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Characterisation of Aureobasidium pullulans isolates from Vitis vinifera and potential biocontrol activity for the management of bitter rot of grapes
Abstract
Aureobasidium isolated from Vitis vinifera (cv Chardonnay) grapevine tissues were characterised using morphological and molecular techniques. Species level identification of 29 isolates was accomplished by partial amplification and sequencing of the ITS region (ITS1–5.8S–ITS2) using universal primers ITS1 and ITS4. A comparison of nucleotide sequences using BLAST followed by phylogenetic analysis revealed that all isolates examined were Aureobasidium pullulans. Strain level discrimination of a total of 100 epiphytic Aureobasidium isolates including three reference strains was successfully carried out using two inter simple sequence repeat (ISSR) primers, (AAC)5 and (GTG)5 and the Intron Splice Junction R1 (ISJ-R1) primer in which 24, 24 and 15 scorable bands were produced for each primer, respectively. The high level of genetic variation recorded among the isolates further highlighted the high levels of strain diversity among A. pullulans residing on grapevines. Thirty-two epiphytic Aureobasidium isolates were examined for their ability to inhibit the growth of Greeneria uvicola, responsible for bitter rot of grapes. Using an in-vitro dual-culture antagonism assay, all isolates inhibited the growth of G. uvicola (Isolates DAR 77272 and DAR 77273) with inhibition ranging from 15 to 85%. Three Aureobasidium isolates were then examined for their ability to inhibit G. uvicola when co-inoculated onto detached berries, leaves and grape bunches growing on potted vines in a glass house. All isolates reduced the severity of bitter rot infection. The results indicate that A. pullulans has the potential to suppress bitter rot of grapes.
... Indeed, it is one of the predominant yeast species found on the surface of the grape berries at all stages of maturity according to several studies [2][3][4][5][6][7] or mostly at veraison time as stated by Renouf at al., 2005 [8]. The widespread occurrence of A. pullulans is attributed to its elevated tolerance to different ecological stresses and its high antagonistic activity against bacteria and fungi [1,9,10]. Because of these characteristics, A. pullulans has been exploited as a biocontrol agent for the management of Botrytis bunch rot (BBR) caused by the fungus Botrytis cinerea, and thus also called grey mould, which is responsible for significant economic damage in vineyards and the postharvest decay of table grape [1,11,12]. ...
... Despite the availability of the A. pullulans-based product currently marketed [10,13,21] which, according to European Union Pesticides Database [22], contains the DSM14940 and DSM14941 strains (Botector ® , BIO-FERM, Austria) [23], new candidates to control BBR in vineyards are being sought. The first step in developing biocontrol agents is the isolation and the screening process and the best sources of antagonistic microbial strains are the natural environments in which they compete with plant pathogens; therefore, they are better adapted to specific ecosystems [11,16]. ...
... The indigenous A. pullulans strain 159-18 exhibited the highest biocontrol activity in the in vitro bio-assay (22%) and it was greater compared to the strains of the Botector ® product, which separately showed a radial B. cinerea growth inhibition of 13% and 19%. Since, as reported by several authors [10,16,32], the main mechanism by which A. pullulans was effective in its biocontrol ability was due to the competition for nutrients and space, the isolates with the best biocontrol potential were expected to possess a rapid growth rate. Indeed, the A. pullulans strain 159-18 was fully grown after 4 days of incubation, inducing the maximum inhibition distance, too. ...
Aureobasidium pullulans is a yeast-like fungus found on the surface of the grape berries that has been proven to act as a biocontrol agent for the management of grey mould disease caused by Botrytis cinerea. In this work, an indigenous strain of A. pullulans isolated from grape berries and selected according to the in vitro activity against B. cinerea, was used in vineyards of the winery where it originated, in comparison with a commercial product containing two A. pullulans strains with the aim of assessing its effectiveness as a biocontrol agent. The experimental design included daily meteorological data registration and the early defoliation of grapevines as treatments. The monitoring of A. pullulans strains on grape berries by plate counts and molecular methods as well as of B. cinerea symptoms on grape bunches was performed in the different trials from the end of flowering to the harvest time. Results highlighted that although no significant differences (p < 0.05) in the occurrence of B. cinerea were detected according to different treatments, the mean incidence of symptomatic berries ranged from 7 to 16%, with the lowest values recorded in bunches treated with the indigenous A. pullulans strain. The efficacy of the biocontrol agent was affected more by meteorological conditions than the defoliation practice.
... In our group's previous work, a suite of VOCs with antifungal properties were quantitatively identified from the volatilome of A. pullulans (isolates A1, A2 and A3, Yalage Don et al., 2020a), a yeast associated with the surface of many plant species that is known for its potential to inhibit the growth of plant pathogenic fungi using several modes of action (Rathnayake et al., 2018) including biosynthesis of antifungal VOCs (Di Francesco et al., 2015;Di Francesco et al., 2020;Yalage Don et al., 2020b). Ethanol (ET), 2-methyl-1-propanol (2MP), 3-methyl-1-butanol (3 MB) and 2-phenylethanol (2PE) synergistically exerted antifungal activities against Botrytis cinerea and Alternaria alternata (Yalage Don et al., 2020a), two plant pathogens responsible for economic loss in a number of horticultural crops worldwide (Steel et al., 2013;Troncoso-Rojas and Tiznado-Hernández, 2014). ...
... P < 0.01, Table 1) and a negative quadratic effect for A. pullulans A1 in experiment 2 (β 33 = 3606.07, P < 0.01, Table 1), making the results inconclusive and underlying the differences in metabolism between both isolates, although both isolates share the same origin, the phyllosphere of grapevines (Rathnayake et al., 2018). ...
... Two isolates of A. pullulans, KR605651.1 (A1) and KR605653.1 (A3), that were previously isolated from wine grapes (Rathnayake et al., 2018), were selected based on the results of two screening experiments conducted for A. pullulans antifungal activity against B. cinerea and A. alternata mycelium growth and conidia germination in our previous study (Yalage Don et al., 2020a). A. pullulans A1 was one of the isolates with the highest inhibitory activity (A1) and A. pullulans A3 showed the lowest inhibitory activity against both B. cinerea and A. alternata (Yalage Don et al., 2020a). ...
The combined biochemical impact of carbon, nitrogen and temperature on the biosynthesis of the antifungal volatile organic compounds (VOCs): ethanol, 2‐methyl‐1‐propanol, 3‐methyl‐1‐butanol and 2‐phenylethanol produced by Aureobasidium pullulans A1 and A3 was investigated using a Box–Behnken experimental design and response surface methodology (RSM). Normalized peak areas derived from solid phase micro extraction‐gas chromatography–mass spectrometry (SPME‐GC–MS) analysis, indicated that initial carbon content had a significant influence on the biosynthesis of ethanol and alcohols with greater than three carbon atoms. This result suggests a dominant activity of the A. pullulans anabolic pathway to biosynthesize three higher alcohols via de novo biosynthesis of amino acids from sugar metabolism. Low concentrations of carbon (3–13 g l−1) with nitrogen as both ammonium and amino acids in the growth medium resulted in a higher number of significant linear and quadratic relationships. Nitrogen availability and growth temperature had significant negative linear and quadratic correlations with VOCs biosynthesis in most instances. Isolate‐dependant metabolic response was evident for all abiotic parameters tested on alcohol production. The findings of this study offer new perspectives to improve the production of key antifungal compounds by antagonists in biological control systems.
... proven to have antifungal properties against Greeneria uvicola in a dual culture system which reduced the radial growth of the pathogen compared to controls 8 . However their ability to produce VOCs has not been investigated. ...
... A. pullulans has been widely studied as a biological control agent 7,8 . A variety of mechanisms have been postulated to describe its antifungal properties including enhanced natural host defences 7 , competition for nutrients 9,35 Content courtesy of Springer Nature, terms of use apply. ...
Volatile organic compounds (VOCs) produced by Aureobasidium pullulans were investigated for antagonistic actions against Alternaria alternata and Botrytis cinerea. Conidia germination and colony growth of these two phytopathogens were suppressed by A. pullulans VOCs. A novel experimental setup was devised to directly extract VOCs using solid-phase microextraction-gas chromatography mass spectrometry (SPME-GC-MS) from antagonist-pathogen culture headspace. The proposed system is a robust method to quantify microbial VOCs using an internal standard. Multivariate curve resolution alternating least squares deconvolution of SPME-GC-MS spectra identified fourteen A. pullulans VOCs. 3-Methyl-1-hexanol, acetone, 2-heptanone, ethyl butyrate, 3-methylbutyl acetate and 2-methylpropyl acetate were newly identified in A. pullulans headspace. Partial least squares discriminant analysis models with variable importance in projection and selectivity ratio identified four VOCs (ethanol, 2-methyl-1-propanol, 3-methyl-1-butanol and 2-phenylethanol), with high explanatory power for discrimination between A. pullulans and pathogen. The antifungal activity and synergistic interactions of the four VOCs were evaluated using a Box-Behnken design with response surface modelling. Ethanol and 2-phenylethanol are the key inhibitory A. pullulans VOCs against both B. cinerea and A. alternata. Our findings introduce a novel, robust, quantitative approach for microbial VOCs analyses and give insights into the potential use of A. pullulans VOCs to control B. cinerea and A. alternata.
... The study focused specifically on the abundances and diversity of yeast populations already known to be natural antagonists against postharvest pathogens, with a particular interest to Botrytis cinerea. The groups of yeasts of interest included Aureobasidium ( Rathnayake et al. 2018), Cryptococcus ( Fu et al. 2015), Rhodotorula ( Zapata et al. 2015) and Sporobolomyces ( Ianiri et al. 2017). ...
... Also coexistence may occur between these two groups, and most members of the grape yeasts are endophytic ( Setati et al. 2015). Furthermore, other yeasts, such as the A. pullulans, a proven biocontrol agent against most common postharvest pathogens ( Rathnayake et al. 2018), can occur as both epiphyte and endophyte (Martini et al. 2009). This yeast like fungus was also found in all the investigated table grape developmental stages. ...
Table grapes harbour a wide diversity of microbes, some of which are potential biocontrol agents that may be responsible for the control of fungal pathogens in the phyllosphere. This study evaluated the diversity of microbial communities associated with naturally present Botrytis cinerea inoculum, with special emphasis on populations of potential biocontrol yeasts during berry development in table grapes. Samples were collected from two agro-ecological habitats in South Africa (Northern Province), characterised by low rainfall (site A) and high rainfall (site B). The phenological development samples included those at full bloom, pea size and mature berry stages. Within the group of yeasts known to be natural antagonists, Aureobasidium, Cryptococcus, Rhodotorula and Sporobolomyces could be cultured, while pathogenic fungal genera from asymptomatic samples included Cladosporium, Alternaria, and Aspergillus. Botrytis cinerea could only be cultured at the harvest stage from symptomatic and asymptomatic berries. Overall, the study showed the highest prevalence of Alternaria (35.6%), Cladosporium (27.2%) and Rhodoturula (21.2%). In conclusion, the study reveals a diverse pathogenic and beneficial naturally-known yeast genera in the presence of B. cinerea. Such information and knowledge can be further explored to manipulate potential antagonistic populations to prevent establishment of pathogenic populations and secure dominance of antagonistic populations at the harvest stage.
