Viruses of faba bean (Vicia faba L.) in Morocco : surveying, identification, and ecological aspects

A systematic virus survey covering the main areas where faba bean ( Viciafaba L.) is grown in Morocco was conducted in 1988 and 1990. From the 240 leaf samples collected on the basis of symptoms suggestive of virus infection from 52 fields, the following viruses were detected by means of electron microscopy, biological indexing, and serology, and their incidence and geographical distribution were assessed: alfalfa mosaic virus (AMV), bean yellow mosaic virus (BYMV), broad bean mottle virus (BBMV), broad bean stain virus (BBSV), broad bean true mosaic virus (BBTMV), peaearly browning virus (PEBV), pea enation mosaic virus (PEMV), pea seed-borne mosaic virus (PSbMV), and luteoviruses. (BBTMV), PEMV, PSbMV, and luteoviruses had not previously been reported in the country. BBMV, considered earlier of limited distribution and the luteoviruses were found to be prevalent (in 50 and 56% of the surveyed fields, respectively; and with field incidences of 20 and 33%, respectively), whereas the opposite held for BBSV and BYMV. More detailed studies concentrated on the actually important BBMV and the luteoviruses, and on the potentially important BYMV-like isolates.The biological indexing of samples revealed considerable variation in symptom severity on test plants among BBMV isolates. Comparison of seven selected Moroccan isolates with isolates from Algeria, Sudan, and Tunisia, revealed a pathogenic variability of the virus on a number of food-legume genotypes. Clusters of isolates differing in virulence could be distinguished as mild, intermediate, and virulent, although they an reacted similarly to the antisera to a Moroccan and a Syrian isolate. When a number of promising ICARDA breeding lines and accessions of faba bean, chickpea, lentil, and pea were tested with the BBMV isolates, different interactions were observed, but all genotypes were found vulnerable to all the isolates investigated, and no immunity could be detected. Some isolates were more pathogenic (often even necrotic) on other food legumes, such as pea and chickpea, than on faba bean. BBMV was found to be seed transmitted in faba bean (at a rate of 1.2%) when occurring on its own, and was detected to be so in chickpea and pea at transmission rates of 0.9 and 0.1%, respectively. Besides transmission by seed, BBMV was found to be transmissible by the curculionid weevils Apion radiolus, Hypera variabilis, Pachytychius strumarius, Smicronyx cyaneus, and the previously reported Sitona lineatus. The latter appeared to be an efficient vector since the first bite was sufficient for acquisition and transmission of the virus, virus retention lasted for at least seven days, and the transmission rate was estimated to be 41%. S.lineatus turned out to transmit BBMV not only from faba bean to the same species, but also to lentil and pea. When searching for natural sources of the virus by testing of 351 samples of food legumes from 24 fields and 102 samples of wild legumes, it was found to occur naturally in chickpea, lentil, and pea, as well as in common bean (Phaseolus vulgaris) in 16, 11, 19, and 16% of the tested samples, respectively; but it was not detected in the samples of wild legumes reported in literature as potential hosts.The problem of virus variability emerged and gradually evolved during these studies. It was encountered with BBMV showing a variation of isolates. It particularly holds for the cluster of potyviruses related to BYMV but also for the luteoviruses, where it leads to difficulties in virus identification.When testing faba-bean samples, showing luteovirus-like symptoms, in DAS-ELISA with polyclonal antisera to bean leafroll virus (BLRV), beet western yellows virus (BWYV), and subterranean clover red leaf virus (SCRLV), and in TAS-ELISA with two monoclonal antibodies discriminating between BLRV and BWYV, various serological reaction patterns were obtained. This pointed to a considerable variation among the luteovirus isolates which could not be identified as one of the known legume luteoviruses. To enable reliable detection of this group of viruses by the polymerase chain reaction (PCR), a pair of designed oligonucleotide primers were found to specifically amplify a 535-bp fragment of the coat- protein gene of known luteoviruses and of all Moroccan isolates tested. In molecular hybridization tests, selected field isolates showed nucleotide sequence homology among them, and with BLRV, but not with BWYV, although some of them behaved BWYV-like serologically. These results support the idea of the involvement of either deviant strains of known luteoviruses or of (a) completely new faba-bean luteovirus(es).On the other hand, BYMV and the closely related clover yellow vein virus (CYVV) could be distinguished by host-range studies including non-legume test species, and by cytopathology and polyacrylamide-gel electrophoresis of their coat proteins. Both viruses could not be distinguished by the N-terminal serology claimed to discriminate between potyviruses. Conflicting reports as to the taxonomic status of the potyviruses infecting legumes showed the need to develop more reliable tools to unambiguously identify these viruses. Recent molecular studies, including the elucidation of nucleotide sequences of legume-potyvirus RNAs, appeared to provide a rational basis for the identification and classification of potyviruses in general. The use of such molecular data in PCR for the distinction of BYMV and CYVV was investigated, and preliminary results showed that a pair of primers could be used in PCR to distinguish between both viruses

