Moniliophthora perniciosa, the Causal Agent of Cacao Witches’ Broom Disease Is Killed in vitro by Saccharomyces cerevisiae and Wickerhamomyces anomalus Yeasts

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fmicb.2021.706675/full#supplementary-materialCacao plantations from South America have been afflicted with the severe fungal disease known as Witches’ Broom Disease (WBD), caused by the basidiomycete Moniliophthora perniciosa. Yeasts are increasingly recognized as good fungal biocides, although their application is still mostly restricted to the postharvest control of plant and fruit decay. Their possible utilization in the field, in a preharvest phase, is nevertheless promising, particularly if the strains are locally adapted and evolved and if they belong to species considered safe for man and the environment. In this work, a group of yeast strains originating from sugarcane-based fermentative processes in Brazil, the cacao-producing country where the disease is most severe, were tested for their ability to antagonize M. perniciosa in vitro. Wickerhamomyces anomalus LBCM1105 and Saccharomyces cerevisiae strains LBCM1112 from spontaneous fermentations used to produce cachaça, and PE2 widely used in Brazil in the industrial production of bioethanol, efficiently antagonized six strains of M. perniciosa, originating from several South American countries. The two fastest growing fungal strains, both originating from Brazil, were further used to assess the mechanisms underlying the yeasts’ antagonism. Yeasts were able to inhibit fungal growth and kill the fungus at three different temperatures, under starvation, at different culture stages, or using an inoculum from old yeast cultures. Moreover, SEM analysis revealed that W. anomalus and S. cerevisiae PE2 cluster and adhere to the hyphae, push their surface, and fuse to them, ultimately draining the cells. This behavior concurs with that classified as necrotrophic parasitism/mycoparasitism. In particular, W. anomalus within the adhered clusters appear to be ligated to each other through roundish groups of fimbriae-like structures filled with bundles of microtubule-sized formations, which appear to close after cells detach, leaving a scar. SEM also revealed the formation of tube-like structures apparently connecThis study was supported by the strategic program UID/BIA/04050/2020 funded by national funds through the FCT I.P., and by the ERDF through the COMPETE2020 – Programa Operacional Competitividade e Internacionalização (POCI) and the project AgriFood XXI (NORTE-01-0145-FEDER-000041). This study was also supported by the Norte Portugal Regional Operational Programme (NORTE 2020). PF was a PhD student of the Doctoral Program in Applied and Environmental Microbiology (DP_AEM; FCT grant no. PD/BD/113814/2015)

High-affinity transport, cyanide-resistant respiration, and ethanol production under aerobiosis underlying efficient high glycerol consumption by Wickerhamomyces anomalus

Wickerhamomyces anomalus strain LBCM1105 was originally isolated from the wort of cachaça (the Brazilian fermented sugarcane juice-derived Brazilian spirit) and has been shown to grow exceptionally well at high amounts of glycerol. This paramount residue from the biodiesel industry is a promising cheap carbon source for yeast biotechnology. The assessment of the physiological traits underlying the W. anomalus glycerol consumption ability in opposition to Saccharomyces cerevisiae is presented. A new WaStl1 concentrative glycerol-H+ symporter with twice the affinity of S. cerevisiae was identified. As in this yeast, WaSTL1 is repressed by glucose and derepressed/induced by glycerol but much more highly expressed. Moreover, LBCM1105 aerobically growing on glycerol was found to produce ethanol, providing a redox escape to compensate the redox imbalance at the level of cyanide-resistant respiration (CRR) and glycerol 3P shuttle. This work is critical for understanding the utilization of glycerol by non-Saccharomyces yeasts being indispensable to consider their industrial application feeding on biodiesel residue.This work was supported by grants from Fundacao de Capacitacao de Pessoal de Nivel Superior from the Ministry of Education-CAPES/Brazil (PNPD 2755/2011; PCF-PVE 021/2012), from FEDER through POFC-COMPETE and by FCT through strategic funding (UID/BIA/04050/2013), from Universidade Federal de Ouro Preto, and a research fellowship from Conselho Nacional de Desenvolvimento Cientifico e Tecnologico-CNPq (Brazil) Process 304815/2012 (research grant) and Process 305135/2015-5 (research fellowship to R.L.B.). C.L. is supported by the strategic program UID/BIA/04050/2013 [POCI-01-0145-FEDER-007569] funded by national funds through the FCT I. P. and by the ERDF through the COMPETE2020-Programa Operacional Competitividade e (POCI). The AUXPE-PVES 1801/2012 (Process 23038.015294/2016-18) from Brazilian Government supported a grant of Visiting Professor to C.L. and a research fellowships to A.C.C. and to F.F.O

