The nucleic acid sequences of the pre-membrane/membrane and envelope protein genes of 23 geographically and temporally distinct dengue (DEN)-3 viruses were determined. This was accomplished by reverse transcriptase-PCR amplification of the structural genes followed by automated DNA sequence analysis. Comparison of nucleic acid sequences revealed that similarity among the viruses was greater than 90%. The similarity among deduced amino acids was between 95% and 100%, and in many cases identical amino acid substitutions occurred among viruses from similar geographical regions. Alignment of nucleic acid sequences followed by parsimony analysis allowed the generation of phylogenetic trees, demonstrating that geographically independent evolution of DEN-3 viruses had occurred. The DEN-3 viruses were separated into four genetically distinct subtypes. Subtype I consists of viruses from Indonesia, Malaysia, the Philippines and the South Pacific islands; subtype II consists of viruses from Thailand; subtype III consists of viruses from Sri Lanka, India, Africa and Samoa; subtype IV consists of viruses from Puerto Rico and the 1965 Tahiti virus. Phylogenetic analysis has also contributed to our understanding of the molecular epidemiology and worldwide distribution of DEN-3 viruses.
Pathogenic fungi belonging to the genera Botrytis, Phaeomoniella, Fusarium, Alternaria and Aspergillus are responsible for vines diseases that affect the growth, grapevine yield and organoleptic quality. Among innovative strategies for in-field plant disease control, one of the most promising is represented by biocontrol agents, including wild epiphytic yeast strains of grapevine berries. Twenty wild yeast, isolated and molecularly identified from three different Malaysian regions (Perlis, Perak and Pahang), were evaluated in a preliminary screening test on agar to select isolates with inhibition against Botrytis cinerea. On the basis of the results, nine yeasts belonging to genera Hanseniaspora, Starmerella, Metschnikowia, Candida were selected and then tested against five grape berry pathogens: Aspergillus carbonarius, Aspergillus ochraceus, Fusarium oxysporum, Alternaria alternata and Phaeomoniella chlamydospora.Starmerella bacillaris FE08.05 and Metschnikowia pulcherrima GP8 and Hanseniaspora uvarum GM19 showed the highest effect on inhibiting mycelial growth, which ranged between 15.1 and 4.3 mm for the inhibition ring. The quantitative analysis of the volatile organic compound profiles highlighted the presence of isoamyl and phenylethyl alcohols and an overall higher presence of low-chain fatty acids and volatile ethyl esters. The results of this study suggest that antagonist yeasts, potentially effective for the biological control of pathogenic moulds, can be found among the epiphytic microbiota associated with grape berries.