Molecular, serological and biological variation among chickpea chlorotic stunt virus isolates from five countries of North Africa and West Asia

Chickpea chlorotic stunt virus (CpCSV), a proposed new member of the genus Polerovirus (family Luteoviridae), has been reported only from Ethiopia. In attempts to determine the geographical distribution and variability of CpCSV, a pair of degenerate primers derived from conserved domains of the luteovirus coat protein (CP) gene was used for RT-PCR analysis of various legume samples originating from five countries and containing unidentified luteoviruses. Sequencing of the amplicons provided evidence for the occurrence of CpCSV also in Egypt, Morocco, Sudan, and Syria. Phylogenetic analysis of the CP nucleotide sequences of 18 samples from the five countries revealed the existence of two geographic groups of CpCSV isolates differing in CP sequences by 8–10%. Group I included isolates from Ethiopia and Sudan, while group II comprised those from Egypt, Morocco and Syria. For distinguishing these two groups, a simple RFLP test using HindIII and/or PvuII for cleavage of CP-gene-derived PCR products was developed. In ELISA and immunoelectron microscopy, however, isolates from these two groups could not be distinguished with rabbit antisera raised against a group-I isolate from Ethiopia (CpCSV-Eth) and a group-II isolate from Syria (CpCSV-Sy). Since none of the ten monoclonal antibodies (MAbs) that had been produced earlier against CpCSV-Eth reacted with group-II isolates, further MAbs were produced. Of the seven MAbs raised against CpCSV-Sy, two reacted only with CpCSV-Sy and two others with both CpCSV-Sy and -Eth. This indicated that there are group I- and II-specific and common (species-specific) epitopes on the CpCSV CP and that the corresponding MAbs are suitable for specific detection and discrimination of CpCSV isolates. Moreover, CpCSV-Sy (group II) caused more severe stunting and yellowing in faba bean than CpCSV-Eth (group I). In conclusion, our data indicate the existence of a geographically associated variation in the molecular, serological and presumably biological properties of CpCSV

Two distinct nanovirus species infecting faba bean in Morocco

Using monoclonal antibodies raised against a Faba bean necrotic yellows virus (FBNYV) isolate from Egypt and a Faba bean necrotic stunt virus (FBNSV) isolate from Ethiopia, a striking serological variability among nanovirus isolates from faba bean in Morocco was revealed. To obtain a better understanding of this nanovirus variability in Morocco, the entire genomes of two serologically contrasting isolates referred to as Mor5 and Mor23 were sequenced. The eight circular ssDNA components, each identified from Mor5- and Mor23-infected tissues and thought to form the complete nanovirus genome, ranged in size from 952 to 1,005 nt for Mor5 and from 980 to 1,004 nt for Mor23 and were structurally similar to previously described nanovirus DNAs. However, Mor5 and Mor23 differed from each other in overall nucleotide and amino acid sequences by 25 and 26%, respectively. Mor23 was most closely related to typical FBNYV isolates described earlier from Egypt and Syria, with which it shared a mean amino acid sequence identity of about 94%. On the other hand, Mor5 most closely resembled a FBNSV isolate from Ethiopia, with which it shared a mean amino acid sequence identity of approximately 89%. The serological and genetic differences observed for Mor5 and Mor23 were comparable to those observed earlier for FBNYV, FBNSV, and Milk vetch dwarf virus. Following the guidelines on nanovirus species demarcation, this suggests that Mor23 and Mor5 represent isolates of FBNYV and FBNSV, respectively. This is the first report not only on the presence of FBNSV in a country other than Ethiopia but also on the occurrence and complete genome sequences of members of two nanovirus species in the same country, thus providing evidence for faba bean crops being infected by members of two distinct nanovirus species in a restricted geographic area

Molecular evidence for the occurrence of beet western yellows virus on chickpea in Morocco.

