The yeast populations associated with the fermentation of Ghanaian cocoa were investigated using denaturing gradient gel electrophoresis (DGGE). Samples were collected at 12-24 h intervals from heap and tray fermentations, at three different fermentation sites and different periods during the season. Eukaryotic universal primers were used to amplify a fragment of the 26S rRNA gene. The DGGE profiles were relatively complex, underlining that the fermentation of cocoa is a complex microbial process. The identities of selected fragments in the denaturing gels were revealed by sequencing. Hanseniaspora guilliermondii, Candida krusei and Pichia membranifaciens were detected from most fermentations, indicating their possible important role in the fermentation of Ghanaian cocoa. Saccharomyces cerevisiae and Candida zemplinina were almost exclusively detected during tray fermentations. The developed DGGE protocol was compared with traditional culture-based isolations. The results were comparable but slightly different, as one yeast species (C. zemplinina) was only detected using DGGE. On the other hand, Trichosporon asahii yielded only faint bands in the denaturing gels, despite the fact that it was detected using culture-based methods. Analysis of pure cultures showed that the targeted region of the 26S rRNA gene was poorly amplified in T. asahii, whereas all other investigated isolates were amplified efficiently using the chosen PCR approach. Cluster analysis revealed that the DGGE profiles clustered according to fermentation method and fermentation site. Furthermore, clustering according to progress in the fermentation was observed. The DGGE technique therefore seems to offer a relatively fast and reliable method for studying yeast population dynamics during cocoa fermentations.

https://www.onlinelibrary.wiley.com/doi/pdf/10.1002/yea.1207

Yeast populations associated with Ghanaian cocoa fermentations analysed using denaturing gradient gel electrophoresis (DGGE)

Nigrospora sphaerica was isolated from date palm leaves which showed severe symptoms of leaf and stem spot. Pathogenicity tests revealed that several isolates of this fungus reproduced symptoms on the cultivar ‘Al-Sayer’. The susceptibility of five different cultivars of date palm showed that ‘Halawii’ and ‘Al-Sayer’ were the most susceptible to infection compared with other cultivars such as ‘Barhee’, ‘Zahdi’ and ‘Khadrawii’. Morphological and molecular characteristics confirmed the identification of N. sphaerica. The phytotoxicity of the culture filtrates were investigated, and shown to induce severe symptoms on date palm leaves.

First report of Nigrospora sphaerica (Sacc.) Mason as a potential pathogen on date palm (Phoenix dactylifera L.)

Compounds in mulberry leaves inducing sporulation of C. dematium, mulberry anthracnose fungus, were detected. Dissolved or suspended aqueous solutions (1%) of 16 amino acids and 10 vitamins occurring in mulberry leaves were applied individually at the margin of the fungal colony growing on PSA plate. Sporulation was induced only where a biotin solution was applied on the mycelium at a concentration of at least 0.01 ppm. This result suggests that biotin, which occurs in mulberry leaves (ca. 0.6 mg/kg of dried leaves), has a role in inducing sporulation of C. dematium.

Biotin Induces Sporulation of Mulberry Anthracnose Fungus, Colletotrichum dematium

Analysis of 49 strains of Fusarium langsethiae originating from northern Europe (Russia, Finland, Sweden, UK, Norway, and Latvia) revealed the presence of spontaneous auxotrophic mutants that reflect natural intraspecific diversity. Our investigations detected that 49.0% of F. langsethiae strains were auxotrophic mutants for biotin, and 8.2% of the strains required thiamine as a growth factor. They failed to grow on vitamin-free media. For both prototrophic and auxotrophic strains, no growth defect was observed in rich organic media. Without essential vitamins, a significant reduction in the growth of the auxotrophic strains results in a decrease of the formation of T-2 toxin and diacetoxyscirpenol. In addition, all analysed F. langsethiae strains were distinguished into two subgroups based on PCR product sizes. According to our results, 26 and 23 strains of F. langsethiae belong to subgroups I and II respectively. We determined that the deletion in the intergenic spacer (IGS) region of the rDNA of F. langsethiae belonging to subgroup II is linked with temperature sensitivity and causes a decrease in strain growth at 30 °C. Four thiamine auxotrophic strains were found in subgroup I, while 21 biotin auxotrophic strains were detected in subgroups II. To the best of our knowledge, the spontaneous mutations in F. langsethiae observed in the present work have not been previously reported.

https://www.mdpi.com/2076-2607/5/2/14/pdf

Identification and Characterization of Spontaneous Auxotrophic Mutants in Fusarium langsethiae

Morphological, molecular and pathological study on Nigrospora oryzae and Nigrospora sphaerica, the leaf spot fungi of date palm

Esposito, R. G. (United Fruit Co., Norwood, Mass.), H. Greenwood, and A. M. Fletcher. Growth factor requirements of six fungi associated with fruit decay. J. Bacteriol. 83:–250–255. 1962.—A study of the growth factor requirements of representatives of six genera of fruit-rotting fungi has been made. Gloeosporium musarum, Nigrospora sphaerica, Thielaviopsis paradoxa, and Verticillium theobromae all required biotin for growth. Under certain conditions in nitrate medium, Fusarium roseum appeared to be partially dependent on biotin. T. paradoxa also required thiamine for growth. In addition to biotin, N. sphaerica required an unknown material contained in extracts of yeast. Twenty-one known growth factors were tested and found to be inactive. Thiamine inhibited the growth of F. roseum, apparently by stimulating the formation of ethanol. Biotin reversed the effect of thiamine on growth and ethanol formation.

H. Greenwood

Papers

Growth factor requirements of six fungi associated with fruit decay.