Monitoring the dynamics of emergence of a non-canonical recombinant of Tomato yellow leaf curl virus and displacement of its parental viruses in tomato.

Recombinant viruses are increasingly being reported but the dynamics of their emergence is rarely documented. A new recombinant Tomato yellow leaf curl virus (TYLCV-IS76) was detected for the first time in 2010 in Southern Morocco (Souss). An original diagnostic tool was needed to fit its unusual recombination profile. Although IS76 was detected following the appearance of Tylc symptoms on tolerant tomato plants, symptoms could not be associated to IS76 or to a synergy with criniviruses. According to infection profiles of Tylc-associated viruses determined on 879 plant samples collected between 1998 and 2014 and a Bayesian inference applied to genomic sequences of representatives of TYLCV, IS76 emerged in Southern Morocco at the end of the 1990s, replaced the parental viruses between 2004 and 2012 in Souss and is spreading towards the North of Morocco. The emergence of IS76 coincides with the increasing use of tolerant cultivars in the 2000s.

First Report of the Plum Pox Virus Recombinant Strain on Peach in Bulgaria.

Plum pox virus (PPV) causes sharka, the most damaging viral disease of stone fruit species. Seven distinct PPV strains are known; PPV-M, PPV-D, and PPV-Rec are the most common (3). PPV-Rec is a unique recombinant (3) between PPV-M and PPV-D and has been reported from plum, apricot, Japanese plum, myrobalan, and blackthorn in eastern and central Europe, but has never been found in peach as a single natural infection (2). A survey was conducted during spring 2009 in eight peach orchards located in the southwest, southeast, and south central regions of Bulgaria to assess the incidence of PPV infection. A total of 98 leaf samples from individual trees showing PPV-like symptoms were collected and analyzed by triple-antibody sandwich (TAS)-ELISA with the universal monoclonal antibody (MAb) 5B (Agritest, Valenzano, Italy). Sixty one samples reacted positive for PPV (optical density 0.161 to 1.267) and these samples were further analyzed with PPV-M (AL) and PPV-D (4DG5) specific MAbs (1). All 61 samples reacted positively with PPV-M specific MAbs. To distinguish PPV-M and PPV-Rec strains, which are serologically identical, immunocapture (IC)-reverse transcription (RT)-PCR was carried out with PPV-M (CIP-M: 5'-GTC GCA GCA TTT GTA GCC CTT GTT-3', CIP-MR: 5'-CCA ACA CGT TAA CGC CAT GCT TCA-3') and PPV-D (CIP-D: 5'-ATG ATG CTG TTT GAC TCG GAG CGA-3', CIP-DR: 5'-TCG CAA CTG CTT GCA CAC ATT CTC-3') specific primers targeting the 6K1-CI genomic region. A PCR fragment of ~880 bp amplified with PPV-M specific primers obtained from 59 samples confirmed that these were PPV-M isolates. However, the remaining two samples (both coming from infected tress located in two different orchards in the southwest region) yielded a 468-bp PCR fragment with PPV-D specific primers, suggesting that these two samples belonged to PPV-Rec strain. These samples together with controls of PPV-M, PPV-D, and PPV-Rec strains were further analyzed by RT-PCR using mD5/mM3 primers spanning the recombination breakpoint (4). Both peach samples and the PPV-Rec strain control produced a single 605-bp PCR product. The two peach amplicons were purified and sequenced directly with the same primers. The nucleotide (nt) sequences obtained were 100% identical to each other. BLAST analysis of the two samples with PPV-Rec (No. AF421118.1) showed maximum nt identity of 98%. Percent maximum nt identity with PPV-M (No. AY324837.1) and PPV-D (No. AB576062.1) were 93 and 87%, respectively. The deduced amino acid sequences of the two isolates were 98% identical to PPV-Rec (No. No. AF421118.1), 93% identical to PPV-M (No. M92280.1), and 84% identical to PPV-D (No. AB576062.1). Analyzed samples were further transmitted from the diseased trees to peach seedlings (GF 305) by chip-budding in a greenhouse during the fall of 2009. Six months later, faint vein clearing on the leaves of inoculated seedlings was observed. The presence of PPV was confirmed by TAS-ELISA and PPV-Rec presence was shown by IC-RT-PCR (mD5/mM3 primers). One of the generated 605-bp products was sequenced and showed 100% nt identity with the isolate used for inoculation. To our knowledge, this is the first identification of PPV-Rec strain in naturally infected peach trees, a finding that calls for further large-scale investigations of PPV-Rec incidence in peach in Bulgaria. References: (1) M. Cambra et al. OEPP/EPPO Bull. 24:569, 1994. (2) S. Dallot et al. Acta Hortic. 781:227, 2008. (3). M. Glasa et al. J. Gen. Virol. 85:2671, 2004. (4) Z. Subr et al. Acta Virol. 48:173, 2004.

Identification of Plum pox virus Determinants Implicated in Specific Interactions with Different Prunus spp.