A luteovirus isolate infecting chickpea in Morocco was experimentally transmitted by Myzus persicae to Physalis floridana, on which it produced mild symptoms. When tested in western blots against antisera to known legume luteoviruses, this isolate reacted strongly to beet western yellows virus (BWYV) antiserum, moderately to bean leafroll virus antiserum, while no reaction was recorded with the antiserum against subterranean clover red leaf virus. In PCR, a fragment of ca. 950 bp was amplified, comprising the 3' end of the open reading frame (ORF) 3, the complete coat protein gene (ORF 4), and the non-translated region in between these ORFs. The nucleotide sequence of the amplified fragment showed high similarity with BWYV (approximately 96%), and lower (50–60%) with other luteoviruses reported to infect legumes. On the basis of these data, the Moroccan isolate was identified as BWYV. This is the first molecular evidence for the occurrence of BWYV on chickpea in Morocco, and on food legumes in general in North Africa

Diversity of luteoviruses infecting faba bean (Vicia faba L) in Morocco, and their detection by the polymerase chain reaction

When surveying faba bean (Vicia faba L) for viruses in Morocco, members of the luteovirus group were found to be economically important. In order to further identify them, a number of faba bean samples showing symptoms indicative of luteovirus infection were serologically tested using polyclonal antisera for bean leafroll virus (BLRV), beet western yellows virus (BWYV), and subterranean clover red leaf virus (SCRLV), and two monoclonal antibodies which discriminate between BWYV and BLRV. Several serological reaction patterns were obtained which pointed towards a large variation in the luteovirus isolates studied. None of these isolates could be clearly identified as one of the luteoviruses known to infect legumes, although a number of them behaved like BWYV serologically. The potential of the polymerase chain reaction in detecting these luteoviruses was investigated, and a set of oligonucleotide primers was designed which specifically amplified a 535 bp fragment of the coat protein gene of known luteoviruses and of all the Moroccan isolates tested. In nucleic acid hybridization tests, the field isolates showed homology in nucleotide sequence among the Moroccan isolates and with BLRV, but not wih BWYV. In polyacrylamide gel electrophoresis, a purified Moroccan isolate was found to be different from BLRV and BWYV in coat-protein migration.Diversité des lutéovirus infectant la fève (Vicia faba L) au Maroc et leur détection par la réaction d'amplification enzymatique. Un inventaire des virus de la fève au Maroc a démontré l'incidence économique des lutéovirus sur cette plante. Dans le but de mieux caractériser ces lutéovirus, des échantillons de fève, présentant des symptômes typiques des lutéovirus, ont été testés sérologiquement en utilisant des antisérums polyclonaux contre le virus de l'enroulement de la fève (BLRV), le virus de la jaunisse occidentale de la betterave (BWYV), le virus de la feuille rouge de trèfle souterrain (SCRL V) et deux anticorps monoclonaux discriminant le BLRV du BWYV. La diversité des profils de réaction sérologique obtenus montre qu'il existe une grande variabilité au sein des isolats étudiés. Aucun d'entre eux n'a pu être strictement assimilé aux lutéovirus infectant la famille des légumineuses, bien que certains isolats se soient comportés sérologiquement comme le BWYV. Le potentiel de la réaction d'amplification enzymatique (PCR) pour la détection de ces lutéovirus a été exploré. Nous avons amplifié spécifiquement un fragment de 535 pb du gène codant pour la protéine de capside, aussi bien chez des lutéovirus connus que chez les isolats marocains testés. Lors des tests d'hybridation moléculaire avec une sonde correspondant au gène de la protéine de capside de différents isolats, des homologies de séquences nucléotidiques ont été montrées, d'une part entre les isolats marocains, et d'autre part entre les isolats marocains et le BLRV. Aucune homologie n'a été trouvée entre les isolats marocains et le BWYV. L'électrophorèse sur gel de polyacrylamide a montré que la migration de la protéine de capside d'un isolat marocain purifié est différente de celles des protéines de capside du BLRV et du BWYV