Low genetic diversity of Banana bunchy top virus, with a sub-regional pattern of variation, in Democratic Republic of Congo.

Banana bunchy top virus (BBTV), belonging to the genus Babuvirus, is the most devastating and widespread banana virus. Banana and plantain are major crops in terms of household income and food security in Democratic Republic of Congo (DRC). Despite the large area under banana and plantain cultivation in the country, before this study, the genetic characterization of BBTV isolates had only been undertaken for two provinces. In the study presented here, genetic variation in BBTV was assessed from 52 BBTV isolates collected in five out of 11 provinces in DRC (Bandundu, Bas-Congo, Katanga, Kinshasa and Kasaï Oriental) and in two provinces using sequences previously described in databases. Full genome sequencing of DNA-R components was performed, revealing low genetic variation (98-100 % nucleotide identity) among the BBTV isolates detected. The phylogenetic analyses showed that all the DRC isolates were clustered in the South Pacific clade of BBTV. Based on the coding region for the replication initiator protein, haplotype diversity was estimated to be 0.944 ± 0.013, with 30 haplotypes from 68 isolates in DRC. Such diversity shows a haplotype distribution mainly at the sub-regional level in DRC. In addition, the sequence determination from the whole genome of selected isolates confirmed low genetic variation among isolates from seven DRC provinces (97-100 % nucleotide identity). This study strengthened the hypothesis of a single BBTV introduction some time ago, followed by the spread of the virus in the country.

NEW BNYVV P25 VARIANTS IN BELGIUM.

Rhizomania is a widespread viral plant disease of major importance in sugar beet cropping and breeding. It is caused by the Beet necrotic yellow vein virus (BNYVV), a Benyvirus transmitted by the soil inhabiting plasmodiophorid Polymyxa betae. This vector also transmits other sugar beet virus such as Beet virus Q (BVQ) and Beet soil-borne virus (BSBV). Despite identification of resistance genes, BNYVV remains a major constraint because of resistance-breaking events as well as its ability to survive for long periods in soils in resting spores of P. betae. During the 2014 growing season, severe rhizomania symptoms were detected in Rz1 resistant beet genotypes in ten Belgian fields suggesting resistance-breaking events. Plants from these fields were sampled and total RNA was extracted from root hairs. The presence of BNYVV, BSBV, BVQ and P. betae was assessed by multiplex RT-PCR. Samples were then tested for the presence of BNYVV RNA5 and RNA3 by RT-PCR respectively targeting P26 and P25 genes. PCR products from P25 gene were then purified and sequenced. The results confirmed the presence of P. betae, BSBV and BVQ in all samples. BNYVV was detected in nine fields. Sequencing of P25 partial cDNA sequences revealed the presence of BNYVV types A and B. Two isolates possessed the amino acids motifs AYPR in the so-called tetrad region aa67-70. This motif was previously associated with resistance-breaking events. The Belgian situation will be discussed in the light of the current situation in neighbouring countries.

Dual infection by cassava begomoviruses in two leguminous species (Fabaceae) in Yangambi, Northeastern Democratic Republic of Congo.

A study on cassava mosaic begomoviruses was conducted around Yangambi (DR Congo) by sampling 10 different leguminous species with or without symptoms similar to cassava mosaic disease. DNA was isolated to amplify CMBs using primers targeting AC2 and AC4 genes for virus detection by PCR. The results showed a dual infection by ACMV and EACMV in two weed species, Centrosema pubescens and Pueraria javanica, associated with mosaic symptoms. The DNA-A genome component of ACMV and EACMV from the infested weeds was sequenced. Seven ACMV and four EACMV isolates are reported. The major ACMV strains were closely related to ACMV-NGogo, ACMV-IC and ACMV-UGMld, whereas all EACMV strains were closely related to a Uganda variant, the most prevalent virus. This study shows that whiteflies may transmit CMBs to non-cassava plants under high epidemic pressure.

Arabidopsis thaliana, a new tool to investigate Polymyxa betae-host interactions.