... Rathnayake et al. [24] reported that the diversity of Aureobasidium isolates from different tissue types was greater than on a regional scale. The authors reported that the vineyards treated with no fungicides were having differences in colonization, having higher genetic variation in the Aureobasidium isolates observed. ...
... Alternatively, the introduction of rootstocks could have co-introduced new isolates. Therefore, it is possible that the genetic variation expressed by the Aureobasidium isolates from different vineyards in close proximity may be the result of evolution of these isolates over time in order to cope with different environmental selection pressures [24]. ...
The saprophytic yeast-like fungus Aureobasidium pullulans has been well documented for over 60 years in the microbiological literature. It is ubiquitous in distribution, being found in a variety of environments (plant surfaces, soil, water, rock surfaces and manmade surfaces), and with a worldwide distribution from cold to warm climates and wet/humid regions to arid ones. Isolates and strains of A. pullulans produce a wide range of natural products well documented in the international literature and which have been regarded as safe for biotechnological and environmental applications. Showing antagonistic activity against plant pathogens (especially post-harvest pathogens) is one of the major applications currently in agriculture of the fungus, with nutrient and space competition, production of volatile organic compounds, and production of hydrolytic enzymes and antimicrobial compounds (antibacterial and antifungal). The fungus also shows a positive role on mycotoxin biocontrol through various modes, with the most striking being that of binding and/or absorption. A. pullulans strains have been reported to produce very useful industrial enzymes, such as β-glucosidase, amylases, cellulases, lipases, proteases, xylanases and mannanases. Pullulan (poly-α-1,6-maltotriose biopolymer) is an A. pullulans trademark product with significant properties and biotechnological applications in the food, cosmetic and pharmaceutical industries. Poly (β-l-malic acid), or PMA, which is a natural biopolyester, and liamocins, a group of produced heavy oils and siderophores, are among other valuable compounds detected that are of possible biotechnological use. The fungus also shows a potential single-cell protein source capacity with high levels of nucleic acid components and essential amino acids, but this remains to be further explored. Last but not least, the fungus has shown very good biocontrol against aerial plant pathogens. All these properties are of major interest in the vitivinicultural sector and are thoroughly reviewed under this prism, concluding on the importance that A. pullulans may have if used at both vineyard and winery levels. This extensive array of properties provides excellent tools for the viticulturist/farmer as well as for the oenologist to combat problems in the field and create a high-quality wine.
... Among Ascomycetes, the most common fungal taxa found in leaves of the three grapevine varieties were A. pullulans and Alternaria spp. Regarding A. pullulans, this is a yeast-like fungus naturally inhabiting the aboveground parts of grapevine tissues, which is also known as black yeast due to its melanin production [6,[35][36][37][38]. This fungus is also known for its antagonistic activity against pathogenic fungi, including B. cinerea [39,40]. ...
The grapevine (Vitis vinifera) is a major fruit crop of economic importance worldwide. Commercial grapevine cultivars are susceptible to infection by pathogenic microorganisms that cause diseases both in leaves and fruits, and it is known that the leaf microbiome plays an important role in plant health and fitness. In this study, shotgun metagenomic sequencing was used to characterize the microbial communities associated with grapevine leaves in three commercial varieties, Cabernet Sauvignon, Garnacha, and Marselan, grown in the same biogeographical unit. Metagenomic data revealed a differential enrichment of the microbial communities living inside grapevine leaves or on the leaf surface in the three varieties. The most abundant fungal taxa associated with grapevine leaves belong to the phylum Ascomycota, which included relevant pathogenic fungi for grapevines, such as Botrytis cinerea, Sclerotinia sclerotium, and Alternaria alternata, as well as several fungal species potentially pathogenic for grapevines (e.g., members of the Colletotrichum, Aspergillus, and Penicillium genera). Basidiomycota constituted a minor fraction of the fungal microbial communities. Grapevine leaves also harbored a diversity of bacterial taxa. At the phylum level, bacterial communities in all three varieties were primarily composed of Pseudomonadata, Bacillota, Bacteroidota, and a lower proportion of Actinomycetota. Differences in the fungal and bacterial community structures were observed between varieties, although they were more important in fungi. In particular, S. sclerotiorum and B. cinerea were found to preferentially colonize leaves in the Marselan and Garnacha varieties, respectively. These findings further support that the host genotype can shape its own microbiome in grapevines. A better understanding of the leaf microbiome in grapevines will provide the basis for the development of tailored strategies to prevent diseases in vineyards while helping to increase sustainability in grapevine production.
... Stable colonization of the grape berry surface by yeasts (e.g., A. pullulans), microcracks and wounds (the pathway for SR microorganisms entering the berry pulp), high tolerance to different ecological stresses (e.g., desiccation and irradiation) [90,91], competition for space and nutrients [92][93][94], and antagonistic activity through the production of extracellular chitinases and β-1-3-glucanases [95] have been proven to be effective against B. cinerea, Penicillium spp., and Aspergillus spp. [96][97][98][99], as well as other microorganisms [100][101][102]. ...
Sour rot (SR) is a disease complex that affects grape berries during ripening and can cause severe yield losses and deterioration of wine quality. The etiology and epidemiology of the disease remain uncertain, which has severely limited the development of specific, targeted management strategies. In this study, a network meta-analysis was applied to data collected through a previous systematic literature review for statistically comparing the efficacy of different methods for the control of SR and some filamentous fungi isolated from rotten berries. Use of either synthetic fungicides (CHEM) and natural compounds or biocontrol microorganisms (BIO) provided partial and variable control of SR; however, the efficacy of BIO was similar to, or higher than, that provided by CHEM. Agronomic practices (AGRO) had a significant but lower effect on SR. The integration of different control methods (IPM) provided better and less variable disease control than any single method. Natural compounds, such as zeolites and bicarbonates, and microorganisms (e.g., yeasts Candida and Aureobasidium) are also promising alternatives to synthetic fungicides in SR control.
... Aureobasidium appears to be particularly suited to the grape juice environment, with a single OTU dominating the abundance associated with this genus. While a species could not be confidently assigned to this OTU, it probably corresponds to Aureobasidium pullulans, given its more frequent isolation from grapes (Grube et al., 2011;Onetto et al., 2020;Prakitchaiwattana et al., 2004;Rathnayake et al., 2018). It is also interesting to note that community types featuring a higher abundance of this genus exhibit an overall greater OTU richness, suggesting that Aureobasidium species may not engage in broad antagonism but rather exhibit targeted antagonism against specific species, as reported in several studies (Castoria et al., 2001;Sharma et al., 2009). ...
Over 6 years, we conducted an extensive survey of spontaneous grape fermentations, examining 3105 fungal microbiomes across 14 distinct grape‐growing regions. Our investigation into the biodiversity of these fermentations revealed that a small number of highly abundant genera form the core of the initial grape juice microbiome. Consistent with previous studies, we found that the region of origin had the most significant impact on microbial diversity patterns. We also discovered that certain taxa were consistently associated with specific geographical locations and grape varieties, although these taxa represented only a minor portion of the overall diversity in our dataset. Through unsupervised clustering and dimensionality reduction analysis, we identified three unique community types, each exhibiting variations in the abundance of key genera. When we projected these genera onto global branches, it suggested that microbiomes transition between these three broad community types. We further investigated the microbial community composition throughout the fermentation process. Our observations indicated that the initial microbial community composition could predict the diversity during the early stages of fermentation. Notably, Hanseniaspora uvarum emerged as the primary non‐ Saccharomyces species within this large collection of samples.
... The effects of this fungus on the grapevine host are also beneficial according to previous studies. Apu has proved to be a potential biocontrol agent against Botrytis cinerea, the causal agent of grapevine bunch-rot (Galli et al., 2021), and Greeneria uvicola, the pathogen of bitter rot in grapes (Rathnayake et al., 2018). Apu is an effective inhibitor of Diplodia seriata (Botryosphaeria dieback), directly inhibiting and possibly elicitating defense responses in the host, as has been suggested by the elevated expression of PR6 pathogenesis-related gene in Apu-treated plants . ...
Esca belongs to the group of grapevine trunk diseases - fungal diseases present worldwide in all wine-growing regions. Some aspects of the disease, like the development of external symptoms, have still not been completely discovered and are believed to be affected by several factors, including interactions within the vine microbiome. The examination of the occurrence of the yeast-like fungus Aureobasidium pullulans in the healthy wood of Esca-diseased grapevines via both isolation and qPCR measurements showed a positive correlation between its abundance and the severity of foliar symptoms, suggesting the contribution of this fungus to Esca pathogenesis via an indirect action. In vitro confrontation tests revealed antagonistic interaction between A. pullulans and the Esca pathogen Phaeomoniella chlamydospora. Mutual growth inhibition and the induction of asexual sporogenesis were observed for both fungi without cytotoxic effects. In planta confrontation tests revealed that A. pullulans in combination with P. chlamydospora can lead to severe foliar damage in a strain-dependent manner. This phenomenon could be explained by the altered metabolism of the Esca pathogen in the presence of A. pullulans, or by the cumulative/synergistic effects of the secreted polysaccharides and/or proteins of the two fungi. The present study shows the importance of microbial interactions in the development of plant diseases, highlighting that even a non-pathogenic microorganism can act as a disease-enhancer.
... Several of the identified microorganisms have already been reported as colonizing the plant phyllosphere, including Epicoccum sp. and Aureobasidium sp. strains in Vitis vinifera (Rathnayake et al. 2018;Del Frari et al. 2019), Bacillus sp. in Zea mays (Abadi et al. 2021), Pseudomonas sp. in Prunus domestica (Janakiev et al. 2020), Sporobolomyces sp. in Lactuca sativa (Haelewaters et al. 2021), and Aspergillus sp. in Oryza sativa (Fan et al. 2019). These studies agree with our results and confirm the widespread presence of these microbial taxa in fruit crops ...
In this study, the diversity of leaf-associated microorganisms of the native Andean ericaceous plants Gaultheria pumila and Empetrum rubrum was screened to identify and characterize microorganisms with plant growth promotion and biocontrol capability against the phytopathogenic fungus Botrytis cinerea affecting Vaccinium corymbosum cultivars. Microbial strains with biocontrol potential against Botrytis cinerea were selected, and in vivo tests were performed to evaluate the biocontrol activity of the selected strains. The fungi Epicoccum nigrum (strains HFE11 and HFG20), Epicoccum layuense (strain HFG13), and Aspergillus sp. (strain HFG1), the yeasts Aureobasidium pullulans (strains BFG22 and BFG24) Sporobolomyces roseus (strains BFE10 and BFE11), and the bacteria Bacillus mycoides (strains BFE4 and BFE14), Bacillus sp. (strain BFG8), Pseudomonas fluorescens (strain BFE6), and Pseudomonas sp. (strain BFG21) were isolated. In vitro biocontrol activity of the selected strains (BFE14, BFE6, and HFG13) showed inhibition percentages ranging from 60 to 80%. Most of the isolates were able to produce Exopolysaccharides, Siderophore, Indole-3-acetic acid, P-solubilization and Ammonia to different levels. The in vivo experiments showed that the inoculation of the isolates BFG22, BFE6, and HFG13 on V. corymbosum leaves before infection avoids severe damage to the infected tissues. Additionally, BFG22 decreases the lipid peroxidation levels (malondialdehyde 36% lower) when the leaves were infected with B. cinerea. Our results provide evidence of beneficial traits of microorganisms inhabiting the phyllosphere of native Ericaceae which can be used as microbial inoculants in agricultural production. These beneficial effects enhance plant growth and avoid damage by B. cinerea in V. corymbosum cultivars.
