Predominant Clonal Reproduction with Infrequent Genetic Recombination of Phaeoacremonium minimum in Western Cape Vineyards.

Phaeoacremonium minimum is an important esca and Petri disease pathogen that causes dieback of grapevines in South Africa. Little is known regarding the reproductive strategy of the pathogen. Sexual reproduction could lead to a better adaptation of the pathogen to disease management strategies by combining alleles through recombination. The study aimed to investigate the genetic diversity and recombination potential of eight populations in the Western Cape, from six commercial vineyards and two nursery rootstock mother blocks. This was achieved by developing and applying nine polymorphic microsatellites and mating-type-specific markers. Thirty-seven genotypes were identified from 295 isolates. Populations were characterised by the same dominant genotype (MLG20 occurring 65.43%), low genotypic diversity (H) and high numbers of clones (81.36% of dataset). However, genotypes from the same sampling sites were not closely related based on a minimum spanning network and had high molecular variation within populations (94%), suggesting that multiple introductions of different genotypes occurred over time. Significant linkage disequilibrium among loci (r̅d) further indicated a dominant asexual cycle, even though perithecia have been observed in these four populations. The two rootstock mother blocks had unique genotypes and genotypes shared with the vineyard populations. Propagation material obtained from infected rootstock mother blocks could lead to the spread of more genotypes to newly established vineyards. Based on our results, it is important to determine the health status of rootstock mother blocks. Management strategies must focus on reducing aerial inoculum to prevent repeated infections and further spread of P. minimum genotypes.

Investigation of Trichoderma species colonization of nursery grapevines for improved management of black foot disease.

BACKGROUND: Black foot disease (BFD) is one of the main fungal diseases associated with young grapevine decline. Trichoderma holds the potential to be used as biocontrol agent against this disease, though variable success of colonization were found when applied to nursery vines in previous studies. Therefore, field experiments were established to evaluate different methods of application of Trichoderma atroviride, and to evaluate the efficacy of different commercial Trichoderma products on BFD in nursery vines post callusing over two seasons. RESULTS: Only in one season of the trial evaluating different products did all of the Trichoderma treatments significantly lower the black foot infections in the rootstock bases of the vines (mean black foot pathogen incidence of 1.00 to 2.50% in Trichoderma treated vines versus 6.50% in the untreated control). When comparing tissue parts, the base of the vine and collar roots had significantly higher Trichoderma colonization than the middle and root tip parts. Significantly less BFD pathogens were isolated from the base in comparison to the roots. These colonization trends were found for both field trials over both seasons. The different application methods showed that dipping of basal ends in the dry formulation followed by monthly soil drenches consistently gave higher colonization [mean Trichoderma incidence in the bases were 39.20% (2016/2017) and 28.00% (2017/2018)], while the 1 h soak of the bases of vines was not effective [mean Trichoderma incidence in the bases were 8.80% (2016/2017) and 4.00% (2017/2018)] and did not differ from the untreated control. CONCLUSION: Even though Trichoderma spp. were not sufficient to prevent infections by BFD pathogens, a certain degree of protection was obtained in the basal ends, which may contribute to longevity of the vines once planted in the vineyard.

FLO gene-dependent phenotypes in industrial wine yeast strains.

Most commercial yeast strains are nonflocculent. However, controlled flocculation phenotypes could provide significant benefits to many fermentation-based industries. In nonflocculent laboratory strains, it has been demonstrated that it is possible to adjust flocculation and adhesion phenotypes to desired specifications by altering expression of the otherwise silent but dominant flocculation (FLO) genes. However, FLO genes are characterized by high allele heterogeneity and are subjected to epigenetic regulation. Extrapolation of data obtained in laboratory strains to industrial strains may therefore not always be applicable. Here, we assess the adhesion phenotypes that are associated with the expression of a chromosomal copy of the FLO1, FLO5, or FLO11 open reading frame in two nonflocculent commercial wine yeast strains, BM45 and VIN13. The chromosomal promoters of these genes were replaced with stationary phase-inducible promoters of the HSP30 and ADH2 genes. Under standard laboratory and wine making conditions, the strategy resulted in expected and stable expression patterns of these genes in both strains. However, the specific impact of the expression of individual FLO genes showed significant differences between the two wine strains and with corresponding phenotypes in laboratory strains. The data suggest that optimization of the flocculation pattern of individual commercial strains will have to be based on a strain-by-strain approach.

Yeast flocculation and its biotechnological relevance.

Adhesion properties of microorganisms are crucial for many essential biological processes such as sexual reproduction, tissue or substrate invasion, biofilm formation and others. Most, if not all microbial adhesion phenotypes are controlled by factors such as nutrient availability or the presence of pheromones. One particular form of controlled cellular adhesion that occurs in liquid environments is a process of asexual aggregation of cells which is also referred to as flocculation. This process has been the subject of significant scientific and biotechnological interest because of its relevance for many industrial fermentation processes. Specifically adjusted flocculation properties of industrial microorganisms could indeed lead to significant improvements in the processing of biotechnological fermentation products such as foods, biofuels and industrially produced peptides. This review briefly summarises our current scientific knowledge on the regulation of flocculation-related phenotypes, their importance for different biotechnological industries, and possible future applications for microorganisms with improved flocculation properties.

