First Report of Banana mild mosaic virus Infecting Plantain in Ivory Coast.

Plantain (Musa sp., genomic group AAB) is an important crop for millions of the world's poorest people. In Ivory Coast, it is the second most consumed food and an important source of income for farmers. Between 2010 and 2011, a survey for viruses infecting plantain (AAB) was conducted in 10 major plantain-growing regions located in eastern (Abengourou), middle-western (Bouaflé, Daloa, Issia, Oumé, Sinfra, Zuenoula), central (Yamoussoukro), and southern (Aboisso, Gagnoa) Ivory Coast. Leaf samples showing yellow streaks or mild chlorotic streaks were collected and dried on CaCl2 for storage. A representative sample from each location was selected and tested for the presence of Cucumber mosaic virus (CMV, genus Cucumovirus), Banana streak virus (BSV, genus Badnavirus), Banana mild mosaic virus (BanMMV, family Flexiviridae), and Banana bract mosaic virus (BBrMV, genus Potyvirus). Immunocapture (IC)-PCR was used for the detection of BSV while reverse transcription (RT)-PCR was used for the detection of CMV, BanMMV, and BBrMV. The following primers sets were used: BSV cl1 and BSV cl2 (1), CMV 3' and CMV 5' (3), BanMMV BanCP1 and BanCP2 (4), BBrMV Bract N2 and Bract NR (2). BanMMV was detected as mixed infections with BSV in the 10 tested samples, one of which also contained CMV. To confirm the identity of the amplification products from the BanMMV primers, one cDNA fragment was directly sequenced in the forward direction (Macrogen Inc., Seoul, South Korea). BLAST search in GenBank revealed that the partial coat protein (CP) sequence of the Ivorian isolate shared 80 to 88% nucleotides and 81 to 92% deduced amino acid similarities with BanMMV isolates. In contrast, partial CP sequence of the Ivorian isolate had less than 40% deduced amino acid sequence identity with other Flexiviridae CP sequence. The partial CP sequence of the Ivorian BanMMV isolate was deposited in GenBank under Accession No. JX014304. To further confirm the identification, all the samples were tested by plate trapped antigen (PTA)-ELISA with rabbit polyclonal antiserum specific to BanMMV (obtained from B. E. Lockhart, University of Minnesota, U.S.A.) and anti-rabbit IgG (Sigma-Aldrich, Belgium/A3687). The 10 samples reacted positive for BanMMV by ELISA. CMV and BSV have been reported in Ivory Coast, but to our knowledge, this is the first report of BanMMV in the country. The detection of BanMMV in association with BSV or CMV in mixed infection in 10 locations which are important plantain growing areas is a first step in the evaluation of the impact of virus diseases on plantain production in this country. References: (1) S. Dallot et al. Arch. Virol. 146:2182, 2001. (2) M.-L. Iskra-Caruana et al. J. Virol. Methods 153:224, 2008. (3) M. Sharman et al. J. Virol. Methods 89:77, 2000. (4) P.-Y. Teycheney et al. J. Gen. Virol. 86:3181, 2005.

Catecholamine biosynthesis pathway potentially involved in banana defense mechanisms to crown rot disease.

Variations in Cavendish bananas susceptibility to crown rot disease have been observed (Lassois et al., 2010a), but the molecular mechanisms underlying these quantitative host-pathogen relationships were still unknown. The present study was designed to compare gene expression between bananas (Musa acuminata, AAA, 'Grande-Naine') showing a high post-harvest susceptibility (S+) and bananas showing a low post-harvest susceptibility (S-) to crown rot disease. This comparison was performed between crowns (S+ and S-) collected one hour before standardized artificial inoculations with Colletotrichum musae. Fruit susceptibility was evaluated through lesion size on the crown 13 days later. Gene expression comparisons were performed with the cDNA-AFLP technique (Lassois et al., 2009). This revealed that a gene showing a strong homology with a dopamine-beta-monooxygenase (DoH) is differently expressed between S+ and S (Lassois et al., 2011). Furthermore, semi-quantitative real-time RT-PCR analyses between S+ and S- were applied to confirm the differential expression results for DoH obtained by cDNA-AFLP. Two biological replicates were tested. These semi-quantitative analyses were performed not only on tissues collected one hour before C. musae inoculation but also on crown tissues collected 13 days after inoculation. The real-time RT-PCR confirmed that DoH was upregulated in the S tissues collected at harvest, just before C. musae inoculation. This gene was also highly upregulated in the S- tissues collected 13 days after crown inoculation. Similar results were obtained for both biological replicates. Our results suggest that catecholamine's could play a role in banana defense mechanisms to crown rot disease.