... Diese Gattung hat die Fähigkeit, atmosphärischen Stickstoff zu fixieren(Dahal et al., 2020) und die Pflanze bei der Stickstoffversorgung zu unterstützen. Aus den Gesamtergebnissen unserer Studie folgt, dass alle im Blutungssaft vorkommenden Gattungen einen wichtigen Faktor für die Pflanzengesundheit darstellen können wie in der Literatur mehrfach beschrieben zum Beispiel als Bioagenten, Stickstofffixierer oder/und Bildner von Pflanzenwachstumsfaktoren(Ippolito et al., 2000;Castoria et al., 2001;Schena et al., 2002;Bencheqroun et al., 2007;Vero et al., 2009;Rathnayake et al., 2018). Die Bakterien sowie Aureobasidium pullulans werden schon zum Teil im Agararsektor zur Pflanzengesundheit eingesetzt, wichtig dabei ist vor allem die Balance der Mikroorgansimen aufrecht zu erhalten, die unter dem Einfluss verschiedener Faktoren gestört werden kann. ...
Zusammenfassung
Die Weinrebe stellt ein natürliches Reservoir ansässiger mikrobieller Ressourcen dar, die in ein komplexes Mikroökosystem eingebettet ist. Ziel dieser Studie war herauszufinden, welche Keime sich im Blutungssaft befinden. Die Gewinnung des Blutungssaftes erfolgte mittels einer sauberen, mit Alkohol desinfizierten PET-Flasche. Nach erfolgter Anreicherung wurde die DNA-Extraktion mit anschließender NGS-Analyse mit der Zielregion V1V3 untersucht und die erhaltenen Sequenzen mit der NCBI-Datenbank abgeglichen. Die dominantesten Gattungen in den Rebstöcken waren Pseudomonas und Massilia , gefolgt von den Gattungen Zoogloea , Bacillus , Idonella , Sphingomonas und Paenibacillus . Zusätzlich konnte der hefeähnliche Mikroorganismus Aureobasidium pullulans bei zwei Rebstöcken bestimmt werden sowie wenige andere Bakteriengattungen, die vereinzelt auftreten. Die literarisch beschriebene hemmende Interaktion zwischen Pseudomonas und Aureobasidium konnte auch in unserer Studie bestätigt werden. Alle im Blutungssaft bestimmten Mikroorganismen haben generell einen pflanzenstärkenden Einfluss und stellen eine Basis für eine Besiedlung in gewebespezifische Pflanzenteile dar.
... VOCs of microbial origin are known to cause morphological abnormalities and conformational changes in microbial cells [9,10]. At the cellular level there is evidence that microbial VOCs alter membrane permeability of fungi via peroxidation of membrane lipids [11]. ...
... Additionally, Sabaghian et al. (2021) reported that three yeasts such as Starmerella bacillaris, Metschnikowia pulcherrima and Hanseniaspora uvarum effectively inhibited the mycelial growth of grapevine pathogens including Aspergillus spp., F. oxysporum, Alternaria alternata and Phaeomoniella chlamydospora. Vargas et al. (2012) and Rathnayake et al. (2018) also reported that several isolates of yeasts isolated from grape leaf surface were able to suppress the growth and spore germination of Botrytis cinerea and Greeneria uvicola, the causative agents of gray mold and grape rot diseases, respectively. ...
... Natural antagonists of B. cinerea have been searched for within the leaf and berry microbiome to develop microbial biocontrol. They include Aureobasidium pullulans, which acts against B. cinerea through nutritive and spatial competition and antibiosis (Carmichael et al., 2019;Martini et al., 2009, p. 200;Rathnayake et al., 2018). They also include some bacteria (Bacillus spp., Actinomycetes) and yeasts (Metchnikovia spp., Pichia spp.) (Loqman et al., 2009;Raspor et al., 2010;Santos and Marquina, 2004). ...
... Natural antagonists of B. cinerea have been searched for within the leaf and berry microbiome to develop microbial biocontrol. They include Aureobasidium pullulans, which acts against B. cinerea through nutritive and spatial competition and antibiosis (Carmichael et al., 2019;Martini et al., 2009, p. 200;Rathnayake et al., 2018). They also include some bacteria (Bacillus spp., Actinomycetes) and yeasts (Metchnikovia spp., Pichia spp.) (Loqman et al., 2009;Raspor et al., 2010;Santos and Marquina, 2004). ...
European grapevine is a complex holobiont composed of two plant genomes, that of the scion (Vitis vinifera L.) and the rootstock (Vitis spp.), and a multitude of microbial genomes that collectively form the microbiome. The grapevine microbiome has been extensively described over the last decade, primarily using metabarcoding approaches. Unfortunately, metabarcoding data alone provide little information on microbial functions and outcomes of plant-microbe interactions. Here we review knowledge about the microorganisms that have a demonstrated influence, positive or negative, on the performance of the grapevine holobiont. Our review encompasses bacteria, filamentous fungi, yeasts, oomycetes and viruses. It covers aboveground and belowground microorganisms, including arbuscular mycorrhizal and ectomycorrhizal fungi. We focus on taxa and functions that protect the plant against pathogens and pests, promote growth, increase tolerance to abiotic stresses and highlight those involved in disease and decline. As the outcomes of plant-microbe interactions are labile, we examine the dynamics and functions of grapevine-microbiome interactions over both the plant lifetime and the plant evolutionary history, beginning with plant domestication. Based on the knowledge and gaps we identify, we suggest field sampling designs, culture-based experiments, molecular tools and theoretical analysis methods, including shotgun metagenomics and network models, that could be used in future research to uncover and leverage the full functional potential of the grapevine microbiome.
... Indeed, among the four A. pullulans strains tested here, only two were more effective in plate tests, and only DiSVA 211 confirmed their significant efficacies in vivo during field treatments. Despite the availability of A. pullulans-based products currently marketed, such as Botector ® or BIO-FERM [38][39][40], new candidates to control gray rot in vineyards are required. In this regard, the results of field treatments indicated that several factors should be evaluated, such as the fast colonisation of potential BCAs and their persistence, which are characteristic of M. pulcherrima DiSVA 269 and A. pullulans DiSVA 211, selected as the best potential BCAs. ...
Background: Botrytis cinerea (B. cinerea) is responsible for grape infection and damage to the winemaking and table grape sectors. Although anti-Botrytis chemicals are available, they are considered unsustainable for resistance phenomenon and adverse effects on the environment and human health. Research is focused on developing alternative approaches, such as exploiting biological control agents (BCAs). In this context, 19 yeasts of the genera Cryptococcus, Aureobasidium, Metschnikowia, Kluyveromyces and Wickerhamomyces were tested as antimicrobial agents against B. cinerea development. Methods: A combination of in vitro tests based on dual-culture methods, volatile organic compound production assay, laboratory tests on grape berries (punctured and sprayed with yeasts) and field experiments based on yeast treatments on grapes in vineyards allowed the selection of two potential BCAs. Results: M. pulcherrima DiSVA 269 and A. pullulans DiSVA 211 exhibited the best ability to contain the development of B. cinerea, showing the severity, the decay and the McKinney index lower than a commercial biological formulation consisting of a mixture of two different A. pullulans strains, which were used as positive controls. Conclusions: The results indicated that the selected strains were effective BCA candidates to counteract B. cinerea in the field, applying them in the partial or total replacement of conventional treatments.
... To verify this hypothesis, we used grape (Vitis vinifera L.) as a model. The indigenous microbiome [22][23][24][25][26] and pathogens [27] are well studied. The grape-associated microbiota was found to be involved in plant health [28]; and, by their volatile organic compounds on the vitivinicultural terroir [29][30][31][32][33][34]. ...
Understanding the plant microbiome is a key for plant health and controlling pathogens. Recent studies have shown that plants are responsive towards natural and synthetic sound vibration (SV) by perception and signal transduction, which resulted in resistance towards plant pathogens. However, whether or not native plant microbiomes respond to SV and the underlying mechanism thereof remains unknown. Within the present study we compared grapevine-associated microbiota that was perpetually exposed to classical music with a non-exposed control group from the same vineyard in Stellenbosch, South Africa. By analyzing the 16S rRNA gene and ITS fragment amplicon libraries we found differences between the core microbiome of SV-exposed leaves and the control group. For several of these different genera, e.g., Bacillus, Kocuria and Sphingomonas, a host-beneficial or pathogen-antagonistic effect has been well studied. Moreover, abundances of taxa identified as potential producers of volatile organic compounds that contribute to sensory characteristics of wines, e.g., Methylobacterium, Sphingomonas, Bacillus and Sporobolomyces roseus, were either increased or even unique within the core music-exposed phyllosphere population. Results show an as yet unexplored avenue for improved plant health and the terroir of wine, which are important for environmentally friendly horticulture and consumer appreciation. Although our findings explain one detail of the long-term positive experience to improve grapevine’s resilience by this unusual but innovative technique, more mechanistic studies are necessary to understand the whole interplay.
... The strain dependence of biocontrol abilities is in agreement with previous findings of isolates of I. terricola (synonymous with P. terricola) and H. uvarum against B. cinerea [60]. Antagonistic activity of strains belonging to I. terricola and A. pullulans isolated from vineyards has been reported against different pathogens such as A. carbonarius and A. niger [41,67]; B. cinerea [60] and Greeneria uvicola, which is responsible for bitter rot [68]. Other isolates such as A. pullulans L30 ( Figure 2C) and Z. meyerae modified the visual appearance of P. glabrum ( Figure 2D). ...
Biocontrol is one of the most promising alternatives to synthetic fungicides for food preservation. Botrytis cinerea, Alternaria alternata, and Aspergillus section Nigri are the most concerning pathogens for grape development. However, frequently, other species, such as Penicillium glabrum in this study, are predominant in spoiled bunches. In this work, 54 native yeasts from vineyards were screened by direct confrontation in potato dextrose agar plates as antagonists against P. glabrum. Isolates of Pichia terricola, Aureobasidium pullulans, and Zygoascus meyerae were selected for their antagonist activity in vitro, plus isolates of Pichia kudriavzevii, Hormonema viticola, and Hanseniaspora uvarum were used as negative controls. However, in vivo, confrontations in wounded grapes showed disagreement with direct confrontation in vitro. P. terricola, P. kudriavzevii, H. viticola, Z. meyerae, and H. uvarum significantly reduced the incidence of P. glabrum on grapes. Nevertheless, P. terricola, H. viticola, and H. uvarum themselves spoiled the wounded grapes. Inhibitions were associated with different mechanisms such as the production of volatile organic compounds (VOCs), lytic enzymes, biofilm formation, and competition for nutrients. The isolates of P. kudriavzevii L18 (a producer of antifungal VOCs which completely inhibited the incidence of P. glabrum) and Z. meyerae L29 (with pectinase, chitinase and β-glucanase activity and biofilm formation which reduced 70% of the incidence of P. glabrum) are proposed as suitable biocontrol agents against P. glabrum.