Little is known about the genome of Polymyxa betae and its interactions with sugar beet, due partly to the obligate nature of the protist and the patents on Beta vulgaris sequences. The identification of an ecotype of Arabidopsis thaliana compatible with the protist would help to improve this knowledge. The infection and development of P. betae in 14 worldwide ecotypes of A. thaliana were studied. The detection of plasmodia and resting spores and the production of zoospores in the roots of A. thaliana were obtained in three bioassays, using automatic immersion systems and individual glass tubes. Detection was done using molecular detection and microscopy. Compatible interactions were established between 13 A. thaliana ecotypes of the 14 that were tested and the monosporosoric Belgian strain of P. betae, A26-41. The ecotype Cvi-0 (N1096), from the Cape Verde Islands, was the most compatible with the protist. This ecotype is also susceptible to Plasmodiophora brassicae, another plasmodiophorid. Polymyxa betae infection in A. thaliana was relatively very low compared with B. vulgaris, but every stage of the life cycle of the protist was present. The spore-forming phase was promoted at the expense of the sporangial phase, probably caused by the stress of this new environment. In addition, the protist revealed a new phenotype. This new model study will allow molecular tools available for A. thaliana to be used in order to gain a better understanding of the P. betae-plant interaction during the spore-forming phase.

Seed, soil and vegetative transmission contribute to the spread of pecluviruses in Western Africa and the Indian sub-continent.

Peanut clump and sugarcane red leaf mottle diseases are caused by viruses of the genus Pecluvirus. Indian peanut clump virus occurs in the Indian sub-continent and Peanut clump virus in West Africa. A feature of these viruses is that they are both seed and soil transmitted. Both modes of transmission contribute to long-term persistence and field spread. Data on seed transmission in pearl millet, virus movement within the plant and virus diversity based on RNA-1 partial sequences are presented. This study emphasizes that pecluviruses are also viruses of cereals infecting sorghum and pearl millet, and highlights a correlation between the countries cultivating these two crops and the virus distribution. Ways of controlling pecluviruses and their vector, Polymyxa graminis, taking into account the virus dissemination routes, are proposed.

Iranian beet necrotic yellow vein virus (BNYVV): pronounced diversity of the p25 coding region in A-type BNYVV and identification of P-type BNYVV lacking a fifth RNA species.

Beet necrotic yellow vein virus (BNYVV) was detected in 288 of the 392 samples collected in Iran. A-type BNYVV was detected most frequently. The p25 coding region on BNYVV RNA-3 was amplified by RT-PCR and sequenced. Nine different variants of the highly variable amino acid tetrad at positions 67-70 of p25 were identified, i.e. ACHG, AHHG, AYHG, ALHG, AFHR, AFHG, AHYG, VLHG and VHHG. These are more different tetrad variants than have been reported from any other country. The first three variants were found most commonly. In 23 out of the 288 BNYVV-positive samples, we detected P-type BNYVV that had previously been identified only in France, Kazakhstan and recently in the UK. Surprisingly, none of these samples contained the fifth RNA species usually associated with P-type BNYVV in other countries. As in other BNYVV P-type sources, the p25 amino acid tetrad in positions 67-70 of the Iranian P-type consists of SYHG.

Peanut Clump virus transmission by Polymyxa graminis under controlled conditions.

More than fifteen soil-borne viruses belonging to the Beny-, Bymo-, Furo- or Pecluvirus, causing diseases on cereals and groundnut, are transmitted by the soil-borne protist root endoparasite Polymyxo graminis. Five special forms are distinguished within this species on the basis of their specific ecological and molecular characteristics, but the specificity of the transmission of the viruses by these forms has been Little investigated. In order to analyse the virus-vector interaction, the transmission assay of the Peanut clump virus by P. graminis f.sp. tropicalis has been conducted under controlled conditions. The major difficulty to be overcome was to combine high levels of PCV and vector infection in the same living plant. This was achieved by using cuttings of PCV-infected sugarcane (variety CP-89327) originating from Burkina Faso showing the typical red leaf mottle symptom as a source for the virus. A culture of P. graminis f.sp. tropicalis isolated from a PCV-infested soil from Niger was used as the vector. Systemic PCV infection in sugarcane at 25-30 degrees C allows plants with new young roots infected by the virus to be produced by placing cuttings of PCV-infected sugarcane in Hoagland nutrient solution for 21 days. These plants were inoculated with aviruliferous zoospores of P. graminis f.sp. tropicalis produced on infected pearl millet roots and maintained in an automatic immersion system. After 21 days' incubation at 25-30 degrees C with a 12 hour photoperiod, zoosporangia were observed in the roots of sugar cane. The zoospores released from these roots were used to infect healthy young pearl millet plants. Twenty days postinoculation, PCV was detected by RT-PCR in roots of inoculated plants. The vector was clearly identified in the roots by microscopy. This result reveals that a PCV isolate from red leaf mottle sugar cane is transmissible by a P. graminis f.sp. tropicalis isolate from a peanut clump infested field. This method should be helpful in the analysis of the specificity of pecluvirus transmission by P. graminis f.sp. tropicalis and P. graminis f.sp. subtropicalis.