Evaluation of three essential oils as potential sources of botanical fungicides.

In previous study, thirty essential oils were evaluated in vitro against two citrus pathogens namely Penicillium italicum Wehmer and Penicillium digitatum Sacc. Essential oils of Cinnamomum zeylanicum, Cinnamomum verum and Eugenia caryophyllus were selected because of their high inhibitory activities against both pathogens. The present study was undertaken to evaluate the in vivo activity of these essential oils. Fresh orange fruits were wounded and treated with different concentrations of essential oil (0.5, 1, and 5%) before being infected at the wound site with conidia suspensions of the tested pathogens. When applied at 5%, essential oils tested controlled totally the infections. Among the three essential oils tested, C. zeylanicum seems particularly interesting because of its high protection activity at 1% compare to the others. It reduced the disease incidence from 40 to 70% and the disease severity from 65 to 82%. Moreover no visible damage burn induced on the orange cuticle or skin was observed up to 5% of essential oil. These results strengthen the potential use of essential oils in postharvest disease management of citrus fruit as alternative to chemical fungicides.

A simple and rapid protocol of crude DNA extraction from apple trees for PCR and real-time PCR detection of 'Candidatus Phytoplasma mali'.

Different PCR protocols have been established for detection of European fruit trees phytoplasmas; however the majority of the procedures for extracting phytoplasma DNA are complex, time consuming, and expensive, with a risk of contamination or loss of target DNA. In present study, a crude extract preparation method previously used to detect other plant pathogens was adapted to samples from apple trees infected by 'Candidatus Phytoplasma mali'. End-point and real-time PCR detection of 'Ca. P. mali' were used to compare this extraction procedure with an established method for efficient extraction of purified DNA. The crude extract proved fully adequate for phytoplasma detection in samples from 86 in vitro and 35 in vivo apple shoots or plants and 10 periwinkle plants. High inter- and intra-run reproducibility was obtained for phytoplasma detection with different TaqMan MGB- or SYBR Green-based real-time PCR protocols applied to the crude extracts. Real-time PCR applied to serially diluted crude and purified extracts revealed the same phytoplasma detection limit (dilution up to 10(5)). All results confirm the suitability of this simple, quick, efficient extraction technique for accurate detection of 'Ca. P. mali' in different types of apple and periwinkle samples.

Preliminary evaluation of antimicrobial activity of some chemicals on in vitro apple shoots infected by 'Candidatus Phytoplasma mali'.

Phytoplasmas are associated with several hundred plant diseases worldwide, including numerous ones with important economical impact. Control of epidemic outbreak of phytoplasma diseases can be theoretically carried out by antibiotics. However, they are expensive, not allowed or prohibited in several countries, and even not always efficient. Presently, effective but safe antimicrobial agents are needed to control severe phytoplasma diseases in field. The aim of the present study was to evaluate the susceptibility of 'Candidatus Phytoplasma mali' to several chemical or synthetic antimicrobial agents. We tested nisin, esculetin, pyrithione and chloramphenicol as molecules having different target activities against micro-organisms. Because of their antimicrobial properties against fungi and bacteria, 4 phyto-essential oils (carvacrol, eugenol, terpineol, alpha-pinene) had also been tested. The activity of these molecules was compared with two antibiotics (tetracycline and enrofloxacin) used as control products. All these compounds were tested in in vitro culture of apples (MM106) infected by 'Ca. P. mall'. All compounds were added to the proliferation medium (modified MS) after autoclaving at 3 concentrations (100, 500, 1,000 ppm), except nisin and pyrithione which were tested at 10, 100 and 500 ppm. Phytoplasma infection was quantified in plant materials by real-time PCR before their transfer and after one or two months of culture in the presence of antimicrobial agents. Primary results showed that phytoplasma were not detectable after one and two months in the presence of pyrithione (at 10 and 100 ppm). Moreover, some other products reduced the concentration of phytoplasma after two months. Shoots died or withered on media enriched with essential oils; that made them impossible to assess, especially when they were used at concentration of 500 and 1,000 ppm.

