmetric RNA-RNA rolling-circle mechanism. Members of the family Pospiviroidae lack the hammerhead ribozymes that are typical of viroids classified in the family Avsunviroidae. The family Pospiviroidae includes the genera Apscaviroid, Cocadviroid, Coleviroid, Hostuviroid and Pospiviroid, with >25 species. This is a summary of the ICTV Report on the family Pospiviroidae, which is available at ictv.global/report/pospiviroidae., Production of this summary, the online chapter, and associated resources was funded by a grant from the Wellcome Trust (WT108418AIA).

The rust fungus Puccinia vincae was found for the first time in Australia on blue periwinkle (Vinca major). Puccinia vincae was confirmed by comparison of the ITS2-LSU region to reference sequences from Europe as well as by morphology.

[EN] Members of the family Avsunviroidae have a single-stranded circular RNA genome that adopts a rod-like or branched conformation and can form, in the strands of either polarity, hammerhead ribozymes involved in their replication in plastids through a symmetrical RNA-RNA rolling-circle mechanism. These viroids lack the central conserved region typical of members of the family Pospiviroidae. The family Avsunviroidae includes three genera, Avsunviroid, Pelamoviroid and Elaviroid, with a total of four species. This is a summary of the ICTV Report on the taxonomy of the family Avsunviroidae, which is available at /http:/www.ictv.global/report/avsunvirodae., Production of this summary, the online chapter and associated resources was funded by a grant from the Wellcome Trust (WT108418AIA).

Coconut foliar decay virus (CFDV) is transmitted to coconut palms by adults of the cixiid plant hopper Myndus taffini. Larvae of this vector develop on the alternate host, Hibiscus tiliaceus, but the way in which the adults become infective has not been studied. We show by in situ hybridization that viral DNA is associated with gut and ovaries but to distinguish between semipersistent, circulative or propagative modes of transmission we looked for evidence of replication in the vector. Larvae raised from eggs laid on the roots of H. tiliaceus, and adults fed on infected coconut leaf, were tested for the presence of the replicative form of CFDV DNA by a single-primer sense-specific PCR assay which distinguishes between virion-sense and complementary-sense CFDV-DNA. We report that only the virion-sense form of CFDV-DNA could be detected in both larvae and adults whereas both senses, indicating the presence of the double-stranded replicative form of the virus DNA, were detected in infected coconut leaf. About 16 % of the larvae were virus positive by this assay. As the larvae had not had access to coconut palm and there was no evidence of virus replication in the larvae or adults, a persistent-circulative mode of transmission is proposed in which the virus is acquired from H. tiliaceus by nymphs and transmitted transstadially to adults. Confirmation of this hypothesis requires the susceptibility of H. tiliaceus to CFDV to be tested.

The International Committee on Virus Taxonomy (ICTV) is the organization responsible for establishing the rules to classify viroids into species, genera, and families, and ultimately to endorse viroid taxonomy. The ICTV recently called for more stringent rules to create new virus and viroid species, which must be based on at least two independent criteria. This chapter discusses the resulting implications and presents an updated viroid classification scheme that takes into account the identification of new viroids by next-generation sequencing and bioinformatics tools, which ICTV may finally accept as new species.

Coconut cadang-cadang viroid (CCCVd) causes the lethal cadang-cadang disease of coconut palm in the Philippines. Variants of CCCVd are associated with a foliar orange spotting syndrome of African oil palm, showing over 90% sequence identity to CCCVd from coconut, which are also mechanically transmissible, but still require testing for pathogenicity. Coconut tinangaja viroid causes the lethal tinangaja disease of coconut palm in Guam, has about 64% sequence identity to CCCVd, but differs from cadang-cadang in symptomatology. Neither disease is under control and their epidemiology is poorly understood. Viroid-like RNAs with sequence identity to CCCVd have been detected by molecular hybridization in palms and other monocotyledonous plants in many South East Asian and South Pacific countries. Their role in palm viroid epidemiology requires thorough investigation.

The ongoing epidemic of the lethal cadang-cadang disease in the Philippines, caused by coconut cadang-cadang viroid (CCCVd), has killed around 40 million (Mn) coconut palms over the last century. A survey in 2012–13 showed that the boundary of cadang-cadang distribution has not expanded markedly over recent decades. The current incidence is about 700,000 diseased palms and rate of spread is predicted to continue at 0.1%–1.0% per annum. Orange leaf-spotting (OS), a nonlethal disorder of oil palm, is associated with infection by variants of CCCVd. OS has a yield penalty of 25%–50% on individual palms, and overall incidence is estimated to be 1%–5%. Malaysia earns approximately $17.24 billion annually from the oil palm industry, and the national loss from OS is estimated at $25.6–256.2 Mn. Provision of CCCVd-free planting material for new plantations is advocated for raising the yield potential of oil palm. The lethal tinangaja disease, caused by coconut tinangaja viroid, remains an uncontrolled disease of coconut palms in Guam.

