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.

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.

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.

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.