Immunogold silver staining associated with epi-fluorescence for cucumber mosaic virus localisation on semi-thin sections of banana tissues.

The immunogold-silver staining (IGSS) technique in combination with epi-fluorescence detection was used to localise cucumber mosaic virus (CMV) particles within banana infected tissues. For this purpose, tissue samples (2 mm3) were excised from CMV-infected and highly proliferating meristem cultures of Williams BSJ banana (ITC. 0570, AAA, Cavendish subgroup). These samples were immediately fixed in a 2% paraformaldehyde/0.25% glutaraldehyde mixture, dehydrated in ethanol, and finally embedded in L.R. White resin. Semi-thin sections were cut, mounted on clean treated glass slides and immunostained for CMV particles using gold-labelled secondary antibodies and silver enhancement. Sections were counterstained with basic fuchsin and examined using laser scanning confocal microscopy. Negative controls included immuno-stained samples excised from non-virus infected material as well as infected material on which primary or secondary antibodies were not applied. Images of autofluorescence (in red) and of epi-reflectance of silver-enhanced immunogold particles (in green) were recorded separately and merged, allowing the specific localisation of CMV particles at the cellular level on semi-thin sections of aldehyde-fixed banana tissues. The main advantage of this analytical approach compared to previously published protocols is that it combines a fast staining procedure, stable preparation, a high resolution, and a narrow plane of focus with the flexibility in generation, processing and analysis of images offered by laser scanning confocal microscopy. Finally, the presence of numerous CMV particles within banana meristems constitutes a clear explanation of the very low CMV elimination efficiency when using meristem-tip culture alone.

Evidence for the assignment of two strains of SPLV to the genus Potyvirus based on coat protein and 3' non-coding region sequence data.

The use of potyvirus-specific primers and subsequent application of the RACE procedure allowed the cloning of the 3' terminal 1088 nucleotides of the genomic RNA of the Taiwan isolate of sweetpotato latent virus (SPLV-T) and the 3' genomic 1085 nucleotides of a SPLV-like virus from China (SPLV-CH). The sequence of an internal part of the presumptive nuclear inclusion b gene was also determined for both isolates. Detailed sequence analyses revealed the presence of consensus motifs which indicated that SPLV-CH and SPLV-T should be regarded as members of the genus Potyvirus. Multiple sequence alignments and phylogenetic analyses were also performed and unambiguously assessed these isolates as strains of a distinct Potyvirus. SPLV was not related to other potyviruses infecting sweetpotato nor to any other sequenced virus. From the presence of the DAG box, SPLV-CH is expected to be a typical aphid transmitted Potyvirus whereas a conceivable explanation is proposed for the non-aphid transmission of SPLV-T.

The nucleotide sequence and genome organization of the whitefly transmitted sweetpotato mild mottle virus: a close relationship with members of the family Potyviridae.

Primers corresponding to conserved regions in the RNA-dependent RNA polymerase and the RACE procedure led to the cloning of the complete sweetpotato mild mottle virus (SPMMV) RNA genome. The assembled SPMMV genomic sequence was 10,818 nucleotides in length with a polyadenylated tract at the 3' terminus. The structure and organization of the SPMMV genome appear to be similar to those of potyviruses and rymoviruses. A 5' untranslated region, rich in A and U residues, is present between nucleotides 1 and 139. A putative initiation codon, at nucleotides 140-142, marks the beginning of a large open reading frame (ORF) which ends in UAA at positions 10,508-10,510. A 308-nucleotide untranslated region is present between the termination codon of the ORF and the beginning of the 3' polyadenylated region. Almost all known potyvirus motifs are present in the polyprotein of SPMMV. However, motifs in the putative helper-component and coat protein of SPMMV are incomplete or missing, which may account for its vector relations. Despite similarities with rymoviruses, potyviruses and, to a lesser extent, bymoviruses, comparative sequence analyses demonstrated that SPMMV belongs to a distinct genus of the family Potyviridae.