Legume-infecting phytoplasmas identified in symptomatic tree medic (Medicago arborea), lucerne (M. sativa) and pigeonpea (Cajanus cajan) growing at a single trial site at Urrbrae, South Australia, were all shown to belong to the 16SrII phytoplasma taxonomic group, but each host species was found to be infected with a different genotype. Within one species (M. sativa) the genotypes were more uniform as demonstrated by comparing five isolates from lucerne plots. These lucerne genotypes differed from the Australian lucerne yellows phytoplasma genotype previously described in New South Wales, and the pigeonpea phytoplasma isolate was more similar to the non-legume infecting Bonamia little leaf (BoLL) than to pigeonpea little leaf phytoplasma. This diversity supports the view that these legumes were infected from different reservoir phytoplasma hosts rather than from a single perennial host such as tree medic.

Genomic mutation in plant viruses of cultivated plants is known to be influenced by virus, host and vector, but the factors influencing mutation in viruses of native plants in natural ecosystems are rarely studied. We have tested the effect of mode of transmission on mutation in Velvet tobacco mottle virus (VTMoV), a mirid-vectored sobemovirus associated with Nicotiana velutina, an Australian native xerophyte growing in a region isolated from anthropogenic influences. Two variants of VTMoV (K1 and R17) were passaged monthly in the alternative experimental plant host, N. clevelandii, for 2 years, either by mechanical inoculation or by transmission with the mirid Cyrtopeltis nicotianae. Sequence variations were scored after 24 passages in regions of the genome containing the open reading frames (ORFs) for the P1 and coat protein (CP). The mean mutation rate was 6.83 × 10(-4) nt/site year, but a higher overall rate was observed for the K1 (satellite -) than the R17 (satellite +) variant. The P1 ORF showed a higher frequency of non-synonymous mutations than the CP. No clear association was found between either mutation site or mutation rate and the mode of transmission, indicating that obligatory mirid transmission had not exerted a specific bottle-neck effect on sequence variation during the experimental time frame. Failure to detect any sequence motifs linked to vector transmission suggests that a specific capsid-stylet interaction is not required for transmission by mirids.

Apple scar skin viroid (ASSVd) is one of the most destructive viroids of pome fruit. We report 12 new sequence variants of ASSVd from apple and pear trees in north-eastern Iran. The Iranian variants ranged in size from 329 to 334 nucleotides. They formed a cluster distinct from other reported ASSVd isolates, and showed sequence variations in specific regions of the viroid RNA. This is the first report of the identification and molecular characterization of ASSVd in Iran.

DNA beta is a single-stranded satellite DNA which encodes a single gene, betaC1. To better understand the role of betaC1 in the pathogenicity of DNA beta, a yeast two-hybrid screen of a tomato cDNA library was carried out using betaC1 from Cotton leaf curl Multan virus (CLCuMV) DNA beta as the bait. A ubiquitin-conjugating enzyme, designated SlUBC3, which functionally complemented a yeast mutant deficient in ubiquitin-conjugating enzymes was identified. The authenticity and specificity of the interaction between betaC1 and SlUBC3 was confirmed both in vivo, using a bimolecular fluorescence complementation assay, and in vitro, using a protein-binding assay. Analysis of deletion mutants of the betaC1 protein showed that a myristoylation-like motif is required both for its interaction with SlUBC3 and the induction of DNA-beta-specific symptoms in host plants. The level of polyubiquitinated proteins in transgenic tobacco plants expressing betaC1 was found to be reduced compared with wild-type plants. These results are consistent with the hypothesis that interaction of betaC1 with SlUBC3 is required for DNA-beta-specific symptom induction, and that this is possibly due to downregulation of the host ubiquitin proteasome pathway.