... The use of biological control is a viable option in the management of rot. Several biological control agents are available, such as Aureobasidium pullulans that showed potential to suppress bitter rot of grapes (Rathnayake et al., 2018) and Candida sake in the control of Botrytis rot (Garrido et al., 2017). ...
The cultivation of grapevine spreads throughout practically the entire world areas. Several species and cultivars are produced for fresh consumption and for industrialization.
... VOCs of microbial origin are known to cause morphological abnormalities and conformational changes in microbial cells [9,10]. At the cellular level there is evidence that microbial VOCs alter membrane permeability of fungi via peroxidation of membrane lipids [11]. ...
Aureobasidium pullulans is a yeast-like fungus that produces volatile organic compounds (VOCs) with antifungal properties. VOCs have the potential to trigger the production of intracellular reactive oxygen species (ROS), lipid peroxidation and electrolyte loss in microorganisms. The relationship among A. pullulans VOCs, induced ROS accumulation and electrolyte leakage was investigated in Botrytis cinerea and Alternaria alternata in vitro. Exposure to a mixture of A. pullulans VOCs: ethanol, 2-methyl-1-propanol, 3-methyl-1-butanol and 2-phenylethanol, resulted in electrolyte leakage in both B. cinerea and A. alternata. Fluorescence microscopy using 2’,7’-dichlorofluorescein diacetate indicated triggered ROS accumulation in exposed fungal mycelia and the presence of the superoxide radical was evident by intense red fluorescence with dihydroethidium. Partial inhibition of enzymes of the mitochondrial respiratory chain complex I of B. cinerea and A. alternata by pre-treatment with rotenone reduced ROS accumulation in hypha exposed to A. pullulans VOCs and reversed the VOCs inhibition of fungal growth. Scanning electron micrographs revealed that B. cinerea and A. alternata hypha exposed to A. pullulans VOCs had altered cell wall structures. Our findings give insights into the potential mechanisms involved in the antifungal properties of A. pullulans in the suppression of B. cinerea and A. alternata growth in vitro.
... Another study revealed that Aureobasidium isolates from cv. Chardonnay inhibited the growth of Greeneria uvicola, a bitter rot agent of bunches, by dual antagonistic tests, detached berries, leaves, and bunches of potted grapevines (Rathnayake et al., 2018). In addition, A. pullulans is mainly reported as an important BCA of an abroad of post-harvest diseases such as in apple fruit (B. ...
... Another study revealed that Aureobasidium isolates from cv. Chardonnay inhibited the growth of Greeneria uvicola, a bitter rot agent of bunches, by dual antagonistic tests, detached berries, leaves, and bunches of potted grapevines (Rathnayake et al., 2018). In addition, A. pullulans is mainly reported as an important BCA of an abroad of post-harvest diseases such as in apple fruit (B. ...
Grapevine trunk diseases (GTDs) are one of the major concern amongst grapevine diseases, responsible for the decline of vineyards and for several economical losses. Since grapevine is naturally colonized by resident microorganisms such as Aureobasidium pullulans, the present challenge is to understand their biocontrol potential and how such microorganisms can be successfully integrated in the control of GTDs. In this context, the first priority consists to exploit the plant-beneficial-phytopathogen interactions in plant model systems, to identify the most prevalent equilibrium limiting expression of GTDs. In the current study, we deep characterized the interaction of a resident and abundant microorganism from grapevine – Aureobasidium pullulans strain Fito_F278 – against D. seriata F98.1, a Botryosphaeria dieback agent, and with plant (cv Chardonnay). Results revealed that A. pullulans strain Fito_F278 was able to reduce significantly the mycelium growth of D. seriata F98.1 at 33.41 ± 0.55%, under in vitro conditions, though this reduction is possibly dependent on a direct interaction between strain Fito_F278 and pathogen. Furthermore, strain Fito_F278 was able to promote an induction of some plant defense responses in cutting plants, 1 week after the D. seriata F98.1 infection. Results evidenced that strain Fito_F278 colonized efficiently grapevine at both epiphyte and endophyte level, could persist on plant roots for long-periods (up to 2 months after its inoculation) and grow at different pH and high salinity conditions. Moreover, a significant decrease of the microbial load from soil and rhizosphere was observed in plants treated with the strain Fito_F278, suggesting its competitivity potential in a microbial ecosystem. Altogether, the present study gives the first insights about the interaction of A. pullulans strain Fito_F278, a resident microorganism, with grapevine, its potential role against a Botryosphaeria dieback agent, and highlights its importance to toward more resilient grapevine.
Grape berry is an ecological niche for a myriad of microbes, whose interactions with one another modulate fruit health and can play a role in fermentation, imparting character and distinctiveness to wines. With the growing concerns about and awareness of the risks associated with the overuse of chemical pesticides in viticulture, microbial-based pest control is increasingly encouraged as a more sustainable and environmentally friendly strategy. The use of yeasts from grape berries is a promising alternative for the control of vineyard diseases and their increasing acceptance is rapidly changing our perception of fungicides. In this paper, we provide an overview of the latest methodologies for characterising the dynamics of grape berry yeasts in the context of grape disease management, and discuss the prospects for their effective use as biocontrol agents in viticulture. Most research has focused on the control of fruit rots produced by Botrytis, Aspergillus, Colletotrichum and Penicillium spp. using formulations that comprise single strains of specific yeast genera, including Aureobasidium, Metschnikowia, Saccharomyces, Pichia, Candida and Rhodotorula. However, the challenging disparity between successful biocontrol outcomes obtained in vitro and their low applicability in the field is a major limitation for the large-scale implementation of these strategies. Novel research approaches for maximising the stability and efficiency of yeast-derived bioactives are discussed in this review.
The grapevine trunk disease, Eutypa dieback (ED), causes significant vine decline and yield reduction. For many years, the fungus Eutypa lata was considered the main pathogen causing ED of grapevines in Australia. Recent studies showed other Diatrypaceous fungi were also associated with vines exhibiting dieback symptoms but there is limited information on how these fungal pathogens spread in vineyards. Thus, information on the spore dispersal patterns of Diatrypaceous fungi in different wine regions will assist in identifying high-risk infection periods in vineyards. Using more than 6800 DNA samples from airborne spores collected from eight wine regions in south-eastern Australia over 8 years using a Burkard spore trap, this study investigated the diversity and abundance of Diatrypaceous species, using multi-faceted molecular tools. A multi-target quantitative PCR (qPCR) assay successfully detected and quantified Diatrypaceous spores from 30% of the total samples with spore numbers and frequency of detection varying between regions and years. The high-resolution melting analysis (HRMA) coupled with DNA sequencing identified seven species, with E. lata being present in seven regions and the most prevalent species in the Adelaide Hills, Barossa Valley and McLaren Vale. Cryptovalsa ampelina and Diatrype stigma were the predominant species in the Clare Valley and Coonawarra, respectively while Eutypella citricola and Eu. microtheca dominated in the Hunter Valley and the Riverina regions. This study represents the first report of D. stigma and Cryptosphaeria multicontinentalis in Australian vineyards. This study further showed rainfall as a primary factor that triggers spore release, however, other weather factors that may influence the spore release in different climatic regions of Australia still requires further investigation.
Leaf-associated microorganisms are the least studied symbiotic microbes in Ericaceous plants and they represent a novel and interesting source of microbial strains for exploring their biocontrol potential against phytopathogenic fungi. In this study, the diversity of leaf-associated microorganisms of the native Andean ericaceous plants Gaultheria pumila and Empetrum rubrum were screened to identify and characterize microorganisms with biocontrol capability against the phytopathogenic fungus Botrytis cinerea affecting Vaccinium corymbosum (highbush blueberry) cultivars. Microbial strains with biocontrol potential against Botrytis cinerea were selected, and in vivo tests were performed to evaluate the biocontrol activity of the selected strains. Four fungal, four yeast, and five bacterial strains were isolated, including the fungi Epicoccum nigrum (strains HFE11 and HFG20), Epicoccum layuense (strain HFG13), and Aspergillus sp. (strain HFG1), the yeasts Aureobasidium pullulans (strains BFG22 and BFG24) Sporobolomyces roseus (strains BFE10 and BFE11), and the bacteria Bacillus mycoides (strains BFE4 and BFE14), Bacillus sp. (strains BFG8), Pseudomonas fluorescens (strains BFE6), and Pseudomonas sp. (strains BFG21). In vitro biocontrol activity of the selected strains (BFE14, BFE6, and HFG13) showed inhibition percentages ranging from 60% to 80. The screening of plant growth-promoting traits detected phosphate solubilization, indole acetic acid production, and ammonium production in most of the isolates. The in vivo experiments showed that the inoculation of the isolates BFG22, BFE6, and HFG13 on V. corymbosum leaves before infection avoids severe damage to the infected tissues. Additionally, inoculation with BFG22 decreases the lipid peroxidation levels (malondialdehyde 36% lower) when the leaves were infected with B. cinerea . Our results provide evidence of beneficial traits of microorganisms inhabiting the phyllosphere of native Andean Ericaceae which can be used as microbial inoculants in agricultural production. These beneficial effects enhance plant growth and avoid damage by B. cinerea in V. corymbosum cultivars.
Research background. Wine yeasts are a heterogeneous microbial group with high enzymatic potential that makes them a useful tool in winemaking. With a better understanding of their oenological properties, selection procedures can be optimised to obtain more efficient strains. The present study aims to isolate and select yeasts from wine grape surface by studying their production of enzymes that hydrolyse plant cell wall polymers and by linking them to different technological parameters and antioxidant activity of wines.
Experimental approach. Yeasts that are able to produce carbohydrolases and related enzymes of oenological importance were firstly selected on plates and subsequently identified. Then, a secondary selection of yeasts was carried out according to technological effects of their extracellular enzyme extracts on short macerations. In this way, the colour extraction, total polyphenol content, clarification, filterability and antioxidant activity were studied. This approach makes it possible to correlate the microorganism capacity to produce cell wall-depolymerizing enzymes with their technological effects.
Results and conclusions. From 366 isolates, 96 strains (26.2%) showed at least one of the polysaccharidase activities and 55 strains (57.3%) of them exhibited activities of multiple enzymes that degrade plant cell wall polymers. Sixteen strains were selected and identified as Aureobasidium, Candida, Debaryomyces, Hanseniaspora, Metschnikowia, Pichia, Saccharomyces and Torulaspora. Pectinolytic enzymes had the highest hydrolytic activity. Aureobasidium pullulans had a broader enzyme blend and higher activity, dominated by pectinases and followed by xylanases and cellulases. Moreover, the Torulaspora delbrueckii m7-2 strain produced high amounts of polysaccharidase and this was strain-dependent. Strains that produced enzyme extracts with a wide range of activities that were also the highest, also had the best chromatic and technological properties. Cluster analysis confirmed that A. pullulans R-22, m11-2, m86-1 and m86-2 and T. delbrueckii m7-2 could be correlated with a better effect on filterability, clarification and extraction of bioactive compounds, encouraging future studies regarding their application in winemaking.