The acyclic nucleoside phosphonate analogues, adefovir, tenofovir and PMEDAP, efficiently eliminate banana streak virus from banana (Musa spp.).

We report that the anti-retroviral and anti-hepadnavirus molecules, adefovir, tenofovir and 9-(2-phosphonomethoxyethyl)-2,6-diaminopurine (PMEDAP), efficiently eradicate the episomal form of Banana streak virus (BSV) from banana plants. Up to 90% of plants regenerated from BSV-infected highly proliferating meristems were virus free following a 6-month treatment period with 10 microg/ml (a non-phytotoxic concentration) of either compounds.

RT-PCR-ELOSA tests on pooled sample units for the detection of virus Y in potato tubers.

A highly sensitive RT-PCR protocol able to detect potato virus Y (PVY) in pooled sample units (tubers) was developed. PVY-specific primers selected in the coat protein gene were found to amplify a 359 bp fragment from diluted crude extract of infected tubers. For the detection of the amplification products, a colorimetric detection procedure in microtiter plates was established. The amplicons are hybridized between a covalently linked capture probe and a specific biotinylated detection probe ELOSA tests. This detection method detects at least 50 pg of virus per reaction for the four cultivars tested. The RT-PCR-ELOSA assay was adapted to pooled units in order to increase the sample size while reducing the number of tests.

Characterization of an Exo-beta-1,3-Glucanase Produced by Pichia anomala Strain K, Antagonist of Botrytis cinerea on Apples.

ABSTRACT The exo-beta-1,3-glucanase (EC 3.2.1.58) activity of Pichia anomala strain K, an antagonistic yeast of Botrytis cinerea on postharvest apples, was studied in a synthetic medium supplemented with laminarin, a cell wall preparation (CWP) of B. cinerea, or glucose. The highest enzyme activity was detected in culture media containing a CWP of B. cinerea as the sole carbon source, whereas the lowest activity was observed in culture media supplemented with glucose. Exoglc1, an exo-beta-1,3-glucanase, was purified to homogeneity from culture filtrates of strain K containing a CWP. The molecular mass of exoglc1 was estimated to be under 15 kDa. Optimum activity of exoglc1 was recorded at 50 degrees C and pH 5.5. The exoglc1 K(m) value was estimated at 22.4 mg/ml. Exoglc1 showed in vitro a stronger inhibitory effect on germ tube growth of B. cinerea than on conidia germination and caused morphological changes such as leakage of cytoplasm and cell swelling. Exo-beta-1,3-glucanase activity was detected on apples treated with strain K and was similar to exoglc1 on the basis of activity on native gel. Moreover, the addition of a CWP to a suspension of P. anomala stimulated both in situ exo-beta-1,3-glucanase activity and protective activity against the pathogen, strengthening the hypothesis that exo-beta-1,3-glucanase activity is one of the mechanisms of action involved in the suppression of B. cinerea by P. anomala strain K.

Possible Role of Colonization and Cell Wall-Degrading Enzymes in the Differential Ability of Three Ulocladium atrum Strains to Control Botrytis cinerea on Necrotic Strawberry Leaves.