A ribonuclease protection assay (RPA) has been developed for detecting Coconut cadang-cadang viroid (CCCVd) sequences. An RNA probe complementary to full-length CCCVd246 was used, terminating at nucleotide 65 in the upper conserved region, and linked to a non-viroid 5′ sequence, which acted as an internal control for ribonuclease activity. Extracts from CCCVd-infected coconut (Cocos nucifera) and African oil (Elaeis guineensis) palms protected three major fragments of approximately 250, 125 and 50 nt and a variable number of minor fragments. Extracts of healthy coconut palms, Potato spindle tuber viroid-infected tomato and transfer RNA did not protect the probe. The approximately 250 nt fragment is predicted to indicate the presence of monomers and dimers of circular CCCVd246, linear CCCVd246 with the same termini as the probe and point mutants of these forms. The origin of smaller protected fragments is discussed. RPA-detected CCCVd sequences in 13 of 18 oil palms surveyed in a commercial plantation in Malaysia. Signal intensity varied between the positive oil palms and was generally lower than in coconut palms infected with CCCVd. An infection phenotype was implied but not confirmed by the observation that in a group of 10 oil palms with orange leaf spotting, 9 contained CCCVd, whereas in a group of 8 palms without orange spotting, the viroid was detected in 4. Of four coconut palms in Sri Lanka shown by dot-blot assay to contain CCCVd-related RNA, one was shown by RPA to be positive for the CCCVd246 sequence. RPA is therefore a robust and sensitive test for CCCVd sequences, and our results show that sequences closely related to CCCVd246 are not confined to the Philippines.

We have investigated the ability of satellite DNA beta to complement mutations in the CP, V2 and C4 genes of the monopartite begomovirus, tomato leaf curl virus, which are potentially involved in movement. A mutation in the coat protein was not complemented by DNA beta. Mutations of the C4 and V2 genes attenuated and abolished symptoms, respectively. In the presence of the C4 mutant, but not the V2 mutant, DNA beta induced typical symptoms, confirming that the satellite encodes a dominant symptom determinant. In contrast to the C4 mutant, DNA beta did not enhance the viral DNA levels of the V2 mutant, suggesting that V2 is required for this phenomenon. The significance of these findings is discussed based on our present understanding of the functions of the viral genes and DNA beta.

A floral organ mutant was observed in transgenic Medicago truncatula Gaertn. plants that had two separate stigmas borne on two separate styles that emerged from a single superior carpel primordium. We propose the name bi-pistil, bip for the mutation. We believe this is the first report of such a mutation in this species. Genetic and molecular analyses of the mutant were conducted. The mutant plant was crossed to a mtapetala plant with a wild-type pistil. Expression of the mutant trait in the F1 and F2 generations indicates that the bi-pistil trait is under the control of a single recessive gene. Other modifying genes may influence its expression. The mutation was associated with the presence of a T-DNA insert consisting of the Alfalfa mosaic virus (AMV) coat protein gene in antisense orientation and the nptII selectable marker gene. It is suggested that the mutation is due to gene disruption because multiple copies of the T-DNA were observed in the mutant. The bi-pistil gene was found to be independent of the male-sterile gene, tap. This novel mutant may assist in understanding pistil development in legumes.

The molecular diversity of 14 isolates of Pea seed-borne mosaic virus (PSbMV) from southern Australia, 13 previously described isolates from Pakistan, and a reference isolate from the United States have been studied to determine whether a relatively simple molecular diagnostic assay and classification scheme could be developed for this virus. The Australian isolates were placed into either pathotype P1 or pathotype P4 by bioassay on differential genotypes of Pisum sativum. The Pakistani isolates represented pathotypes P1, P4, U1, and U2, and an undetermined pathotype. The reference US isolate was pathotype P1. A reverse transcription-polymerase chain reaction (RT-PCR) assay based on an amplicon from the variable HC-Pro coding region of potyviruses was shown to distinguish PSbMV from seven other legume infecting potyviruses. Restriction fragment length polymorphisms (RFLPs) generated from the HC-Pro RT-PCR products of all 28 isolates using seven restriction endonucleases placed them into eight groups. A phylogenetic tree based on a Bray-Curtis similarity comparison placed the groups into three clusters. The groups and clusters had no clear association with either pathotype or geographic source. It is concluded that within the range of viruses and isolates tested, the RT-PCR-RFLP method will both specifically identify PSbMV and provide a simple, qualitative, and rapid means for placing PSbMV isolates into groups. Applications could include mapping and tracking isolates in space and time.

Variants of Coconut cadang-cadang viroid have been identified in a plantation oil palm growing in Malaysia. Three size classes are described, comprising 297, 293, and 270 nt. Compared with the 296-nt form of coconut cadang-cadang viroid (CCCVd), all variants substituted C31 --U in the pathogenicity domain and A175 --C in the right-hand terminus. Other mutations and deletions accounted for the different sizes. These are the first sequences reported for variants of Coconut cadang-cadang viroid in a species other than coconut palm, and this is the first evidence that variants closely related to CCCVd occur outside the Philippines.