Novelty and scientific contribution. The study of yeast multi-enzymatic systems impacting the grape maceration process enables a proper selection criterion for wine yeasts to improve colour extraction, technological parameters and antioxidant activity of Malbec wine. This work shows that A. pullulans and T. delbruekii have a high enzymatic potential for oenological purposes.
Oszust and Frąc (2020). "Apple pomace microbiome," BioResources 15(1), 945-966. This experiment evaluated the taxonomic diversity of the fungal community in conventional (AP) and organic (OAP) apple pomace using high-throughput sequencing, applying fungal genetic barcodes to functional guilds. The most abundant taxonomic groups identified in both AP and OAP were the genera Aureobasidium, Cladosporium, and Alternaria, classified into the pathotroph-saprotroph-symbiotroph guild. The phenotype microarray provided insight into the role of the apple pomace fungal community in the ecosystem. It is theorized that adding apple pomace to the soil may improve the bioavailability of bioresource-based polyols. Evaluation of the antagonistic ability of the AP fungal community and Trichoderma atroviride G79/11 strain against pathogenic fungi was performed. Trichoderma G79/11 developed well on apple pomace and revealed the antagonistic mode against tested fungal plant pathogens. Therefore, it could be applied to soil as a formulation of AP with spores or AP with metaferm biopreparation.
Botryosphaeria dieback, caused by species of Botryosphaeriaceae, is an important grapevine trunk disease in Australia. Inocula produced by the pathogens are primarily dispersed by rain splash and wind and infect pruning wounds leading to cankers, dieback, and eventually death of vines. The objective of this study was to develop molecular tools to detect and quantify Botryosphaeriaceae inocula from the environment. These tools are essential for investigating spore dispersal patterns of Botryosphaeriaceae pathogens in Australian vineyards. DNA extraction protocols were evaluated and one modified protocol was found suitable for extracting Botryosphaeriaceae DNA from artificially and naturally inoculated Burkard volumetric spore sampler tapes. Multispecies primers and a hydrolysis probe for quantitative PCR (qPCR) were further developed to detect and quantify Botryosphaeriaceae inocula from environmental samples. Specificity tests showed that the multispecies primers were able to amplify the DNA of 10 Botryosphaeriaceae species (58 isolates) found in Australia while none of the 27 non target fungal species (90 isolates) tested were amplified. The qPCR assay was suitable for amplifying purified DNA, synthetic DNA fragments (gBlocks), and mixed DNA from spore trap tapes. The qPCR method developed in this study was shown to be rapid and sensitive in detecting Botryosphaeriaceae inocula from the environment using spore traps.
Fire blight (FB), caused by Erwinia amylovora, is one of the most important pome fruit pathogens worldwide. To control this devastating disease, various chemical and biological treatments are commonly applied in Switzerland, but they fail to keep the infection at an acceptable level in years of heavy disease pressure. The Swiss authorities therefore currently allow the controlled use of the antibiotic streptomycin against FB in years that are predicted to have heavy infection periods, but only one treatment per season is permitted. Another strategy for controlling Erwinia is to breed resistant/tolerant apple cultivars. One way of accelerating the breeding process is to obtain resistant cultivars by inserting one or several major resistance genes, using genetic engineering. To date, no study summarizing the impact of different FB control measures on the environment and on human health has been performed. This study consequently aims to compare different disease-control measures (biological control, chemical control, control by antibiotics and by resistant/tolerant apple cultivars obtained through conventional or molecular breeding) OPEN ACCESS Int. J. Environ. Res. Public Health 2015, 12 11423 applied against E. amylovora, considering different protection goals (protection of human health, environment, agricultural diversity and economic interest), with special emphasis on biosafety aspects. Information on each FB control measure in relation to the specified protection goal was assessed by literature searches and by interviews with experts. Based on our results it can be concluded that the FB control measures currently applied in Switzerland are safe for consumers, workers and the environment. However, there are several gaps in our knowledge of the human health and environmental impacts analyzed: data are missing (1) on long term studies on the efficacy of most of the analyzed FB control measures; (2) on the safety of operators handling streptomycin; (3) on residue analyses of Equisetum plant extract, the copper and aluminum compounds used in apple production; and (4) on the effect of biological and chemical control measures on non-target fauna and flora. These gaps urgently need to be addressed in the near future.
Background and Aim: Members of this genus Aureobasidium are ubiquitous
microorganisms which can be isolated from wide ranges of substrates such as
plant materials (phyllosphere, plant debris, bark, roots, fruits and wood), soil, dead
wood, air, and as rare etiologic agent of pheohyphomycosis, keratomycosis,
septicemia, peritoneal sepsis, and dermatological infections in human. Very little
is known on the identity, substrates and distribution of Aureobasidium spp. in Iran.
Methods: 14 Aureobasidium isolates were recovered from vascular tissues of
pome and stone fruit trees displaying decline symptoms in orchards of West and
East Azarbaijan provinces, Iran. Pure cultures were established by using a single
spore technique. The identity of the isolates were determined using sequence data
from ITS-rDNA region. Phylogenetic relationship among isolates was inferred
based on sequence data from ITS-rDNA.
Results: A megablast search analysis of ITS sequence data at NCBI revealed the
identity of Aureobasidium isolates as A. pullulans. A phylogeny inferred using
sequence data from ITS region placed our isolates together with the other A.
pullulans var. pullulans in GenBank. Morphological and cultural characteristics
were in agreement with the description for A. pullulans var. pullulans.
Conclusion: Our results represent new report on the occurrence of A. pullulans
var. pullulans in Iran. As A. pullulans is known as rare etiologic agent of
pheohyphomycosis, keratomycosis, septicemia, peritoneal sepsis, and
dermatological infections in human, possible occurrence and involvement of A.
pullulans in human infections should be taken into account.
KeyWords: ITS-rDNA; Endophytes; Black yeast; Pullulan; Human pathogen
A comprehensive, but simple-to-use software package for executing a range of standard numerical analysis and operations used in quantitative paleontology has been developed. The program, called PAST (PAleontological STatistics), runs on standard Windows computers and is available free of charge. PAST integrates spreadsheettype data entry with univariate and multivariate statistics, curve fitting, time-series analysis, data plotting, and simple phylogenetic analysis. Many of the functions are specific to paleontology and ecology, and these functions are not found in standard, more extensive, statistical packages. PAST also includes fourteen case studies (data files and exercises) illustrating use of the program for paleontological problems, making it a complete educational package for courses in quantitative methods.
The polymerase chain reaction (PCR) can be used to detect polymorphisms in the length of amplified sequences between the annealing sites of two synthetic DNA primers. When the distance varies between two individuals then the banding pattern generated by the PCR reaction is essentially a genetic polymorphism and can be mapped in the same way as other genetic markers. This procedure has been used in a number of eukaryotes. Here we report the use of PCR to detect genetic polymorphisms in cereals. Known gene sequences can be used to design primers and detect polymorphic PCR products. This is demonstrated with primers to the α-amylase gene family. A second approach is to use semi-random primers to target diverse regions of the genome. For this purpose the consensus sequences at the intron-exon splice junctions were used. The targeting of the intronexon splice junctions in conjunction with primers of random and defined sequences, such as α-amylase, provides a source of extensive variation in PCR products. These polymorphisms can be mapped as standard genetic markers.
We announce the release of an advanced version of the Molecular Evolutionary Genetics Analysis (MEGA) software, which currently contains facilities for building sequence alignments, inferring phylogenetic histories, and conducting molecular evolutionary analysis. In version 6.0, MEGA now enables the inference of timetrees, as it implements our RelTime method for estimating divergence times for all branching points in a phylogeny. A new Timetree Wizard in MEGA6 facilitates this timetree inference by providing a graphical user interface (GUI) to specify the phylogeny and calibration constraints step-by-step. This version also contains enhanced algorithms to search for the optimal trees under evolutionary criteria and implements a more advanced memory management that can double the size of sequence data sets to which MEGA can be applied. Both GUI and command-line versions of MEGA6 can be downloaded from www.megasoftware.net free of charge.
A bunch rot disorder of Rhine Riesling grapes was investigated during the 1976-77 and 1977-78 seasons in south-west of Western Australia. Fungi isolated from immature berries at or just after flowering were not identical to those isolated from rotting berries at harvest. A wide range of fungi, including Aureobasidium pullulans (de Bary) Arnaud, Botrytis cinerea Pers. ex Fr., Rhizopus, Aspergillus, Penicillium and Alternaria spp., was isolated from rotted berries at harvest. More than 98% of rotting berries showed berry splitting or cracking, or both, at the pedicel end of the grape. Berry thinning of bunches by hand resulted in the almost complete elimination of berry splitting and cracking and a corresponding decline in bunch rot levels. The yield of unrotted bunches from the thinned vines was more than twice that for the unthinned ones; however, thinning reduced overall total vine yields by 63% compared with yields from unthinned vines.
Bunch rot of grape berries causes economic loss to grape and wine production worldwide. The organisms responsible are largely filamentous fungi, the most common of these is Botrytis cinerea (gray mold), however there are a range of other fungi responsible for the rotting of grapes such as Aspergillus spp., Penicillium spp. and fungi found in sub-tropical climates (e.g. Colletotrichum spp. (ripe rot) and Greeneria uvicola (bitter rot)). A further group more commonly associated with diseases of the vegetative tissues of the vine can also infect grape berries (e.g. Botryosphaeriaceae, Phomopsis viticola ). The impact these fungi have on wine quality is poorly understood as are remedial practices in the winery to minimise wine faults. Compounds found in bunch rot affected grapes and wine are typically described as having mushroom, earthy odours and include geosmin, 2-methylisoborneol, 1-octen-3-ol, 2-octen-1-ol, fenchol and fenchone. This review examines the current state of knowledge about bunch rot of grapes and how this plant disease complex impacts on wine chemistry. Current wine industry practices to minimise wine faults, and gaps in our understanding of how grape bunch rot diseases affect wine production and quality are also identified.
Purpose of review: Drawing on several representative studies, this review focuses on the interaction between antagonist microorganisms and fungicides and provides related suggestions for more efficient control of postharvest diseases (PHDs). Findings: Lack of knowledge of the compatibility of antagonist microorganisms with agrochemicals may contribute to the failure of biocontrol to perform as expected, as pre- and postharvest fungicide applications can affect the survival and population of natural and artificially introduced antagonists on fruits and vegetables. A more rational application schedule of substances that inhibit antagonists would optimise both the colonisation and the efficacy of biocontrol agents (BCAs). The combination or alternation of synthetic fungicides with antagonist microorganisms may enhance and stabilise the efficacy of BCAs. In addition, this strategy may display even better control of resistant strains of fungal pathogens and may enable commercial growers and packinghouses to reduce the amount of fungicides used, thus lowering the amount of chemical residue on marketed products. Combined applications of BCAs with small quantities of fungicides may also reduce the biomass of antagonists and the relative costs of formulation. Directions for future research: The integration of fungicides with antagonist microorganisms has the potential to make BCAs more attractive for large-scale applications against PHDs. Therefore, compatibility with chemicals is an important factor for more appropriate manipulation/application of naturally occurring antagonists or selected BCAs. However, in environments where fungicide resistance has already reached high levels, use of old fungicides should be suspended or replaced with new effective compounds which should be registered for postharvest treatments. However, integrating BCAs with chemicals remains a necessary prerequisite to optimise the management of PHDs and to maintain effectiveness of existing or new emerging fungicides for as long as possible. Therefore, future research projects should be aimed at both improving knowledge of antagonist-pathogen-fungicide interaction and mechanisms involved.