ABSTRACT Ulocladium atrum (strain 385) consistently reduced Botrytis cinerea sporulation on necrotic fragments of strawberry leaves. On these tissues, two strains of U. atrum (isolates 18558 and 18559) showed lower antagonistic activities than the reference strain 385. Colonization of strawberry leaflets by the three U. atrum strains appeared similar in the absence of B. cinerea, whether quantified by chitin or immunological assays. The second method (based on anti-U. atrum antibodies) revealed that strawberry leaflet colonization by U. atrum 385 was better than by the other U. atrum strains in the presence of B. cinerea. An immunoassay using anti-B. cinerea antibodies revealed that the colonization of B. cinerea in tissues was lower in the presence of U. atrum 385 than with the two other U. atrum strains. The enzymatic activities produced by U. atrum 385 during the colonization phases of necrotic tissues were compared to B. cinerea and U. atrum strains 18558 and 18559. U. atrum 385 had the highest lipase, pectate lyase, and cellobiase activities while B. cinerea had the highest endo-beta-1,4-glucanase activity. The study of lytic activities hydrolyzing the fungal cell wall revealed higher beta-1,3-glucanase activity with U. atrum 385, which was stimulated by B. cinerea on necrotic strawberry leaflets. These results suggest that plant and fungal cell wall-degrading enzymes produced by U. atrum 385 may play a complementary role in the competitive colonization of dead strawberry leaves against B. cinerea.

Development of Real-Time PCR for the Rapid Detection of Episomal Banana streak virus (BSV).

A real-time assay for the detection of episomal Banana streak virus (BSV; strain OL) in banana and plantains that carry integrated BSV sequences is described. Primers specific to the viral DNA were designed using the viral sequence integrated into the cv. Obino l'Ewai genome and the sequence of the genomic DNA of the infecting virus strain OL. They amplify a sequence of 1,336 bp that is detected in real-time by a short fluorogenic 3' minor groove binder DNA probe. This method enables reproducible and specific detection of episomal BSV from purified DNA as well as from crude extracts from infected plants. The assay is rapid, adaptable for large-scale experiments, and circumvents carryover problems.

Differentiation Among Potyviruses Infecting Sweet Potato Based on Genus-and Virus-Specific Reverse Transcription Polymerase Chain Reaction.

Knowledge of virus diseases affecting sweet potato has been complicated due to the frequent occurrence of mixed infections and difficulties in isolating and purifying sweet potato viruses. A combined assay of reverse transcription and polymerase chain reaction (PCR) utilizing degenerate genus-specific primers POT1 and POT2 was applied to 18 sweet potato clones from China. The primers were designed to amplify the variable 5' terminal region of the potyvirus coat protein gene. Molecular analysis of the amplified fragments identified the Chinese strains of sweet potato feathery mottle virus (SPFMV-CH), sweet potato latent virus (SPLV-CH), and sweet potato virus G (SPVG-CH). Among the detected potyviruses, a distantly related strain of SPFMV-CH, tentatively named SPFMV-CH2, was identified in sweet potatoes from China. On the basis of sequence identity, SPFMV-CH2 was closely related to the common (-C) strain of that virus. Identification of a closely related strain of SPVG-CH in one sweet potato clone from China further illustrated the usefulness of broad-spectrum PCR for detecting uncharacterized viruses. The acquisition of sequence information permitted the design of virus-specific primers for detecting and differentiating SPFMV, SPLV, and SPVG.

Development of Real-Time RT-PCR Assay for Detection of Prunus necrotic ringspot virus in Fruit Trees.

A real-time fluorescent reverse-transcriptase polymerase chain reaction (RT-PCR) assay using a short fluorogenic 3' minor groove binder (MGB) DNA hydrolysis probe was developed for the detection of Prunus necrotic ringspot virus (PNRSV) in stone fruit trees. The covalent attachment of the minor groove binder moiety at the 3' end of the probe increased the probe target duplex stability and raised the melting temperature to a range suitable for real-time analysis. The real-time RT-PCR assay correlated well with conventional RT-PCR results for the detection of PNRSV. This assay reliably detects PNRSV in bark tissues of dormant cherry and plum trees. Furthermore, it is well adapted for the routine detection of PNRSV because it eliminates one risk of contamination by performing the whole test in a single closed tube. This system may replace the commonly used diagnostic techniques (e.g., woody indicators and immunological tests) to detect this virus.