The incidence of Rupestris stem-pitting-associated virus (RSPaV) in two vineyardsin South Australia was studied by comparing symptoms with the resultsobtained from biological indexing and from virus detection by reverse-transcription-polymerase chain reaction (RT-PCR). Vineyard 1 was an experi-mental block comprising a number of varieties grafted onto Vitis rupestris cv.St George rootstocks for the detection of Rupestris stem pitting disease. Noswelling at graft union or decline was observed at 3 years, but some vinesshowed pitting on the rootstock trunk. Vineyard 2 contained Vitis vinifera cv.Shiraz (Syn. Syrah) vines on Paulsen 1103 rootstock, showing a range ofsymptoms including swelling of the graft union, pitting on the rootstock, leafreddening and vine decline, resembling those in Syrah Decline, a disorderknown to occur in this variety. RT-PCR using the coat-protein-specific pri-mers detected RSPaV in all samples from either vineyard including symp-tomless V. rupestris cv. St George, which is used as the biological indicator forthe virus. In contrast, a pair of primers designed from the replicase genedetected the virus only in symptomatic vines, whereas the symptomlessSt George control and nonsymptomatic vines in both vineyards tested nega-tive. An assay for 12 other major grapevine viruses showed that none wereassociated with either type of symptom. Comparison of a 628-bp amplicon onthe replicase gene in 13 RSPaV isolates showed that while they had 96–99%nucleotide sequence identity to each other, their identity to the Americanisolates from California and New York was around 65%. This low homologymay indicate that a different virus species is present. The variability was morepronounced between the two vineyards than among the virus isolates in theShiraz vines within the same vineyard. However, even the vines of the samevineyard did not contain viruses with exactly the same sequence homology.We found no association between the type of symptom expressed and thesequence variation in the amplicons in either vineyard. The sequence var-iants in Vineyard 1 showed a closer cluster than in Vineyard 2.IntroductionRupestris stem pitting (RSP) is a widespread viral diseaseof the grapevine and has been reported from all overthe world (Martelli, 1993). Flexuous virus particles of800 nm were isolated from infected vines and grape-vine extracts (Conti et al., 1980; Terlizzi & Credi, 2002).Although the aetiology of RSP is not well understood,a virus, Rupestris stem-pitting-associated virus (RSPaV), hasbeen isolated from diseased plants and fully sequencedby two independent laboratories in the USA (Meng et al.,1998, 2005; Zhang et al., 1998). In both labs, grapevine

Geminivirus replication enhancer (REn) proteins dramatically increase the accumulation of viral DNA species by an unknown mechanism. In this study, we present evidence implicating SlNAC1, a new member of the NAC domain protein family from tomato (Solanum lycopersicum), in Tomato leaf curl virus (TLCV) REn function. We isolated SlNAC1 using yeast (Saccharomyces cerevisiae) two-hybrid technology and TLCV REn as bait, and confirmed the interaction between these proteins in vitro. TLCV induces SlNAC1 expression specifically in infected cells, and this upregulation requires REn. In a transient TLCV replication system, overexpression of SlNAC1 resulted in a substantial increase in viral DNA accumulation. SlNAC1 colocalized with REn to the nucleus and activated transcription of a reporter gene in yeast, suggesting that in healthy cells it functions as a transcription factor. Together, these results imply that SlNAC1 plays an important role in the process by which REn enhances TLCV replication.

Analysis of the protein composition of tomato spotted wilt virus (TSWV), purified by an improved procedure, by polacrylamide gel electrophoresis, revealed three major structural proteins (of MW 84,000, 50,000, and 29,000d) and a minor one of MW 220,000d. The three major proteins constitute about 98% of the total viral protein and all three were shown to be glycoproteins. One of the major proteins (MW 29,000d) and the minor protein were shown to be associated with subviral particles isolated by treatment of virus with the nonionic detergent Nonidet P-40. Only traces of the other two proteins were detected in the subviral particles. Synthesis of virus-induced proteins in TSWV-infected tobacco leaves was studied by labeling infected and healthy tissue with [ 3 H]and [ 14 C]valine, respectively. The labeled tissues were then fractionated into crude subcellular fractions and protein patterns of healthy and infected tissues were compared by coelectrophoresis on polyacrylamide gels. Only one virus-specific protein (of MW 49,000d) was detected in the virus-enriched fractions; this corresponded with the viral structural protein of MW 50,000d.