ABSTRACT The characterization of pathogenic properties of two infectious clones of Plum pox virus (PPV) isolates, pGPPV (D group) and pGPPVPS (M group), was investigated in their woody hosts (seedlings of Prunus spp.). The two clones differed in their ability to infect plum and peach cultivars, from no infection to local and systemic infection. The phenotype determinants were located with a set of chimeric viruses from the two clones. In plum, determinants of systemic infection were located in a genomic fragment encoding the P3 and 6K1 proteins, which might influence genome amplification or virus movement. The capacity of pGPPVPS to induce stable local and systemic infections in peach was not located accurately and might be influenced by multiple determinants carried by different regions of the genome, excluding those encoding the protein 1, the majority of helper component, nuclear inclusions a and b, and coat protein. We conclude that PPV infections of plum and peach are governed by different determinants.

Evidence that the proliferation stage of micropropagation procedure is determinant in the expression of banana streak virus integrated into the genome of the FHIA 21 hybrid (Musa AAAB).

Banana streak virus (BSV) is causing increasing concern in almost every producing area of banana and plantain (Musa spp.) worldwide. This situation appeared partially linked to some breeding lines and micropropagated hybrids. A complete BSV sequence integrated into the genome of a triploid plantain has been recently characterised and it has been hypothesised that it could give rise to infectious virus via recombination. In this study, we evaluated the effect of a routine micropropagation procedure on the expression of BSV in the FHIA 21 tetraploid hybrid. The widespread presence of integrated sequences and the absence of episomal BSV in thirty FHIA 21 "mother plants" selected for micropropagation were first confirmed by specific PCR and IC-PCR tests. The proliferation stage of the procedure, characterised by an intensive production of neoformed buds, appeared determinant in BSV expression whereas the rooting and acclimatisation stages had little or no effect. The duration in culture and the way of subdividing the clumps of proliferation influenced greatly the percentage of episomal BSV infections, reaching 58% of infected micropropagated lines after six in vitro subcultures. These data suggest that the expression of episomal BSV observed during the in vitro procedure is correlated with the presence of an integrated form.

Occurrence of Potyviruses on Yam (Dioscorea spp.) in Colombia and First Molecular Characterization of Yam mild mosaic virus.

A survey to determine the prevalence of potyviruses on yams, Dioscorea alata and D. cayenensis-rotundata, was undertaken in Colombia. Two hundred fifty leaf samples showing mottling symptoms were collected on the Atlantic coast and analyzed by antigen-coated plate enzyme-linked immunosorbent assay with universal potyvirus monoclonal antibodies (Agdia, Elkhart, IN). Potyviruses were detected in 70% (165/235) of the D. alata and in 66% (10/15) of the D. cayenensis-rotundata samples. The presence of Yam mild mosaic virus (YMMV) was indicated in some of these samples by immunocapture reverse-transcriptase polymerase chain reaction performed as previously reported (1). A 600-bp fragment that included the core and C-terminal region of the coat protein gene (CP) and the 3' untranslated region (3'UTR) was amplified from a D. alata isolate using universal potyvirus primers (1), cloned, and sequenced (EMBL Acc. AJ311725). Comparison with the two previously published YMMV sequences revealed 96.1 and 97.4% identity for the deduced amino acid sequence in the CP region, 74.1 and 83.2% nucleotide identity in the 3'UTR for Papua New Guinea (AB022424 [2]) and Martinique (AJ250336) isolates, respectively. YMMV is known to be widespread on D. alata in Africa and the South Pacific and has been recently identified in the Caribbean (1). To our knowledge, this is the first report of its occurrence in Colombia. A study of its incidence and genetic diversity in South America has been undertaken. References: (1) M. Bousalem and S. Dallot. Plant Disease 84:200, 2000. (2) S. Fuji et al. Arch Virol. 144:1415, 1999.

The use of phylogenetic data to develop molecular tools for the detection and genotyping of Yam mosaic virus. Potential application in molecular epidemiology.

Molecular detection of Yam mosaic virus (YMV), the most important potyvirus for yam plants (Dioscorea spp.) has been limited by its high genetic diversity. According to recent phylogenetic data, suitable molecular tools were developed for detection and genotyping. A reliable and rapid molecular test is described using single-tube immunocapture (IC)-RT-PCR combining the use of a monoclonal antibody able to recognise all YMV serotypes and primers designed to take into account the whole variability of YMV. Phylogenetic data were then used to develop an uncoupled IC-RT-PCR procedure adapted to the genotyping of the three major phylogenetic groups and the two recombinants implicated in the YMV epidemics in the Caribbean and French Guiana. A third exploitation of phylogenetic data was carried out with the development of a typing test based on the analysis of short nucleotide sequences. The direct sequencing of a 450 nts fragment constituted a reliable complementary typing tool as the resulting information was quite similar to that obtained with sequences of the complete coat protein gene. The approach, consisting in a robust study of YMV variability followed by the use of phylogenetic data to develop reliable detection and genotyping tools, offers new perspectives for powerful molecular epidemiological studies.