Detection of Apple Stem Grooving Virus in Dormant Apple Trees with Crude Extracts as Templates for One-Step RT-PCR.

Partial nucleotide sequences of amplification products obtained from four European apple stem grooving virus (ASGV) isolates using degenerate primers showed 80 to 85% similarity with the published ASGV sequence of a Japanese strain but 98 to 100% identities among themselves. Based on these sequences, two ASGV-specific primers (ASGV4F-ASGV4R) were designed to amplify a 574-bp fragment located in the putative viral RNA polymerase. With these primers, six European and five American ASGV isolates, maintained in herbaceous hosts (Chenopodium quinoa, Nicotiana glutinosa, and N. occidentalis) or in apple trees, were readily detected by reverse transcription-polymerase chain reaction (RT-PCR). Using these specific ASGV primers, dsRNA preparations have been shown to constitute good templates for reliable amplification of ASGV sequences from leaves and bark tissues of apple trees, both in a two-step RT-PCR protocol and in the one-step Titan One-Tube RT-PCR. System. Furthermore, the one-step RT-PCR system allowed a specific amplification of ASGV sequences directly from clarified crude extracts of leaves and bark tissues of apple trees during both active growth and the dormant season.

First Report of Banana mild mosaic virus Isolated from Plantains (Musa AAB) in Colombia.

Plantains (Musa AAB) are important sources of food and income for millions of people in Colombia and other developing countries. Colombia is the largest producer of plantains (2) and the third largest exporter of bananas in the world. In 2001, plants of 'Dominico-Hartón' plantain showing mild chlorotic streak symptoms were observed in northwestern Colombia. Electron microscopy of symptomatic tissue extracts revealed the presence of filamentous virus-like particles approximately 800 nm long. Immunocapture reverse-transcription polymerase chain reaction was performed to test for the presence of Banana mild mosaic virus (BanMMV) as described by J. E. Thomas (unpublished, Queensland Department of Primary Industries, Australia) and Sharman et al. (3). For polymerase chain reaction (PCR), the upstream primer No. 193 (5'-CAC TTA GGT TTG TGT GAT GT-3') (designed in this study by using the computer Program DNAMAN Version 4.13) and the downstream primer Poty1 (5'-GGA TCC CGG GTT TTT TTT TTT TTT TTT V-3') (1,3; J. E. Thomas, unpublished, Queensland Department of Primary Industries, Australia) were used. Amplification products of the expected size (approximately 900 bp) were obtained and sequenced after cloning in a pCR2.1 plasmid vector. Analyses of nucleic acid sequences using the international sequence databases and the BLAST program yielded nucleotide and amino acid sequence similarities of 80 to 83% and 90 to 92%, respectively, with an Australian isolate of BanMMV (GenBank Accession No. AF314662). The coat protein (CP) gene of the Colombian BanMMV isolate consists of 717 nucleotides. When the CP of the Colombian BanMMV isolates (GenBank Accession Nos. AY319331, AY319332, and AY319333) was compared with the CP of the Australian isolate, a highly variable region was observed in the N-terminus region. To our knowledge, this is the first report of BanMMV isolated from plantains in Colombia and the presence of molecular variability in the CP of BanMMV isolates. BanMMV has been found in Colombia associated with Banana streak virus and Cucumber mosaic virus in plantain. References: (1) A. Gibbs and A. Mackenzie. J. Virol.Methods 63:9, 1997 (2) N. S. Price. Infomusa 8(2):26, 1999. (3) M. Sharman et al. J. Virol. Methods 89:75, 2000.

Development of molecular tests for the detection of ILAR and latent viruses in fruit trees.

The detection throughout the year of latent and ILAR viruses in fruit tress by classical serological tests appear to be unreliable. We have developed RT-PCR tests for a reliable detection of latent and ILAR viruses in fruit trees. These assays were then simplified to allow the direct use of crude plant extracts instead of total RNA preparations, and the analyses of pooled samples. In this way, such RT-PCR protocols are suitable for a routine diagnosis of latent and ILAR viruses in fruit tree certification.