The six open reading frames of Tomato leaf curl virus (TLCV) were expressed in host Nicotiana species using a Tobacco mosaic virus vector. Each of the genes, except that encoding the viral coat protein, produced a phenotypic effect when expressed in planta, but the corresponding untranslatable mutant genes were asymptomatic. The C1 (Rep) gene invoked a hypersensitive response in Nicotiana clevelandii that restricted the viral construct to sites of infection. Expression of the C2 gene in N. benthamiana produced necrotic lesions on inoculated leaves as well as severe veinal necrosis on systemically infected leaves. This gene was also able to suppress post-transcriptional gene silencing in N. tabacum. C4 induced viruslike symptoms in host plants tested, providing further evidence for the involvement of this gene in symptom expression. Expression of the V1 and C3 genes caused severe stunting of N. benthamiana plants, indicating they may also have a role in symptom development. These results reveal that a complex set of interactions between the TLCV gene products and host factors occurs in planta, and these are discussed in relation to our current understanding of TLCV gene function.

Allocasuarina muelleriana shrubs growing in natural sclerophyll roadside vegetation near Willalooka in the upper south-east of South Australia have a high incidence of a yellowing disorder in either all or part of the foliage, combined in some cases with a shortening and curling of the leaf-bearing stems. Samples from symptomatic and adjacent asymptomatic plants were tested for phytoplasmas by the polymerase chain reaction (PCR) assay. All but one asymptomatic plant were negative for phytoplasmas, whereas about half of the symptomatic plants were positive. Restriction fragment polymorphism analysis of PCR products indicated that the phytoplasma was related to the buckthorn witches'-broom (BWB) and apple proliferation (AP) groups of phytoplasmas, members of which have not been previously reported in Australia. Further evidence from the sequence of the 16S rRNA gene and the use of PCR primers specific to the AP and pear decline (PD) phytoplasmas confirmed the close relationship to the BWB and AP group phytoplasmas.

Promoters isolated from the Tomato leaf curl virus (TLCV) drive both constitutive and tissue-specific expression in transgenic tobacco. Following systemic TLCV infection of plants stably expressing TLCV promoter:GUS transgenes, transgene expression driven by all six TLCV promoters was silenced. Silencing in the TLCV coat protein promoter:GUS plants (V2:GUSdeltaC) was characterized in more detail. Transgene silencing observed in leaf, stem, and pre-anthesis floral tissue occurred with the continued replication of TLCV in host tissues. Infection of the V2:GUSdeltaC plants with heterologous geminiviruses did not result in transgene silencing, indicating that silencing was specifically associated with TLCV infection. Nuclear run-on assays indicated that silencing was due to the abolition of transcription from the V2:GUSdeltaC transgene. Bisulfite sequencing showed that silencing was associated with cytosine hypermethylation of the TLCV-derived promoter sequences of the V2:GUSdeltaC transgene. Progeny derived from V2:GUSdeltaC plants silenced by TLCV infection were analyzed. Transgene expression was silenced in progeny seedlings but was partially reactivated in the majority of plants by 75 days postgermination. Progeny seedlings treated with the nonmethylatable cytosine analog 5-azacytidine or the histone deacetylase inhibitor sodium butyrate exhibited partial reactivation of expression. This is the first report of the hypermethylation of a virus-derived transgene associated with a DNA virus infection.

Viroids are the smallest autonomous infectious nucleic acids known so far. With a small circular RNA genome of about 250-400 nt, which apparently does not code for any protein, viroids replicate and move systemically in host plants. Since the discovery of the first viroid almost forty-five years ago, many different viroids have been isolated, characterized and, frequently, identified as the causal agents of plant diseases. The first viroid classification scheme was proposed in the early 1990s and adopted by the International Committee on Taxonomy of Viruses (ICTV) a few years later. Here, the current viroid taxonomy scheme and the criteria for viroid species demarcation are discussed, highlighting the main taxonomic questions currently under consideration by the ICTV Viroid Study Group. The impact of correct taxonomic annotation of viroid sequence variants is also addressed, taking into consideration the increasing application of next-generation sequencing and bioinformatics for known and previously unrecognized viroids.

Sugarcane striate mosaic associated virus (SCSMaV) has slightly flexuous 950 nm x 15 nm filamentous particles and is associated with sugarcane striate mosaic disease in central Queensland, Australia. We report the full sequence of its RNA genome, which comprises 5 open reading frames representing the polymerase, movement function proteins encoded in a triple gene block and coat protein. Phylogenetic analyses based on either the full nucleotide sequence, the polymerase protein, or the coat protein all placed SCSMaV in an intermediate position between the genera Foveavirus and Carlavirus, but outside both genera. In addition, the absence of a sixth open reading frame excludes it from the genus Carlavirus, and the coat protein is approximately half the size of the type member for the genus Foveavirus. Although SCSMaV was most closely allied to Cherry green ring mottle virus by genome analysis, the two viruses are morphologically and biologically dissimilar. SCSMaV may therefore represent a new plant virus taxon.

