Thermotherapy, chemotherapy, and meristem culture in banana.

Bananas that provide a staple food to the millions of people are adversely affected by several viruses such as Banana bunchy Top Virus (BBTV), Banana Streak Virus (BSV), and Cucumber Mosaic Virus (CMV). These viruses are known to have a devastating effect on crop production and constraint to the international exchange and conservation of banana germplasm-a cornerstone for breeding new cultivars. The viruses are particularly problematic in vegetative propagated crops, like bananas, because of their transmission in the planting material. Different virus eradication techniques have been developed, such as thermotherapy, chemotherapy, and meristem culture for providing virus-free planting material. Meristem culture proved to be the most effective procedure to eradicate phloem-associated viruses. This method requires isolation of meristematic dome of plant under the aseptic conditions and culture in an appropriate nutrient medium to develop new virus-free plants. Thermotherapy is another widely used virus eradication technique, which is initially carried out on in vivo or in vitro plants and eventually combined with meristem culture technique. The plantlets are initially grown at 28°C day temperature and increase it by 2°C per day until reaches 40°C and the night temperature at 28°C; maintain plants at 40°C for 4 weeks; excise meristem and culture onto the regeneration medium. In chemotherapy technique, antiviral chemical compound Virazole(®) is applied on meristem culture. Combination of these techniques is also applied to improve the eradication rate.

Identification of genes involved in the response of banana to crown rot disease.

Variations in banana susceptibility to crown rot disease have been observed but the molecular mechanisms underlying these quantitative host-pathogen relationships are still unknown. This study was designed to compare gene expression between crowns of banana fruit showing a high susceptibility (S(+)) and crowns showing a low susceptibility (S(-)) to the disease. Comparisons were performed at two situation times: i) between crowns (S(+) and S(-)) collected 1 h before inoculation and ii) between crowns (S+ and S-) collected 13 days after inoculation. Gene expression comparisons were performed with cDNA-amplified fragment length polymorphism (AFLP) and results were confirmed by real-time reverse-transcription polymerase chain reaction. Among genes identified as differentially expressed between S(+) and S(-) crowns, two were involved in signal transduction, three in proteolytic machinery, two had similarity to pathogenesis-related protein 14, one to a CCR4-associated factor protein, and one to a cellulose synthase. Paradoxically, the overexpression of the cellulose synthase gene was associated with banana showing a high susceptibility in both pre- and post-inoculation situations. Finally, the cDNA-AFLP identified a gene that seems to be associated with the quantitative banana responses to crown rot disease; this gene encodes a dopamine-ß-monooxygenase, which is involved in the catecholamine pathway. To our knowledge, this work is the first to address both pre- and post-infection gene expression with the same host-pathogen combination and distinct susceptibility levels.

Development of a Quick Quantitative Real-Time PCR for the In Vivo Detection and Quantification of Peach latent mosaic viroid.

Viroids are plant pathogens infecting a broad range of herbaceous and tree crops. Among them, the Peach latent mosaic viroid (PLMVd) infects mainly peach trees, causing a loss of production with no curative options. Detecting this viroid is thus important for certification procedures aiming to avoid the release of infected material into orchards. Presented here is a complete detection method based on reverse transcription (RT) followed by a quantitative real-time polymerase chain reaction (PCR). New primers were selected and optimal reaction conditions determined for routine application of the method. The technique is 105 times more sensitive than the endpoint RT-PCR used for PLMVd detection, and permits earlier detection of PLMVd in infected plants. The quick, low-cost extraction procedure used and the quality of the results obtained make this method suitable for routine testing.

Characterization of the Exoglucanase-Encoding Gene PaEXG2 and Study of Its Role in the Biocontrol Activity of Pichia anomala Strain K.

ABSTRACT The PaEXG2 gene, encoding an exo-beta-1,3-glucanase, was isolated from the biocontrol agent Pichia anomala strain K. PaEXG2 has the capacity for coding an acidic protein of 427 amino acids with a predicted molecular weight of 45.7 kDa, a calculated pI of 4.7, and one potential N-glycosylation site. PaEXG2 was disrupted by the insertion of the URA3 marker gene, encoding orotidine monophosphate decarboxylase in strain KU1, a uracil auxotroph derived from strain K. Strain KU1 showed inferior biocontrol activity and colonization of wounds on apples, compared to the prototrophic strain. Antagonism and colonization were recovered after the restoration of prototrophy by transformation with the URA3 gene. Integrative transformation was shown to be mostly ectopic in strain K descendants (only 4% of integration by homologous recombination). PaEXG2 disruption abolished all detectable extracellular exo-beta-1,3-glucanase activity in vitro and in situ but did not affect biocontrol of Botrytis cinerea on wounded apples.

Detection of apple chlorotic leaf spot virus using a 5' nuclease assay with a fluorescent 3' minor groove binder-DNA probe.

The development of a real-time 5' nuclease RT-PCR assay for the detection of apple chlorotic leaf spot virus (ACLSV) from infected plant material is described. A short fluorogenic 3' minor groove binder-DNA hydrolysis probe was used to circumvent genome variability between isolates and target a short conserved sequence. 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 method is rapid, sensitive and takes place within a single tube without post-PCR handling of the amplification products.