Controlled expression of the dominant flocculation genes FLO1, FLO5, and FLO11 in Saccharomyces cerevisiae.

In many industrial fermentation processes, the Saccharomyces cerevisiae yeast should ideally meet two partially conflicting demands. During fermentation, a high suspended yeast count is required to maintain a satisfactory rate of fermentation, while at completion, efficient settling is desired to enhance product clarification and recovery. In most fermentation industries, currently used starter cultures do not satisfy this ideal, probably because nonflocculent yeast strains were selected to avoid fermentation problems. In this paper, we assess molecular strategies to optimize the flocculation behavior of S. cerevisiae. For this purpose, the chromosomal copies of three dominant flocculation genes, FLO1, FLO5, and FLO11, of the haploid nonflocculent, noninvasive, and non-flor-forming S. cerevisiae FY23 strain were placed under the transcriptional control of the promoters of the ADH2 and HSP30 genes. All six promoter-gene combinations resulted in specific flocculation behaviors in terms of timing and intensity. The strategy resulted in stable expression patterns providing a platform for the direct comparison and assessment of the specific impact of the expression of individual dominant FLO genes with regard to cell wall characteristics, such as hydrophobicity, biofilm formation, and substrate adhesion properties. The data also clearly demonstrate that the flocculation behavior of yeast strains can be tightly controlled and fine-tuned to satisfy specific industrial requirements.

Many Saccharomyces cerevisiae Cell Wall Protein Encoding Genes Are Coregulated by Mss11, but Cellular Adhesion Phenotypes Appear Only Flo Protein Dependent.

The outer cell wall of the yeast Saccharomyces cerevisiae serves as the interface with the surrounding environment and directly affects cell-cell and cell-surface interactions. Many of these interactions are facilitated by specific adhesins that belong to the Flo protein family. Flo mannoproteins have been implicated in phenotypes such as flocculation, substrate adhesion, biofilm formation, and pseudohyphal growth. Genetic data strongly suggest that individual Flo proteins are responsible for many specific cellular adhesion phenotypes. However, it remains unclear whether such phenotypes are determined solely by the nature of the expressed FLO genes or rather as the result of a combination of FLO gene expression and other cell wall properties and cell wall proteins. Mss11 has been shown to be a central element of FLO1 and FLO11 gene regulation and acts together with the cAMP-PKA-dependent transcription factor Flo8. Here we use genome-wide transcription analysis to identify genes that are directly or indirectly regulated by Mss11. Interestingly, many of these genes encode cell wall mannoproteins, in particular, members of the TIR and DAN families. To examine whether these genes play a role in the adhesion properties associated with Mss11 expression, we assessed deletion mutants of these genes in wild-type and flo11Δ genetic backgrounds. This analysis shows that only FLO genes, in particular FLO1/10/11, appear to significantly impact on such phenotypes. Thus adhesion-related phenotypes are primarily dependent on the balance of FLO gene expression.

The regulation of Saccharomyces cerevisiae FLO gene expression and Ca2+ -dependent flocculation by Flo8p and Mss11p.

The ability of many microorganisms to modify adhesion-related properties of their cell surface is of importance for many processes, including substrate adhesion, cell-cell adhesion, invasive growth, pathogenic behaviour and biofilm formation. In the yeast Saccharomyces cerevisiae, a group of structurally related, cell-wall associated proteins encoded by the FLO gene family are directly responsible for many of the cellular adhesion phenotypes displayed by this organism. Previous research has suggested that the differential transcription of FLO genes determines specific adhesion phenotypes. However, the transcriptional regulation of most FLO genes remains poorly understood. Here we show that the transcriptional activator Mss11p, which has previously been shown to be involved in the regulation of starch degradation, the formation of pseudohyphae and haploid invasive growth, also acts as a strong inducer of flocculation. The data indicate that Mss11p induces flocculation together with Flo8p, and that FLO1 is the dominant target gene of the two factors in this process. The deletion of MSS11 leads to a non-flocculent phenotype, and specific domains of Mss11p that are critical for the induction of flocculation are identified. The data clearly show that several essential transcription factors are shared by at least two flocculation genes that control different adhesion phenotypes.

Mss11p is a transcription factor regulating pseudohyphal differentiation, invasive growth and starch metabolism in Saccharomyces cerevisiae in response to nutrient availability.

In Saccharomyces cerevisiae, the cell surface protein, Muc1p, was shown to be critical for invasive growth and pseudohyphal differentiation. The transcription of MUC1 and of the co-regulated STA2 glucoamylase gene is controlled by the interplay of a multitude of regulators, including Ste12p, Tec1p, Flo8p, Msn1p and Mss11p. Genetic analysis suggests that Mss11p plays an essential role in this regulatory process and that it functions at the convergence of at least two signalling cascades, the filamentous growth MAPK cascade and the cAMP-PKA pathway. Despite this central role in the control of filamentous growth and starch metabolism, the exact molecular function of Mss11p is unknown. We subjected Mss11p to a detailed molecular analysis and report here on its role in transcriptional regulation, as well as on the identification of specific domains required to confer transcriptional activation in response to nutritional signals. We show that Mss11p contains two independent transactivation domains, one of which is a highly conserved sequence that is found in several proteins with unidentified function in mammalian and invertebrate organisms. We also identify conserved amino acids that are required for the activation function.