Identification and Quantification of Polymyxa graminis f. sp. temperata and P. graminis f. sp. tepida on Barley and Wheat.

Polymyxa graminis f. sp. temperata and P. graminis f. sp. tepida are distinguished on the basis of their specific ribosomal DNA sequences. In order to evaluate whether or not host specialization is associated with the special form, the occurrence of infection of both forms on barley and wheat was studied. P. graminis inocula were obtained from soils collected in Belgium and France. Their ribotypes were characterized using molecular tools specific to P. graminis f. sp. temperata or P. graminis f. sp. tepida such as restriction fragment length polymorphism (RFLP) analysis of polymerase chain reaction (PCR)-amplified rDNA, nested and multiplex PCR. Both special forms were found in each country and coexisted in some soils. The host specificity of P. graminis special forms for barley and wheat was studied from two soils collected at Gembloux (Belgium) and Chambon-sur-Cisse (France), each infested by bymo- and furoviruses. P. graminis f. sp. temperata is more frequent on barley and P. graminis f. sp. tepida on wheat. Furthermore, the quantification of each form on barley and wheat by two separated real-time quantitative PCR assays confirms the observations on the vector specialization. These results suggest a certain but not exclusive host specificity of P. graminis special forms.

A certain but non-exclusive association between Polymyxa graminis special forms and cereals.

Polymyxo graminis, a ubiquitous plasmodiophorid obligate root endoparasite, is recognized as the vector of about 15 viruses on cereals and groundnut in temperate and tropical areas. Within the species, five special forms have been distinguished on the basis of specific ribotypes. Three of them occur in tropical areas: P. graminis f.sp. colombiana on rice, P. graminis f.sp. subtropicalis on cereals cropped in the tropics such as maize, pearl millet and sorghum but also on barley and/or wheat, and P. graminis f.sp. tropicalis mainly on maize, pearl millet and sorghum. Their particular host ranges distinguish them significantly from P. graminis f.sp. temperata and P. graminis f.sp. tepida found in temperate areas on barley and wheat. In order to assess whether these special forms commonly infect these cereals, barley and wheat plants were grown under controlled conditions on two soils from Belgium and France and both infested by P. graminis f.sp. temperata and P. graminis f.sp. tepida. The infection of each cereal species by each form was quantified by real-time quantitative PCR with specific primers and Taqman probes. The infection of P. graminis f.sp. temperata was significantly higher on barley than on wheat, whereas the quantities of P. graminis f.sp. tepida on wheat were higher than on barley. These results show that the distinction between these special forms, based on the ribotype, reflects differences in ecological features.

Barley yellow mosaic virus is overcoming RYM4 resistance in Belgium.

Barley yellow mosaic virus (BaYMV) is the causal agent of a soil-borne systemic mosaic disease on barley. It has been reported in Belgium since the 1980s. The control of this disease is managed almost exclusively through the use of resistant varieties. The resistance of most commercial barley cultivars grown in Europe is conferred mainly by a single recessive gene, rym4. This monogenic resistance provides immunity against BaYMV pathotype 1 and has been mapped on barley chromosome 3HL and shown to be caused by mutations in the translation initiation factor eIF4E. Another pathotype, BaYMV pathotype 2, which appeared in the late 1980s (in Belgium, in the early 1990s), is able to overcome the rym4-controlled resistance. Until recently, this pathotype remained confined to specific locations. During a systematic survey in 2003, mosaic symptoms were observed only on susceptible barley cultivars collected in Belgian fields. BaYMV was detected by ELISA and RT-PCR on the susceptible cultivars and only by RT-PCR on the resistant cultivars. In 2004, mosaic symptoms were observed on susceptible and resistant cultivars. BaYMV was detected by ELISA and RT-PCR on both cultivars. In addition to developing RT-PCR methods for detecting and identifying BaYMV and Barley mild mosaic virus (BaMMV), an RT-PCR targeting the VPg/NIa viral protein part of the genome, known to discriminate the two BaYMV pathotypes, was set up to accurately identify the pathotype(s) now present in Belgium. The sequences from the generated amplicons revealed the single nucleotide substitution resulting in an amino acid change from lysine to asparagine specific to BaYMV pathotype 2. The possible reasons for the change in the BaYMV pathotype situation in Belgium, such as climatic change or a progressive build-up of soil inoculum potential, will be discussed, as well as the use of eIF4E-based resistance.