Identification of a pathogenicity determinant of Plum pox virus in the sequence encoding the C-terminal region of protein P3+6K(1).

A full-length genomic cDNA clone of a plum pox potyvirus (PPV) isolate belonging to the M strain (PPV-PS) has been cloned downstream from a bacteriophage T7 polymerase promoter and sequenced. Transcripts from the resulting plasmid, pGPPVPS, were infectious and, in herbaceous hosts, produced symptoms that differed from those of virus progeny of pGPPV, a full-length genomic cDNA clone of the D strain PPV-R. Viable PPV-R/-PS chimeric viruses were constructed by recombination of the cDNA clones in vitro. Analysis of plants infected with the different chimeras indicated that sequences encoding the most variable regions of the potyvirus genome, the P1 and capsid protein coding sequences, were not responsible for symptom differences between the two PPV isolates in herbaceous hosts. On the contrary, complex symptomatology determinants seem to be located in the central region of the PPV genome. The results indicate that a genomic fragment that encodes 173 aa from the C-terminal part of the P3+6K(1) coding region is enough to confer, on a PPV-R background, a PS phenotype in Nicotiana clevelandii. This pathogenicity determinant also participates in symptom induction in Pisum sativum, although the region defining the PS phenotype in this host is probably restricted to 74 aa.

First Report and Molecular Characterization of Yam mild mosaic virus in Dioscorea alata on the Island of Martinique.

Naturally infected Dioscorea alata plants showing mild mosaic were collected in 1998 on the island of Martinique in the Caribbean. Isolates were first screened by double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) with monoclonal antibodies raised against Yam mosaic virus (YMV) and antigen-coated plate ELISA with universal potyvirus monoclonal antibodies (Agdia, Elkhart, IN). A positive reaction was obtained only with the universal potyvirus antiserum. Immunocapture reverse-transcriptase polymerase chain reaction was performed for specific detection of Yam mild mosaic virus (YMMV [3]) and YMV. A product with the predicted size of 249 bp was obtained with YMMV primers. YMMV is a recently recognized distinct potyvirus infecting D. alata in West Africa and the South Pacific (2-4). It was originally described as Yam virus I and is synonymous with Dioscorea alata virus (4). To characterize the YMMV Martinique isolate, total RNA was extracted, and universal potyvirus degenerate primers (1) were used to amplify a 700-bp fragment that included the core and C-terminal region of the coat protein (CP) and 3' untranslated region (3'UTR). Sequence information generated (EMBL AJ250336) from the cloned fragment was compared with sequences of other yam potyviruses. Sequence comparisons of the partial CP (453 nt) showed a similarity of 94.6% (amino acids [aa]) with the YMMV isolate from Papua New Guinea (EMBL AB022424 [2]); 72.2% (aa) with the Japanese yam mosaic virus (JYMV) isolate (EMBL AB016500); and 67 to 73% (aa) with 27 YMV isolates. These sequences are most diverse in the 3'UTR, which showed a similarity of 72.8% with the YMMV Papua New Guinea isolate, 30% with the JYMV isolate, and 26% with the YMV isolates. These results confirm, as previously shown by S. Fuji et al. (2), that YMMV should be classified as a new potyvirus of yam. This is the first report of the natural occurrence of YMMV in the Caribbean. References: (1) Colinet et al. Phytopathology 84:65, 1994. (2) S. Fuji et al. Arch Virol. 144:1415, 1999. (3) R. A. Munford and S. E. Seal. J. Virol. Methods 69:73, 1997. (4) B. O. Odu et al. Ann. Appl. Biol. 134:65, 1999.

Comparison of monoclonal antibodies and polymerase chain reaction assays for the typing of isolates belonging to the d and m serotypes of plum pox potyvirus.

ABSTRACT Plum pox potyvirus (PPV) isolates may be divided into four groups separated by serological, molecular, and epidemiological differences. Monoclonal antibodies specific for the two major groups of isolates, represented by the D and M serotypes of the virus, have been obtained. Polymerase chain reaction (PCR)-based assays allowing the direct detection and differentiation of PPV isolates have also been developed. We now report on a large-scale comparison of these two typing approaches. The results obtained show an overall excellent correlation between the results obtained in indirect double-antibody sandwich enzyme-linked immunosorbent assay using PPV-D- and PPV-M-specific monoclonal antibodies and those derived from either specific PCR assays or restriction fragment length polymorphism analysis of PCR fragments. Without exception, all isolates reacting positively with the PPV-M-specific monoclonal antibody were found to belong to the M serotype using the PCR-based assays, while 51 out of 53 isolates recognized by the D-specific monoclonal antibodies belonged to the D serotype according to the PCR typing results. However, failure to react with a specific monoclonal antibody did not prove as effective a predictor of the serotype of the isolate analyzed. In a few cases, the results obtained with the various techniques diverged, indicating low level variability of the epitopes recognized by the serotype-specific monoclonal antibodies. Isolates belonging to the two minor groups of PPV (El Amar and Cherry) also gave divergent results, indicating that the current typing assays are not suited for the analysis of such isolates.