During the initiation of fermentation overexpression of hexokinase PII in yeast transiently causes a similar deregulation of glycolysis as deletion of Tps1

In the yeast Saccharomyces cerevisiae a novel control exerted by TPS1 (=GGS1=FDP1=BYP1=CIF1=GLC6=TSS1)-encoded trehalose-6-phosphate synthase, is essential for restriction of glucose influx into glycolysis apparently by inhibiting hexokinase activity in vivo. We show that up to 50-fold overexpression of hexokinase does not noticeably affect growth on glucose or fructose in wild-type cells. However, it causes higher levels of glucose-6-phosphate, fructose-6-phosphate and also faster accumulation of fructose-1,6-bisphosphate during the initiation of fermentation. The levels of ATP and Pi correlated inversely with the higher sugar phosphate levels. In the first minutes after glucose addition, the metabolite pattern observed was intermediate between those of the tps1Δ mutant and tile wild-type strain. Apparently, during the start-up of fermentation hexokinase is more rate-limiting in the first section of glycolysis than phosphofructokinase. We have developed a method to measure the free intracellular glucose level which is based on the simultaneous addition of D-glucose and an equal concentration of radiolabelled L-glucose. Since the latter is not transported, the free intracellular glucose level can be calculated as the difference between the total B-glucose measured (intracellular + periplasmic/extracellular) and the total L-glucose measured (periplasmic/extracellular). The intracellular glucose level rose in 5 min after addition of 100 mM-glucose to 0.5-2 mM in the wild-type strain, ± 10 mm in a hxk1Δ hxk2Δ glk1Δ and 2-3 mM in a tps1Δ strain. In the strains overexpressing hexokinase PII the level of free intracellular glucose was not reduced. Overexpression of hexokinase PII never produced a strong effect on the rate of ethanol production and glucose consumption. Our results show that overexpression of hexokinase does not cause the same phenotype as deletion of Tps1. However, it mimics it transiently during the initiation of fermentation. Afterwards, the Tps1-dependent control system is apparently able to restrict Properly up to 50-fold higher hexokinase activity

Cyclic AMP and low molecular weight effector(s) present in yeast extract are involved in pectin lyase production by Penicillium griseoroseum cultured on sucrose

Pectin lyase (PL) induction by organic and inorganic components of yeast extract (YE) was evaluated in Penicillum griseoroseum, cultured in a mineral medium containing sucrose, by determining PL activity (A 235) and mycelial growth (mycelial dry weight). The lowest YE concentration that promoted significant PL induction without acting as a carbon source for the fungus corresponded to 0.0075%. Neither calcined YE nor a nutrient solution containing micronutrients induced PL production, indicating that the inducer was an organic compound. Vitamins, phospholipid components, amino acids, and nitrogenous bases were tested in place of YE and promoted no significant PL induction. APL inducer compound was found to be soluble in the nucleotide fraction obtained during extraction of YE. The inducer was shown to be a thermostable polar substance dialyzable at 2000 Daltons, hydrolyzable by HCl, and activated by boiling for up to 60 min. Cyclic AMP (cAMP) exogenously added to the culture medium at 5 and 10 mM was capable of inducing PL in P. griseoroseum grown on sucrose, suggesting that at least one compound may be present in YE acting in a cooperative fashion for the maintenance of high levels of cAMP in to the cell. PL activity and the level of cAMP inside the fungal cells increased after the addition of YE to the culture medium, suggesting the participation of this messenger in this enzyme's synthesis

Biochemical and Molecular Characterization of Saccharomyces cerevisiae Strains Obtained from Sugar-Cane Juice Fermentations and Their Impact in Cachaça Production▿