Sugarcane striate mosaic (ScSM)-affected sugarcane leaves contain a disease-associated 9-kilobase (kb) double-stranded RNA (dsRNA), usually together with 6- and 2.6-kb dsRNAs. The purified 9-kb dsRNA was amplified by the randomly primed polymerase chain reaction (PCR) and cloned. The nucleotide sequences of three separate regions, representing about 2.55 kb (28%) of the dsRNA sequence, were found to have significant similarities to viruses in the genera Capillo-, Carla-, Fovea-, Potex-, Poty-, Tricho-, and Tymovirus. Greatest overall similarity was found to apple stem pitting virus, with less similarity to blueberry scorch virus and potato virus M. A standard virus purification procedure was used to identify slightly flexuous filamentous particles that copurified with the disease-associated RNA. Particle modal lengths were approximately 950 and 1,900 nm with a diameter of 15 nm. Preparations contained a 51-kDa putative capsid protein and a 9-kb single-stranded RNA with a probable 3′ polyadenylate tract. These ScSM-associated virus particles differ physically from viruses in existing genera because of their relative rigidity, length, and putative coat protein size. Reverse-transcription PCR with a primer pair designed from the sequenced segments amplified a 820-base pair fragment from ScSM-affected but not healthy sugarcane plants.

Hodgson, R. A. J., Wall, G. C., and Randles, J. W. 1998. Specific identification of coconut tinangaja viroid for differential field diagnosis of viroids in coconut palm. Phytopathology 88:774-781. Tinangaja is a widespread lethal disease of putative viroid etiology affecting coconut palm on the island of Guam. Determination of its distribution and mode of spread requires a simple and reliable diagnostic procedure that is specific for the associated coconut tinangaja viroid (CTiVd). A method of extracting tissue followed by analytical agarose gel electrophoresis for CTiVd detection has been developed and used to identify the viroid in leaf samples of suspect symptomatic palms growing in the field. Two-dimensional polyacrylamide gel electrophoresis showed that the viroid band contained circular molecules that are typical for viroids. Confirmation of the identity of CTiVd and detection of low levels of viroid below the threshold of detection by agarose gel electrophoresis was achieved either by diagnostic oligonucleotide-probe (DOP) hybridization assay or by reverse-transcription polymerase chain r eaction (RT-PCR) with the oligonucleotide probe as one of the two PCR primers. RT-PCR was not substantially more sensitive than DOP-hybridization assay. This procedure also was applicable to coconut cadang-cadang viroid (CCCVd), and oligonucleotide probes designed to be specific for either CTiVd or CCCVd distinguished between these two viroids in coconut leaf extracts. This strategy provides a rapid and specific indexing procedure for the two characterized viroids of coconut palm and w ill be applicable to further studies on the viroid-like sequences previously reported in tropical monocotyledons.

Viroids are the only class of autonomously replicating subviral pathogens whose molecular structure is well defined. Their structural and functional properties, as well as their evolutionary origin differ fundamentally from those of viruses (see below), posing specific problems for the classification of these subviral pathogens. For example, some of the criteria suggested by the Executive Committee of the International Committee on Taxonomy of Viruses (ICTV) for the definition of species, which include those regarding morphological characteristics, protein characteristics and antigenic properties, are not applicable to viroids. The purpose of this note is to present a consensus taxonomic proposal (see Fig. 1) that has emerged from consultations among the members of the Viroid Study Group of the Plant Virus Subcommittee of the ICTV. This proposal updates and expands the previous classification, which appeared in the Sixth Report of the ICTV [9], and is open to discussion and suggestions.

Spontaneous cleavage of the less abundant form of tobacco ringspot virus satellite RNA is readily reversible. Capitalizing on earlier observations by Feldstein and Bruening that small 'mini-monomer' RNAs derived from this molecule and containing little more than covalently attached ribozyme and substrate cleavage products are able to efficiently circularize, we have constructed a series of self-circularizing RNAs of precisely known size. Mixtures of linear and circular RNAs synthesized in vitro and containing 225-1132 nt could be completely resolved using a novel two-dimensional denaturing polyacrylamide gel electrophoresis system. Similar analyses of a complex mixture of coconut cadang-cadang viroid RNAs revealed the presence of relatively large amounts of a previously undescribed 'fast-slow' heterodimeric RNA species in infected palms. Only a single DNA template is required to prepare each pair of circular and linear RNA markers.