During the storage of harvested commodities, environmental parameters are quite stable. For this and other reasons, it is generally believed that biological control by means of microbial antagonists may have a greater potential for success when applied postharvest. However, one of the major obstacles to the development of postharvest biocontrol agents is that they are unable to control previously established infections, such as latent and quiescent infections and incipient infections occurring through wounds resulting from harvesting operations. Field application of biocontrol agents may enable early colonisation of fruit surfaces, thus protecting from these infections. Moreover, preharvest applications can be an appropriate strategy for fruits and vegetables subject to damage in postharvest handling. To be successful in preharvest applications, putative biocontrol agents must be able to tolerate low-nutrient availability, UV radiation, high temperature and dry conditions. Some reports of postharvest biological control accomplished by preharvest applications, include apples, avocados, sweet cherries, grapes, and strawberries. This paper provides a brief overview on particular aspects of preharvest application of biocontrol agents to reduce postharvest decay. Research areas relevant for the development of this strategy are also indicated.
Fifty-one endophytic isolates of Aureobasidium pullulans were obtained from the flesh of sweet cherries and extensively screened to evaluate their biocontrol activity against postharvest rots of sweet cherries and table grapes. Preliminary analysis of all isolates by randomly amplified polymorphic DNA (RAPD) with three different primers showed the presence of a high genetic variability and enabled isolates not showing any genetic difference to be discarded. Thirty-five isolates with different RAPD electrophoresis patterns had a wide range of biocontrol activity against Botrytis cinerea and Monilinia laxa on single-wounded berries of sweet cherries and table grapes with a reduction of decay from 10 to 100%. Two isolates (533 and 547) significantly reduced B. cinerea on table grape berries also when applied 6, 12, and 24 h after the pathogen inoculation. In a 2-year period of investigation (1998–1999), a reduction of total rots ranging from 32 to 80% (sweet cherries) and from 59 to 64% (table grape) was achieved with isolates 533 and 547 applied after harvest. Preharvest applications of isolate 547 significantly reduced postharvest rots of sweet cherries and table grapes by 47 and 38%, respectively. On the whole, isolates 533 and 547 were more effective than A. pullulans L47, a biocontrol agent of postharvest diseases with a known activity. Population studies demonstrated that isolate 547 was able to survive under field conditions, to increase its population during cold storage, and to penetrate the flesh of sweet cherries when applied during flowering.
This paper presents PyElph, a software tool which automatically extracts data from gel images, computes the molecular weights of the analyzed molecules or fragments, compares DNA patterns which result from experiments with molecular genetic markers and, also, generates phylogenetic trees computed by five clustering methods, using the information extracted from the analyzed gel image. The software can be successfully used for population genetics, phylogenetics, taxonomic studies and other applications which require gel image analysis. Researchers and students working in molecular biology and genetics would benefit greatly from the proposed software because it is free, open source, easy to use, has a friendly Graphical User Interface and does not depend on specific image acquisition devices like other commercial programs with similar functionalities do.
PyElph software tool is entirely implemented in Python which is a very popular programming language among the bioinformatics community. It provides a very friendly Graphical User Interface which was designed in six steps that gradually lead to the results. The user is guided through the following steps: image loading and preparation, lane detection, band detection, molecular weights computation based on a molecular weight marker, band matching and finally, the computation and visualization of phylogenetic trees. A strong point of the software is the visualization component for the processed data. The Graphical User Interface provides operations for image manipulation and highlights lanes, bands and band matching in the analyzed gel image. All the data and images generated in each step can be saved. The software has been tested on several DNA patterns obtained from experiments with different genetic markers. Examples of genetic markers which can be analyzed using PyElph are RFLP (Restriction Fragment Length Polymorphism), AFLP (Amplified Fragment Length Polymorphism), RAPD (Random Amplification of Polymorphic DNA) and STR (Short Tandem Repeat). The similarity between the DNA sequences is computed and used to generate phylogenetic trees which are very useful for population genetics studies and taxonomic classification.
PyElph decreases the effort and time spent processing data from gel images by providing an automatic step-by-step gel image analysis system with a friendly Graphical User Interface. The proposed free software tool is suitable for researchers and students which do not have access to expensive commercial software and image acquisition devices.
Using media with low water activity, a large numbers of aureobasidium-like black yeasts were isolated from glacial and subglacial ice of three polythermal glaciers from the coastal Arctic environment of Kongsfjorden (Svalbard, Spitsbergen), as well as from adjacent sea water, sea ice and glacial meltwaters. To characterise the genetic variability of Aureobasidium pullulans strains originating from the Arctic and strains originating pan-globally, a multilocus molecular analysis was performed, through rDNA (internal transcribed spacers, partial 28 S rDNA), and partial introns and exons of genes encoding β-tubulin (TUB), translation elongation factor (EF1) and elongase (ELO). Two globally ubiquitous varieties were distinguished: var. pullulans, occurring particularly in slightly osmotic substrates and in the phyllosphere; and var. melanogenum, mainly isolated from watery habitats. Both varieties were commonly isolated from the sampled Arctic habitats. However, some aureobasidium-like strains from subglacial ice from three different glaciers in Kongsfjorden (Svalbard, Spitsbergen), appeared to represent a new variety of A. pullulans. A strain from dolomitic marble in Namibia was found to belong to yet another variety. No molecular support has as yet been found for the previously described var. aubasidani. A partial elongase-encoding gene was successfully used as a phylogenetic marker at the (infra-)specific level. Taxonomic novelties: Aureobasidium pullulans var. subglaciale Zalar, de Hoog & Gunde-Cimerman, var. nov.; Aureobasidium pullulans var. namibiae Zalar, de Hoog & Gunde-Cimerman, var. nov.
Wine contamination with ochratoxin A (OTA) is due to the attack of wine grapes by ochratoxigenic Aspergillus carbonarius and Aspergillus spp. section Nigri. Four A. pullulans strains, AU14-3-1, AU18-3B, AU34-2, and LS30, are resistant to and actively degrade ochratoxin A in vitro. The less toxic ochratoxin alpha and the aminoacid L-beta-phenylalanine were the major degradation products, deriving from the cleavage of the amide bond linking these two moieties of OTA. The same strains were studied further as biocontrol agents of A. carbonarius on wine grapes in laboratory experiments. Three of the four strains significantly prevented infections by A. carbonarius. Berries pretreated with the biocontrol agents and infected with A. carbonarius contained lower amounts of OTA as compared to the untreated infected control berries. Two of these strains were shown to degrade OTA to ochratoxin alpha in fresh grape must, but the mechanisms of the decrease of OTA accumulation in infected berries pretreated with the biocontrol agents remain to be elucidated. Assessment of one strain carried out in the vineyard during the growing season of 2006 showed that the tested strain was an effective biocontrol agent, reducing both severity of Aspergillus rots and OTA accumulation in wine grapes. To our knowledge this is the first report describing the positive influence of biocontrol agents on OTA accumulation in this crop species.
Biocontrol activities of different yeast species and strains isolated from grape/must/wine environments have been compared to those of commercially available antagonistic yeast species of Candida oleophila. A total of 591 yeast isolates were tested in a preliminary screening on agar to select isolates showing inhibitory effect against Botrytis cinerea, the plant pathogen causing grey mould disease on grape. Yeast species Aureobasidium pullulans, Metschnikowia pulcherrima and Pichia guilliermondii showed, on average, higher biocontrol activity than commercially used yeast Candida oleophila. Furthermore, these three species and Saccharomyces cerevisiae, which is potentially interesting biocontrol agent against grey mould of grapes, were selected for their inhibitory effects and assayed in vitro on different solid synthetic media for their antagonistic capacity towards B. cinerea. The results indicate that the composition of the medium had an impact on the biocontrol activity of yeast species and strains, as Saccharomyces cerevisiae showed the highest antagonistic activity against B. cinerea when tested on media with increased concentrations of glucose. The antagonistic activity of selected yeast strains was finally determined on wounded and sound grape berries of cultivars Rebula and Chardonnay for their ability to inhibit infection by B. cinerea moulds. Results suggest that antagonist yeasts with the potential to control B. cinerea on grape can be found among the microflora associated with the berries.
Antagonistic yeasts were isolated from the surface of grape berries cv. “Cabernet sauvignon” and “Maratheftiko” from six vineyards in Cyprus and identified at species level using molecular methods. The identification revealed that the yeast isolates belonged to seven taxonomically distinct groups: Aureobasidium pullulans, Cryptococcus magnus, Hanseniaspora uvarum, Candida zeylanoides, Candida sake, Rhodotorula mucilaginosa and Pseudozyma aphidis. A total of 55 yeast isolates were evaluated in a preliminary screening test on agar to select isolates exhibiting inhibition against an ochratoxigenic strain of Aspergillus tubingensis. Thirty-three yeast isolates were selected for their antisporulant activity on A. tubingensis and their ability to reduce the growth of fungal mycelium. These isolates were assayed by a detached berry test for their ability to inhibit infection by the ochratoxigenic strain of A. tubingensis. Twenty-eight yeast isolates belonging to three species, namely 25 isolates of Aureobasidium pullulans, 2 isolates of Cryptococcus magnus and 1 isolate of Candida sake, reduced the A. tubingensis colonization of grape berries. The highest antagonistic activity was shown by the Aureobasidium pullulans isolates, with biocontrol efficacies ranging between 17.1% and 95.7%. The results of this study suggest that antagonist yeasts potentially effective for biological control of A. tubingensis on grape can be found among the microbiota associated with grape berries in Cyprus vineyards.
The first and second editions of Fungi and Food Spoilage established a reputation as the foremost book on foodborne fungi. This completely revised and updated third edition is an invaluable reference for food microbiologists investigating fungal spoilage and sources of mycotoxin contamination in foods. The introductory chapters of the book deal with the ecology of food spoilage and give an overview of how food processing, packaging and storage affect fungal growth. Subsequent chapters cover the fundamentals of classifying and naming fungi and current methods for isolation and enumeration, including general and special purpose media, incubation conditions, etc. The major part of the book provides keys, descriptions and illustrations of all yeasts and moulds commonly encountered in foods. Characteristics of the species, including their ecology and potential for mycotoxin production, are also included. The broad and practical nature of the coverage will appeal to microbiologists, mycologists and biotechnologists in the food industry, academic, research and public health institutions. Dr John Pitt and Dr Ailsa Hocking are both Honorary Research Fellows at CSIRO Food Science Australia, North Ryde, NSW, Australia. © Springer Science+Business Media, LLC 2009. All rights reserved.