[Etiology of the sugar beet rhizomania]. / Étiologie de la rhizomanie de la betterave sucrière.

Beet necrotic yellow vein virus is responsible for sugar beet rhizomania. Root proliferation is characteristic of the viral infection and lead to sugar losses. Pathogenicity is particularly linked to the expression of RNA-3-encoded p25. The extensive use of viral tolerant crops allows maintenance of sugar yields but also permits viruliferous vector to be maintained and therefore the appearance of resistance breaking isolates. The resistance breaking isolates present some amino acid variations within the p25 protein sequence, a key determinant in BNYVV pathogenicity. Here, we will review the molecular biology of BNYVV, of its vector and the antiviral strategies that may be used against rhizomania.

Widespread Occurrence of Wheat spindle streak mosaic virus in Belgium.

In order to assess the occurrence of Wheat spindle streak mosaic virus (WSSMV) in Belgium, a reverse-transcription polymerase chain reaction (RT-PCR) was developed, targeting WSSMV isolates from Canada, France, Germany, Italy, and the United States. The primers also were designed for virus quantification by real-time RT-PCR with SYBR-Green. No cross-reaction with soilborne cereal viruses such as Barley mild mosaic virus, Barley yellow mosaic virus, Soilborne cereal mosaic virus, and Soil-borne wheat mosaic virus was observed. The RT-PCR and real-time quantitative RT-PCR allowed a more sensitive detection of WSSMV than enzymelinked immunosorbent assay. The incidence of WSSMV in Belgium was evaluated using a bioassay with wheat cvs. Cezanne and Savannah and rye cv. Halo, grown in 104 Belgian soils. The presence of WSSMV was detected from plants grown in 32% of the soils. The RT-PCR methods developed here, combined with large sampling, allowed WSSMV to be detected for the first time in Belgium. The real-time quantitative RT-PCR was developed as a tool for evaluating the resistance to WSSMV by quantifying the virus concentration in wheat cultivars.

Xanthomonas translucens from Small Grains: Diversity and Phytopathological Relevance.

ABSTRACT Sixty-eight presumptive Xanthomonas translucens strains isolated from 15 small grains or grass species were studied by pathogenicity tests on barley, bread wheat, oat, and bromegrass species, and also by AFLP, analysis of fatty acid methyl esters (FAME), and sodium dodecyl sulfate-polyacrylamide gel electrophoresis of protein extracts. The X. translucens strains were divided into three pathogenicity types based on differences observed on barley and bread wheat. Two unspeciated strains producing atypical symptoms formed a fourth pathogenicity type. Pathogenicity on oat and bromegrass species varied within these types. Clusterings observed by AFLP analysis and, to a lesser extent, by FAME analysis were consistent with these pathogenicity groupings. The current results, as well as those of previous restriction fragment length polymorphism analyses of the same strains, support the recent reclassification of X. translucens pv. translucens and X. translucens pv. hordei as true synonyms. X. translucens pv. cerealis, X. translucens pv. translucens, and X. translucens pv. undulosa cluster in different groups by AFLP and FAME analyses. Even though distinction by simple biochemical tests is not clear-cut, the data indicate that the pathovars cerealis, translucens, and undulosa correspond to true biological entities.

First report of Beet virus Q in Belgium.