Sixty-two commercial pea fields or experimental plots located in eight districts of the major pea-growing areas of North West Frontier Province (NWFP) of Pakistan were surveyed for pea viruses in the early winter growing season of 1995. Samples were analyzed by dot-immunobinding assay (DIBA) using antisera to 14 different viruses. Of the 713 plants sampled, 82 were positive for either bean yellow mosaic potyvirus, cucumber mosaic cucumovirus, or pea seedborne mosaic potyvirus (PSbMV), with an average incidence of 9.4, 0.57, and 1.5%, respectively. PSbMV was also detected in 1 to 5% of dry seed from five of the 12 pea varieties tested and in 8 to 20% of seedlings raised from seed of three of these varieties. The infectivities of 12 PSbMV isolates found in the survey of pea varieties from Pakistan were compared using a standard range of pea differential genotypes, and the isolates were classified into four distinct pathotypes. Four isolates were classified as pathotype P-1 and two as P-4. The remainder did not fit into the existing PSbMV pathotype classification and were tentatively placed into two other groups named U-1 and U-2.

Virus diseases are serious constraints to the productivity and profitability of a wide range of tropical crops. Identification of the causal viruses and understanding their epidemiology is the key to estimating the incidence and economic impact of the diseases they cause, and to devising virus management strategies. Epidemics result from interactions between virus, host plant, vector and environmental factors, and every epidemic can be considered to be a unique pathosystem in which each of the components contributes to the epidemic, and in which none are limiting. Pathogen diagnosis is the key to managing diseases, and we list 18 crops, their main virus/viroid diseases and pathosystem descriptors for each. Six types of pathosystem are described on the basis of the mode of spread, and the different management strategies applicable to each are discussed. The Ninth Report of the International Committee on Taxonomy of Viruses (2012) is an essential reference for virologists working with tropical crops. Key Concepts: Identification of viruses or viroids is the key to managing the diseases they cause. Koch's rules are applied to establish causal associations between viruses/viroids and disease. Viruses/viroids are identified by their biological, morphological and genetic properties using classical and molecular methods. The universal database for viruses/viroids published by the International Committee on Taxonomy of Viruses is the resource for classification. Rapid diagnostic methods rely on nucleotide sequence comparisons with publicly available data bases. Epidemics are driven by virus, vector, host, environment, time and human activity. Vector–virus interaction is the main determinant of the rate, range, timing and pattern of disease spread. Viruses/viroids of tropical plants have unique pathosystems. A full description of a pathosystem is required to sustainably manage virus epidemics. Management of epidemics can be achieved by destabilising or down-regulating one or more components of the relevant pathosystem. Keywords: Geminiviridae; Potyviridae; Tospovirus; Caulimoviridae; Nanoviridae; Pospiviroidae; Sobemovirus; taxonomy; pathosystem; management

Summary The relationship between time of inoculation with cucumber mosaic cucumovirus (CMV) and the growth, seed production and rate of seed transmission of virus in lupin (Lupinus angustifolius cv. Illyarrie) was studied in field-grown plants. Plants inoculated at the seedling stage (2 days post-emergence) showed 45% mortality. Plants infected through the seed were more stunted than plants inoculated at the seedling stage. Plants inoculated up to the mid-vegetative growth stage (58 days post-emergence) yielded ≤ 27% of the dry matter and ≤ 9% of the seed of healthy plants. Late inoculation (114 days post-emergence) did not affect dry matter yield, but reduced seed yield to 75% of that of healthy plants. Rate of seed transmission depended on the time of inoculation of plants. The maximum rate was 24.5% for plants that were inoculated at the mid-vegetative growth stage (58 days post-emergence). However, early inoculation caused a large reduction in seed yield, and it was shown that plants inoculated at the beginning of flowering (94 days post-emergence) produced greater numbers of infected progeny than plants inoculated at earlier or later times. No relationship was observed between seed weight and transmission of CMV. Infectious CMV was recovered from the embryo, but not from the testa. A simple seed transmission model was used to evaluate several hypothetical epidemics and to determine the time of inoculation which results in greatest rates of seed transmission of CMV. For example, when fewer than 73% of plants in a crop become infected with CMV, then the rate of transmission of virus in crop seeds will be greatest when inoculations are at the beginning of flowering.

DNA β satellites are circular single-stranded molecules associated with some monopartite begomoviruses in the family Geminiviridae. They co-infect with their helper viruses to induce severe disease in economically important crops. The βC1 protein encoded by DNA β is a pathogenicity determinant and has been reported to suppress post-transcriptional gene silencing (PTGS). The βC1 proteins from various DNA β molecules show low levels of amino acid sequence conservation. We show here that the βC1 from DNA β associated with Cotton leaf curl Multan virus (CLCuMV) is a suppressor of systemic PTGS. When this DNA β satellite co-inoculated with a heterologous helper virus, Tomato leaf curl virus (ToLCV), reduced the level of ToLCV siRNA and this was associated with a higher level of virus accumulation in infected tobacco plants. This may be a mechanism by which βC1 protects a heterologous virus from host gene silencing.