Greeneria uvicola causes bitter rot on Vitis vinifera (bunch grapes) and Muscadinia rotundifolia (muscadine grapes) in warm moist temperate and subtropical regions. This study investigated the phylogenetic relationship of G. uvicola representatives from Australia (67 isolates), the USA (31 isolates), India (1 isolate) and Costa Rica (1 isolate) and compared their pathogenicity and fungicide sensitivity. Differences in cultural and conidial morphology were observed between the isolates from Australia and the USA. Phylogenetic relationships were determined based on three gene regions: the ribosomal DNA (rDNA) internal transcribed spacer 1 (ITS1–58S–ITS2), 28S large subunit (LSU) nuclear rDNA and β‐tubulin‐2. Greeneria uvicola isolates were clearly differentiated into four groups: isolates from Australia and India; USA isolates from V. vinifera; USA isolates from M. rotundifolia; and the isolate from Costa Rica. All isolates were pathogenic on V. vinifera (cv. Chardonnay) berries although those originating from M. rotundifolia were not as aggressive as isolates from V. vinifera, irrespective of geographical origin. Sensitivity to pyraclostrobin and salicylhydroxamic acid (SHAM) was studied. Despite differences in fungicide applications, hyphal growth inhibition was not significantly different for geographical location, cultivar, tissue, year of collection or different spray regimes. For the Australian and USA isolates, fungal growth inhibition was significantly greater for pyraclostrobin than for SHAM, and was significantly greater for the combined treatment than for each of the fungicides applied singly. The aetiological and epidemiological knowledge of bitter rot collected through this study will aid better prediction and management strategies of this pathogen.
Background
Aureobasidium pullulans is a black-yeast-like fungus used for production of the polysaccharide pullulan and the antimycotic aureobasidin A, and as a biocontrol agent in agriculture. It can cause opportunistic human infections, and it inhabits various extreme environments. To promote the understanding of these traits, we performed de-novo genome sequencing of the four varieties of A. pullulans.
Results
The 25.43-29.62 Mb genomes of these four varieties of A. pullulans encode between 10266 and 11866 predicted proteins. Their genomes encode most of the enzyme families involved in degradation of plant material and many sugar transporters, and they have genes possibly associated with degradation of plastic and aromatic compounds. Proteins believed to be involved in the synthesis of pullulan and siderophores, but not of aureobasidin A, are predicted. Putative stress-tolerance genes include several aquaporins and aquaglyceroporins, large numbers of alkali-metal cation transporters, genes for the synthesis of compatible solutes and melanin, all of the components of the high-osmolarity glycerol pathway, and bacteriorhodopsin-like proteins. All of these genomes contain a homothallic mating-type locus.
Conclusions
The differences between these four varieties of A. pullulans are large enough to justify their redefinition as separate species: A. pullulans, A. melanogenum, A. subglaciale and A. namibiae. The redundancy observed in several gene families can be linked to the nutritional versatility of these species and their particular stress tolerance. The availability of the genome sequences of the four Aureobasidium species should improve their biotechnological exploitation and promote our understanding of their stress-tolerance mechanisms, diverse lifestyles, and pathogenic potential.
We studied the epiphytic yeast species of the plants of the Negev Desert and the Dead Sea region, Israel, which are considered one of the most extreme hyper-arid lands in the world. For this purpose, we developed isolation protocols; we performed morphological, cultural and molecular identification tests, and compared yeast diversity between the locations and the plants. The composition of the yeast populations present in the study's plants underwent seasonal fluctuations, whereas differences in community compositions were significant within sites. The maximum number of species of yeast occurred in autumn and Cryptococcus spp. were predominant year round. The isolated yeast strains showed an unusual tolerance to extreme growth conditions, such as high temperatures (up to 72% viability at 50 °C), lethal hydrogen peroxide (H2O2), and NaCl concentrations. These results suggest that the epiphytic yeasts that inhabit the plants of the Dead Sea region and the Negev Desert have a community structure that is unique to the plant species and have a high tolerance to the harsh conditions that enables them to adapt to a hyper-arid ecosystem.
The activity of two biological control agents, strains L1 and L8, previously identified as Aureobasidium pullulans, was tested on apple, artificially inoculated with Botrytis cinerea (grey mould), Colletotrichum acutatum (bitter rot) or Penicillium expansum (blue mould). The washed cells of antagonists controlled over 86% of three types of decay. The cell concentration of both antagonists was highly correlated with their efficacy, the R2 ranging from 0.93 to 0.99. The highest concentration (108 CFU mL−1) of L1 and L8 provided the best control of B. cinerea, C. acutatum and P. expansum, although grey mould was completely inhibited also by a concentration one log lower (107 CFU mL−1). The population dynamics of L1 strain in ‘Gala’ apple increased almost 8-fold during the first 48 h after treatment and remained elevated until 7 d, revealing that, although the antagonist was isolated from the carposhere of peach fruit, it showed good adaptation in other wound environments. Preliminary in vitro trials were conducted in order to investigate the mechanisms of action of L1 and L8 strains and in a dual culture dish assay, the antifungal effects observed on pathogen mycelium growth could be attributed to the production of volatile organic compounds (VOCs) generated by the antagonists. The VOCs significantly inhibited the growth of all three tested pathogens compared to the control, albeit with a different rate. When L1 strain was introduced into the wound 12 h from inoculum, it showed post-inoculation activity, the incidence of blue mould and bitter rot being reduced by 38% and 50% respectively, while the greatest inhibition of grey mould was observed when fruit were treated with the antagonist 6 h from the inoculum. In conclusion, A. pullulans L1 and L8 strains could be considered good candidates for the development of biofungicides for postharvest application in the pomefruit industry.
Phyllosphere yeasts were isolated from leaves of vine (Vitis vinifera L.) canes and evaluated in a detached berry assay for their ability to suppress Aspergillus carbonarius (Order: Eurotiales, Family: Trichocomaceae) growth. Seventeen of the 21 yeast isolates significantly reduced A. carbonarius growth, i.e. sour rot infection compared to untreated controls in laboratory tests. The most effective yeast isolate Aureobasidium pullulans (Order: Dothideales, Family: Dothioraceae), isolate Y-1, was field tested on two varieties of red grape, Grenache Rouge and Agiorgitiko located on the Island of Rhodes and in Corinthos County, Greece. It was demonstrated that A. pullulans Y-1 was as effective as the commercial fungicide fludioxonil+cyprodinil, in reducing sour rot infection, A. carbonarius presence on berries at harvest and ochratoxin A contamination in must.
Restriction fragment length polymorphisms (RFLPs) were identified among total DNA from clonal lines of Uncinula necator when cloned sequences of total U. necator DNA were used as probes. Four probes, pUnP14, pUnP27, pUnE21 and pUnE4, hybridized to multiple-copy sequences and, with the exception of pUnE4, detected genetic variation among clonal lines of U. necator. Clones pUnP14, pUnP27 and pUnE4 produced banding patterns that were stable for DNA extracted from different asexual generations of U. necator clonal lines over at least 15 months. In addition, clones were evaluated for species specificity. Clones pUnP27 and pUnE4 detected only U. necator sequences in total DNA from infected grapevine leaves. Clones pUnP14 and pUnE4 did not hybridize to total DNA from a range of fungi.
Genetic diversity in a sample of the Australian U. necator population was investigated; 15 genotypes were identified among 29 U. necator clonal lines examined. Genetic variation was detected in samples collected within micro-geographical areas, for example, different genotypes representing both mating types of U. necator were detected on a single plant of Vitis amurensis. RFLP analysis of the banding patterns produced using pUnP14, pUnP27 and pUnE21, identified two broad genetic groups, designated A and B. Analysis of DNA fragment patterns obtained using the polymerase chain reaction (PCR) and the plant intron splice junction (ISJ) primer R1 also supported the allocation of U. necator clonal lines into groups A and B.
In small-scale experiments Aureobasidiumpullulans (isolate LS-30) displayed significant antagonistic activity against Botrytis cinerea, Penicillium expansum, Rhizopus stolonifer and Aspergillus niger on table grapes, and B. cinerea and P. expansum on apple fruit. To improve the performance of this yeast-like fungus, possible modes of action were investigated. Competition for nutrients appeared to play a role in the activity of this antagonist. Extracellular exochitinase [N-acetyl-β-d-glucosaminidase (Nagase)] and β-1-3-glucanase activities were also detected both in vitro and in apple wounds, which are the main sites of penetration of postharvest fungal pathogens, suggesting that these enzymes may actually be involved in the antagonistic activity of this microorganism. Neither antibiosis nor direct physical interaction of LS-30 cells with the hyphae of B. cinerea appeared to be involved in the activity of this antagonist.
The efficacy of Aureobasidium pullulans PL5 against different postharvest pathogens of fruits (Monilinia laxa on plums and peaches, Botrytis cinerea and Penicillium expansum on apples) were evaluated under storage conditions when applied at 108cellsml−1 and their interactions were studied in vitro and in vivo to discover the possible modes of action. Under 1.2°C and 95% relative humidity (RH) for 28days, the efficacy of PL5 against M. laxa on plums was 45%, reducing disease incidence from 78% to 43%. Under 1°C and 95% RH for 21days, the efficacy against M. laxa on peaches was 63%, reducing disease incidence from 79% to 29%. Under 4°C and 95% RH for 45days, the efficacy against B. cinerea and P. expansum on apples was 56% and 46%, respectively. In Lilly–Barnett minimal salt medium with the fungal cell walls of pathogens as sole carbon source, PL5 produced β-1,3-glucanase, exo-chitinase and endo-chitinase. Nutrient concentrations had significant effect on pathogen growth reduction by PL5. No attachment was observed in antagonist–pathogen interactions in vitro or in vivo. PL5 completely inhibited pathogen spore germination in PDB at 108cellsml−1, whereas at 106cellsml−1 the efficacy was significantly decreased. However, inactivated cells and culture filtrate of PL5 had no effect on pathogen spore germination and germ tube elongation. Our results showed that A. pullulans PL5 could be introduced in commercial formulations to control postharvest pathogens on fruits and its activity was based on secretion of lytic enzymes and competition for nutrients.
Keywords: Durum wheat (Triticum durum L.) Powdery mildew Biological and integrated control Biocontrol agents (BCAs) Calcium silicate Sulphur Grain yield components a b s t r a c t Powdery mildew, caused by Blumeria graminis f. sp. tritici, has been recognized as the main and widespread disease of wheat in the growing areas of this crop. In the present study, to set up new effec-tive and eco-compatible control methods against the pathogen, the activity of three biological control agents (BCAs), the yeasts Rhodosporidium kratochvilovae, strain UM350, and Cryptococcus laurentii, strain UM108, and the yeast-like fungus Aureobasidium pullulans, strain LS30, previously selected for their high activity against different pathogens on various crops, were tested for two consecutive years on durum wheat in the field. The BCAs were tested alone or in combination with two mineral salts (calcium silicate and calcium chloride) or with low doses of wettable sulphur or with synthetic fungicides (azoxystrobin, tebuconazole or tetraconazole) currently used for controlling this disease. Treatments were applied twice, at the flag leaf and at pre-flowering stages. Disease severity, total yeast population dynamic on wheat leaves, grain yield components (grain yield, grain number, grain weight) were recorded. In both years, the results of the investigations showed that (i) at the flag leaf phenological stage, disease severity on untreated wheat leaves was very high (up to 63%); (ii) the sprayed BCAs colonized wheat plant phyl-loplane at high rate; (iii) the BCAs, applied alone or in combination with the two mineral additives or with fungicides, compared with untreated control, in general not only reduced the disease severity (up to 89%), but also increased grain yield (up to 35.3%) and grain weight (up to 13.2%) of durum wheat. Among the integrated treatments, the highest level of disease reduction as well as better levels of grain yield components were supplied by BCAs combined with calcium silicate or sulphur. Collectively, our results demonstrated that integrated approaches based on the combination of biocontrol yeasts with suitable additives or low doses of fungicides have a high potential for large-scale applications in eco-compatible agriculture for both controlling wheat powdery mildew and improving grain yield components.