Saccharomyces cerevisiae strains from different regions of Minas Gerais, Brazil, were isolated and characterized aiming at the selection of starter yeasts to be used in the production of cachaça, the Brazilian sugar cane spirit. The methodology established took into account the screening for biochemical traits desirable in a yeast cachaça producer, such as no H2S production, high tolerance to ethanol and high temperatures, high fermentative capacity, and the abilities to flocculate and to produce mycocins. Furthermore, the yeasts were exposed to drugs such as 5,5′,5"-trifluor-d,l-leucine and cerulenin to isolate those that potentially overproduce higher alcohols and esters. The utilization of a random amplified polymorphic DNA-PCR method with primers based on intron splicing sites flanking regions of the COX1 gene, as well as microsatellite analysis, was not sufficient to achieve good differentiation among selected strains. In contrast, karyotype analysis allowed a clear distinction among all strains. Two selected strains were experimentally evaluated as cachaça producers. The results suggest that the selection of strains as fermentation starters requires the combined use of biochemical and molecular criteria to ensure the isolation and identification of strains with potential characteristics to produce cachaça with a higher quality standard

New Lager Brewery Strains obtained by crossing techniques using cachaça (Brazilian Spirit) yeasts

The development of hybrids has been an effective approach to generate novel yeast strains with optimal technological profile for use in beer production. This study describes the generation of a new yeast strain for lager beer production by direct mating between two Saccharomyces cerevisiae strains isolated from cachaça distilleries: one that was strongly flocculent, and the other with higher production of acetate esters. The first step in this procedure was to analyze the sporulation ability and reproductive cycle of strains belonging to a specific collection of yeasts isolated from cachaça fermen- tation vats. Most strains showed high rates of sporulation, spore viability, and homothal- lic behavior. In order to obtain new yeast strains with desirable properties useful for la- ger beer production, we compare haploid-to-haploid and diploid-to-diploid mating procedures. Moreover, an assessment of parental phenotype traits showed that the seg- regant diploid C2-1d generated from a diploid-to-diploid mating experiment showed good fermentation performance at low temperature, high flocculation capacity, and de- sirable production of acetate esters that was significantly better than that of one type la- ger strain. Therefore, strain C2-1d might be an important candidate for the production of lager beer, with distinct fruit traces and originating using a non-genetically modified organism (GMO) approach. Recent work has suggested the utilization of hybridization techniques for the generation of novel non-genetically modified brewing yeast strains with combined properties not commonly found in a unique yeast strain. We have observed remarkable traits, especially low temperature tolerance, maltotriose utilization, flocculation ability, and production of volatile aroma compounds, among a collection of Saccharomyces cerevisiae strains isolated from cachaça distilleries, which allow their utilization in the production of beer. The significance of our research is in the use of breeding/hybridization techniques to generate yeast strains that would be appropriate for producing new lager beers by exploring the capacity of cachaça yeast strains to flocculate and to ferment maltose at low temperature, with the concomitant production of flavoring compounds

Biotechnological potential of yeast isolates from cachaça: the brazilian spirit

This study identified phenotypic traits appropriate for biotechnological applications of 118 yeasts isolated from cachaça distilleries. Different properties were verified: capacity to use alternative carbon sources; ability to tolerate high concentrations of sucrose, ethanol, methanol, aluminum and zinc as well as different pH values and foam production. Pichia guilliermondii and Pichia anomala strains were identified as the most promising ones for application in the second-generation biofuel industry, showing ability to grow on high glycerol concentrations. Other isolates, identified as Saccharomyces cerevisiae, produced bioethanol comparable to the industrial strains, and were therefore ideal for use in the first-generation ethanol industry. Some of these strains also showed high resistance to aluminum, as observed in sugarcane juice, and to inter-cycle washings with diluted sulphuric acid, as performed in the industrial bioethanol production process. In summary, yeast isolates from cachaça distilleries displayed robustness and phenotypic plasticity, which makes them interesting for biotechnological applications.This work was supported by grants from Fundação de Capacitação de Pessoal de Nível Superior from the Ministry of Education, CAPES/Brazil (PNPD 2755/2011; PCF-PVE 021/2012) and from Universidade Federal de Ouro Preto, Fundação de Amparo à Pesquisa do Estado de Minas Gerais, FAPEMIG (Process APQ-00263-10) and a research fellowship from Conselho Nacional de Desenvolvimento Científico e Tecnológico, CNPq (Brazil) Process 304815/2012. C. Lucas is supported by FCT/MEC through Portuguese funds (PIDDAC), PEst-OE/BIA/UI4050/2014 and by a grant of Visiting Professor from the programme “Ciência sem Fronteiras”, CAPES, Brazil, Process 2021/2012.info:eu-repo/semantics/publishedVersio