Peach latent mosaic viroid isolates from peach and plum in Iran have been compared with an Australian isolate from nectarine. Thirteen sequence variants 336-338 nt in size were obtained. All variants clustered phylogenetically with variants reported from several hosts and countries. A total nucleic acid extract, a slightly longer than full-length RT-PCR amplicon, and a recombinant plasmid clone from the Australian isolate were all infectious to, and symptomatic in, mechanically inoculated peach seedlings. The infectious clone generated two progeny viroid molecules, which each showed 10 different mutations compared with the parent clone inoculated 30 days previously.

Summary Four isolates of pea seed-borne mosaic virus (PSbMV) representing pathotypes P1 (isolates US and Q) and P4 (isolates S4 and S6), and groups III (US and Q) and V (S4 and S6) have been used in a study of the survival and partitioning of PSbMV under conditions of continuous seed transmission in the commercial pea cultivar Dundale. Assays suitable for detecting virus in small tissue samples were developed, and included dot-immunobinding assay with antisera to both PSbMV and cytoplasmic inclusion body (CIB) protein, and dot hybridisation assay (DHA) with cDNA transcribed from virus RNA. Under the conditions of our experiments, seed transmission occurred at rates exceeding 90% for all virus isolates. Virus was detectable by serology and symptoms in inoculated plants, and in all vegetative tissue of second generation plants raised from seed of the inoculated plants. However, in the third, fourth and fifth sequential generations raised from seed, all plants were symptomless. Neither virus nor CIB were detectable in leaf, stem or roots by serology, but both were readily detectable in some floral parts, and in immature and mature seed. Mature seed contained virus and CIB antigen in the testa, cotyledon and embryo. Inoculum prepared from whole seeds was infectious. The testa was shown not to be involved in transmission between generations, thus implicating the embryo alone in vertical transmission. Virus antigen could not be detected in the emerging cotyledons of germinating seed and all true leaves by serology, but the leaves contained PSbMV RNA detectable by DHA. These results show that PSbMV infection can be transferred through the vegetative phase at a subliminal level, and reaches relatively high concentrations in floral parts and seeds. Thus PSbMV may be maintained at a high level of infection in seed in the absence of any apparent symptoms in the plant, and without a requirement for horizontal transmission between plants by vectors. Such a mechanism may explain the high levels of infection commonly reported in pea breeding lines.

Summary Fractionation, electroblotting and molecular hybridisation of nucleic acids extracted from tissue of African oil palm and coconut palm and some other monocotyledonous species, collected in several areas of the south-west Pacific region, demonstrated the presence of small nucleic acids with nucleotide sequences and secondary structure similar to coconut cadang-cadang viroid (CCCVd). The oil palms which contained CCCVd-related molecules showed orange leaf spots resembling those described for oil palm naturally infected with CCCVd in the Philippines, and also characteristic of a condition known as “genetic orange spotting” (GOS). We provide preliminary evidence that GOS is an infectious disorder caused by a viroid. The coconut palms did not show symptoms typical of cadang-cadang disease, but sometimes were chlorotic, stunted, or had a reduced yield. The possibility that the isolates represent variants of CCCVd is discussed. The data suggest that viroids with nucleotide sequences similar to CCCVd occur widely in palms and other monocotyledons outside the Philippines.

DNA beta is a circular single-stranded satellite DNA associated with certain monopartite begomoviruses (family Geminiviridae) which causes economically important diseases such as cotton leaf curl disease. DNA beta contains a single gene, betaC1, which encodes a pathogenicity protein responsible for symptom production. Transient expression studies in Nicotiana tabacum using the beta-glucuronidase reporter gene driven by a betaC1 promoter-deletion series of the DNA beta associated with cotton leaf curl Multan virus identified a 68 nt region (between -139 and -207) which is important for betaC1 transcription. This 68 nt region contains a G-box (CACGTG) located 143 nt upstream of the betaC1 start codon. Mutation of the G-box resulted in a significant reduction in betaC1 promoter activity and DNA beta replication efficiency. In addition, the G-box motif was found to bind specifically to a protein(s) in nuclear extracts prepared from tobacco leaf tissues. Our results indicate that interaction of the G-box motif with host nuclear factors is important for efficient gene expression and replication of DNA beta.