Beet necrotic yellow vein virus (BNYVV), the causal agent of rhizomania disease on sugar beet, has been reported in Belgium for more than 16 years. Other soilborne viruses belonging to the genus Pomovirus, such as Beet soilborne virus (BSBV) (3) and Beet virus Q (BVQ) (1), are suspected pathogens of sugar beets grown in Belgium. During the 2000 growing season, more than 20 fields showing rhizomania-like and yellowing symptoms on sugar beet leaves were investigated for the presence of BVQ, BNYVV, and BSBV. All samples were checked by enzyme-linked immunosorbent assay (ELISA) using commercial BNYVV (Sanofi Diagnostics Pasteur, Marnes-La-Coquette, France) and BSBV/BVQ (DSMZ, Braunschweig, Germany - AS-0576 polyclonal, AS-0576/2 MAb) antisera. RNA was extracted from sugar beet rootlets using an RNeasy extraction kit (Qiagen, Hilden, Germany), before performing a reverse transcription-polymerase chain reaction (RT-PCR) using primers (5'-GCTGGAGTATATCACCGATGAC-3' and 5'-AAAATC TCGGATAGCATCCAAC-3') designed to specifically amplify a 510-bp region of BVQ RNA-1. The presence of BSBV and BNYVV was also checked by RT-PCR using previously described primers (1,2). The BVQ-derived PCR product was sequenced and proved to be more than 99% identical to the Wierthe BVQ isolate nucleotide sequence. Soil transmission of BVQ was demonstrated through a bioassay using soil dilutions with quartz and sugar beet cv. Cadyx as bait. After 6 weeks, BVQ was detected by RT-PCR in bait plants. The putative vector, Polymyxa betae, was identified by lactophenol-cotton blue staining of the roots followed by microscopic examination. BVQ produces irregularly shaped local lesions that appear ≈5 days after mechanical inoculation and tend to spread along veins. BVQ was detected in six fields located in the Polders Region and Brabant Province of Belgium. BVQ was always found in sugar beet samples coinfected with BNYVV and BSBV. The economic significance of BVQ and its interaction with other viruses is not known. References: (1) R. Koenig et al. J. Gen. Virol. 79:2027, 1998. (2) M. Saito et al. Arch. Virol. 141:2163, 1996. (3) M. Verhoyen and M. Van den Bossche. Parasitica. 44:71, 1987.

Status and prospects of plant virus control through interference with vector transmission.

Most plant viruses rely on vector organisms for their plant-to-plant spread. Although there are many different natural vectors, few plant virus-vector systems have been well studied. This review describes our current understanding of virus transmission by aphids, thrips, whiteflies, leafhoppers, planthoppers, treehoppers, mites, nematodes, and zoosporic endoparasites. Strategies for control of vectors by host resistance, chemicals, and integrated pest management are reviewed. Many gaps in the knowledge of the transmission mechanisms and a lack of available host resistance to vectors are evident. Advances in genome sequencing and molecular technologies will help to address these problems and will allow innovative control methods through interference with vector transmission. Improved knowledge of factors affecting pest and disease spread in different ecosystems for predictive modeling is also needed. Innovative control measures are urgently required because of the increased risks from vector-borne infections that arise from environmental change.

Effect of household and industrial processing on the levels of pesticide residues and degradation products in melons.

Two varieties of melons (Cucumis melo) were treated with two fungicides (carbendazim and maneb) and four insecticides (acetamiprid, cyromazin, imazalil and thiamethoxam) to quantify the effect of household processing on the pesticide residues. To ensure sufficiently high levels of residues in flesh and peel, the most concentrated formulations were applied observing good agricultural practice. The peeling step decreased the concentration of pesticide residues for maneb, imazalil and acetamiprid by more than 90%. Cyromazin, carbendazim and thiamethoxam were reduced by approximately 50%. The reduction of the pesticides could not be fully explained by the systemic character of the pesticides. However, the agricultural practices (time of application), solubility and mode of action (systemic versus contact pesticide) of the pesticides could be used to explain the difference in processing factors for the studied pesticides. Degradation products (melamine and ethylenethiourea) were also investigated in this study, but were not detected.

Targeting highly conserved 3'-untranslated region of pecluviruses for sensitive broad-spectrum detection and quantitation by RT-PCR and assessment of phylogenetic relationships.

The 3'-end region of many virus isolates has been shown to possess conserved sequences in addition to the presence of numerous genomic and subgenomic RNAs. Utilizing these sequences, a broad-spectrum reverse transcription-polymerase chain reaction protocol has been developed to detect all the known Indian peanut clump virus and Peanut clump virus isolates, that cause peanut clump diseases in West Africa and India. The primers were targeted at the highly conserved 3'-untranslated regions of the PCV RNA-1 and RNA-2. The conservation was confirmed by sequencing these untranslated regions of RNA-1 for six isolates and RNA-2 for one isolate. The conserved structure of the RNA-1 and RNA-2 was observed and the importance of this region for the virus survival was confirmed. The primers were also designed for virus quantitation using a Taqman(®)-based real-time RT-PCR. The use of RT-PCR and real-time quantitative RT-PCR improved the sensitivity of PCV detection compared to ELISA. RT-PCR also led to the detection of IPCV and PCV on two new natural hosts: Oldenlandia aspera and Vigna subterranea. Real-time RT-PCR is considered to be an ideal tool for identifying resistant sources to both IPCV and PCV.

Genetic Diversity among Xanthomonas campestris Strains Pathogenic for Small Grains.

A collection of 51 Xanthomonas campestris strains from throughout the world was studied to detect and assess genetic diversity among pathogens of small grains. Isolates from barley, bread wheat, bromegrass, canary grass, cassava, maize, orchard grass, rice, rough-stalked meadow grass, rye, timothy, and triticale were analyzed by pathogenicity tests on bread wheat cv. Alondra and barley cv. Corona, indirect immunofluorescence, and restriction fragment length polymorphism (RFLP). Three probes were used for the RFLP analysis. They were an acetylaminofluorene-labelled 16S+23S rRNA probe from Escherichia coli and two (sup32)P-labelled restriction fragments from either plasmidic (pBSF2) or chromosomal (pBS8) DNA of X. campestris pv. manihotis. Strains clustered in 9 and 20 groups with the rRNA probe and the pBSF2 DNA probe, respectively. Strains of X. campestris pv. graminis, X. campestris pv. phleipratensis, and X. campestris pv. poae are shown to be related but are also distinguishable by RFLP patterns, serology, and pathogenicity on bread wheat. Strains pathogenic only for barley and not for wheat grouped together. Another group is temporarily designated deviant X. campestris pv. undulosa. These South American isolates from bread wheat did not react by indirect immunofluorescence and produced atypical lesions in pathogenicity tests. The results stress the need to perform pathogenicity tests before strains are named at the pathovar level. The importance of the different probes used for epidemiological studies or phylogenetic studies of closely related strains is underlined.

The Beet soilborne pomovirus in Belgium and relationship with rhizomania.

The objective of this study was to determine the extend of the Beet soilborne pomovirus (BSBV) and the Beet virus Q in sugar beet fields in Belgium. During the 2000 sugar beet growing season, more than 80 fields located in Belgium were investigated for the presence of the Beet necrotic yellow vein benyvirus (BNYVV), the BSBV and Polymyxa betae, the plasmodiophorid vector of both viruses. Fields investigated were identified either using previous recorded data or by visual identification of yellow leaves on sugar beets or root symptoms. Sampling position (longitude-latitude) was recorded using the global positioning system (G.P.S.) with the view to follow-up infested fields in the following years. Three different techniques were used to evidence the aforementioned biological agents: enzyme-linked immunosorbent assay (ELISA), a RT-PCR assay to detect the viruses and direct coloration of Polymyxa betae in plant root tissues, using lactophenol-aniline blue. ELISA allowed the detection of 43 BSBV-infested soils, largely distributed in all Belgian sugar beet growing areas. These results were largely confirmed by RT-PCR using two different primers pairs targeting respectively a 400 bp fragment of the 145K ORF located on virus RNA-1 and a 970 bp fragment of the conserved 3' end of the viral genome. Five other primer's pairs have also been evaluated for BSBV identification. The detection of BSBV-infested soils without BNYVV, as well as BNYVV-infested soils without BSBV allowed the design of a competition assay between both viruses. Among the samples, 21 were selected randomly and tested for the presence of Beet virus Q by RT-PCR. Here also, six fields were detected positive for this virus. Sequence data reveal a clonal population of BSBV isolates in Belgium though a high level of diversity is observed for the coat protein region. Compared to BSBV, BVQ isolates show less diversity at sequence level.

Virus-like particles assemble in plants and bacteria expressing the coat protein gene of Indian peanut clump virus.

cDNA copies of the coat protein (CP) gene of Indian peanut clump virus (IPCV)-H were introduced into cells of Nicotiana benthamiana or Escherichia coli by transformation with vectors based on pROKII or pET respectively. In both plant and bacterial cells, IPCV CP was expressed and assembled to form virus-like particles (VLP). In plant extracts, the smallest preponderant particle length was about 50 nm. Other abundant lengths were about 85 and about 120 nm. The commonest VLP length in bacterial extracts was about 30 nm. Many of the longer VLP appeared to comprise aggregates of shorter particles. The lengths of the supposed 'monomer' VLP corresponded approximately to those expected for encapsidated CP gene transcript RNA. Immunocapture RT-PCR, using primers designed to amplify the CP gene, confirmed that the VLP contained RNA encoding IPCV-H CP. The results show that encapsidation does not require the presence of the 5'-terminal untranslated sequence of the virus RNA and suggest that if there is an 'origin of assembly' motif or sequence, it lies within the CP gene. When transgenic plants expressing IPCV-H CP were inoculated with IPCV-L, a strain that is serologically distinct from IPCV-H, the virus particles that accumulated contained both types of CP.