Sour rot on White Riesling grapes increased oBrix, glucose to fructose ratio, titratable acidity, tartaric acid, glycerol and glu-conic acid while reducing berry weight. Sour rot did not influence pH, acetic acid, ethanol or laccase activity. Two classes of grape aroma components, potentially volatile terpenes (PVT) and free volatile ter-penes (FVT), were not influenced by sour rot. However, quantitative differences in selected free aroma compounds between 'clean' and rot-degradated fruit were observed. Rot reduced free geraniol, nerol, and linalool concentrations, while increasing trans-furan linalool oxide, benzyl alcohol, 2-phenylethanol, 2-methyl-1-propanol, and 3-methyl-1-bu-tanol.
The influence of temperature and relative humidity (RH) on the activity of three biocontrol agents-the yeast Metschnikowia pulcherrima LS16 and two strains of the yeast-like fungus Aureobasidium pullulans LS30 and AU34-2-against infection by A. carbonarius and ochratoxin A (OTA) accumulation in wine grape berries was investigated in lab-scale experiments. The presence of wounds on grape skin dramatically favored infection of berries by A. carbonarius strain A1102, since unwounded berries showed very low levels of infection at all conditions of RH and temperature tested. Artificially wounded berries pre-treated with the biocontrol agents were inoculated with the ochratoxigenic A. carbonarius strain A1102 and were incubated for 5days at two levels of RH (60% and 100%) and three different temperatures (20, 25 and 30°C). The three biocontrol agents were able to prevent infections at 60% RH and 20°C. At 60% RH and 25°C only strain AU34-2 achieved some protection on day 5, whereas at 30°C a limited biocontrol efficacy was evident only up to day 2. At 100% RH, LS16, LS30 and AU34-2 showed effective protection of grape berries at 20°C until the 5th day of incubation. The three biocontrol agents achieved significant protection at higher temperatures only until the 2nd day after the beginning of the experiment: all three strains at 25°C, and only strain LS16 at 30°C. After 5days, the three biocontrol agents were able to significantly reduce the level of OTA in berries at all the conditions tested. This occurred even when protection from infection was not significant, except at 30°C and 100% of RH for all the three strains, and at 25°C and 100% of RH for strain LS16. The biocontrol agents displayed a higher rate of colonization on grape berries at 20 and 25°C than at 30°C. The higher value of RH (100%) appeared to increase the rate of colonization, in particular at 20 and 25°C. Taken together, our results emphasize the significant influence of environmental factors on the effectiveness of biocontrol against A. carbonarius as well as on OTA contamination in wine grape berries, and the need for biocontrol agents that can cope with the environmental conditions that are conducive to attack by A. carbonarius.
The foregoing paper by Lorenz et al. (1995), translated into English by P. May, describes a new system for the identification of grapevine growth stages called the BBCH system. This is an adaptation, for the grapevine, of a basic scale developed to cover all monocot and dicot crops. Appraisal of this and two other systems has led to a preference for that by Eichhorn and Lorenz (1977) but with some amendments. These amendments are discussed and a new system of measurement and description of stages of the grapevine is proposed which copes with the dual needs for a simple listing of major stages and, at the same time, provides intermediate detailed stages. It is called the Modified E-L system.
Efficacy and mode of action were investigated fox Aureobasidium pullulans, a potential biocontrol agent for grey mould on strawberry fruit. Wound inoculation of detached green strawberry cv. Elsanta
fruit with Aureobasidium pullulans prevented grey mould rot on fruit inoculated 2 days later with Botrytis cinerea. Treatment of white, pink and red-ripe fruit did not, however, control grey mould. Treatment of wound sites on green fruit
with both live and heat-killed A. pullulans cells reduced B. cinerea infection compared to controls. Dip-inoculation of unwounded green strawberry fruit with A pullulans when still attached to the plant delayed the development of grey mould after harvest at the fully ripe stage. The (i) efficacy
of A. pullulans on green but not on ripening fruit, (ii) partial inhibitory effect of both live and heat-killed A. pullulans cells and (iii) absence of evidence for antibiotic production from in vitro competition tests suggest that control of grey mould on green fruit is at least partly due to a mechanism other than antagonism
and / or competition. Bioassays showed that skin tissue from green fruit treated with A. pullulans had greater antifungal activity than control tissue. Thus, enhanced natural disease resistance in green strawberry fruit
contributed to grey mould rot suppression by A. pullulans.
Additional keywords
Botrytis cinerea
–postharvest
Many yeasts, including yeast-like fungi, were selectively isolated from fruits and vegetables. In several assays performed on strawberries, table grape berries and kiwifruit, the yeast-like fungus Aureobasidium pullulans L47 and the yeasts Candida vanderwaltii L60 and C. oleophila L66 were the most effective antagonists of Botrytis cinerea and Rhizopus stolonifer. Isolates L47 and L66 were utilized in trials on strawberries grown under plastic tunnels. They were applied at flowering (full bloom and late petal fall) and at fruit maturity (just before or after harvest). Isolate L47 was the most effective against both B. cinerea and R. stolonifer. Both antagonists were more active when applied at the flowering stage, with isolate L47 more effective than vinclozolin. The antagonist population was consistently high on flowers and developing fruits, and on cold-stored strawberries. Isolates L47 and L66 showed a low sensitivity towards some fungicides in culture and were able to grow at temperatures between those of cold storage and 33 °C. Competition for nutrients seems to be the main mode of action.
Molecular fingerprinting of biocontrol agents is pivotal both for environmental monitoring and registration purposes. Fluorescent amplified fragment length polymorphism (fAFLP) analysis was utilised for the first time to investigate the intraspecific variability of the yeast-like fungus Aureobasidium pullulans, in order to identify specific molecular markers for its strain LS30, an effective biocontrol agent against major postharvest pathogens on different crops, and to pave the way to the development of molecular-based tools for unequivocal tracking of this agent after its release in the environment. Forty-eight isolates of A. pullulans from phyllosphere and carposphere of several crops from different sites of Southern Italy and Greece were analyzed by using four couples of primers. A pairwise comparison of fAFLP patterns was performed, for each primer pair, by using Dice similarity coefficient (SD). Four matrices were generated and, subsequently, averaged and combined for constructing a single dendrogram, in which clustering of fingerprints was performed with the unweighted pair groups (UPGMA). In the combined dendrogram, most of the isolates grouped into three main fAFLP clusters with levels of similarity ranging from 0.18 to 0.35. Only two isolates (AU73 and AU91) were very similar in all fAFLP patterns. Only primers AC/CA yielded three DNA sized fragments that appeared to be specific for LS30.
Aureobasidium pullulans, a cosmopolitan yeast-like fungus, colonises leaf surfaces and is a potential biocontrol agent for plant pathogens. Forty-one isolates of the fungus from the surface of several fruits and vegetables cultivated in Southern Italy were compared by molecular analysis and biocontrol activity. Characterisation of the isolates by using arbitrarily primed PCR (ap-PCR) confirmed the presence of a high genetic variability within the species. All the isolates were evaluated for their ability to control postharvest grey mold of apples, and two of them (SL250 and SL236), plus a proven antagonist (isolate L47), were able to control Penicillium digitatum on grapefruit, Botrytis cinerea, Rhizopus stolonifer and Aspergillus niger on table grape and B. cinerea and R. stolonifer on cherry tomato. Furthermore, preharvest application of isolate L47 on table grape resulted in a significant reduction of grey mold ranging from 27.1 to 49.5% compared to the untreated control. Random amplified polymorphic DNA technique (RAPD) was a useful method for the identification and evaluation of the survival rate of the applied antagonist.
An alkaline protease gene was amplified from genomic DNA and cDNA of the antagonistic yeast-like fungus Aureobasidium pullulans PL5, a biocontrol agent effective against Monilinia laxa on stone fruit and Botrytis cinerea and Penicillium expansum on pome fruits. An open reading frame of 1248 bp encoding a 415-amino acid (aa) protein with a calculated molecular weight (M(r)) of 42.9 kDa and an isoelectric point (pI) of 4.5 was characterized. The cDNAALP5 gene had an 18-amino acid signal peptide, one N-gylcosylation, one histidine active site, and one serine active site. The ALP5 gene with a M(r) of 1351 bp contained two introns. One intron was of 54 bp, while the other was of 50 bp. Protein BLAST and phylogenetic tree analysis of the deduced amino sequences from the cDNAALP5 gene showed that the encoded protein had 100% homology to a protease enzyme (ALP2) of a sea strain of A. pullulans, suggesting that the protein ALP5 was an alkaline serine protease. Expression of ALP5 in Escherichia coli BL21 (DE3), followed by identification with Western-blotting, purification with Ni-NTA and analysis of enzymatic activity, yielded an homogeneous recombinant ALP5 which hydrolysed the substrate casein and inhibited the mycelial growth of the pathogens. At its optimal pH of 10.0 and reaction temperature of 50°C, the recombinant protease exhibited the highest activity towards the substrate casein, though the highest stability was at lower temperatures and pH between 7.0 and 9.0. This study provided the direct evidence that extracellular proteases secreted by the antagonist A. pullulans PL5 played a role in the biocontrol activities against some postharvest pathogens of apple and peach.
Vineyards in the Hunter Valley and Hastings Valley (sub-tropical NSW) were examined by unaided eye for visible symptoms of bunch rot diseases at berry maturity in 2003 (for a total of five varieties over seven vineyards). Grey mould (Botrytis cinerea) was evident in three vineyards, and ripe rot (Colletotrichum acutatum) was evident in four of those seven vineyards surveyed. Other bunch rots at six of the vineyards could not be readily identified by visual inspection. The incidence of fungi on grapevine reproductive structures (and potentially bunch-rot fungi) was then recorded for a Cabernet Sauvignon vineyard in the Hastings Valley during the 2004/5 and 2005/6 growing seasons, and outcomes are reported here in some detail. By berry maturity, C. acutatum and Greeneria uvicola (bitter rot) were the predominant pathogens isolated from those structures, and constitute the first such report of bitter rot fungi on wine grapes in Australia. Indeed, the frequency of latent infection by C. acutatum and G. uvicola increased with berry development during the growing season. Other fungi isolated included Alternaria spp., Botryosphaeria spp., Cladosporium spp., Epicoccum sp., Fusarium spp., Nigrospora spp., Pestalotia spp., Phomopsis viticola and Trichoderma spp. Isolation of B. cinerea from this vineyard was rare. Infection of various wine grape varieties in vitro with C. acutatum and G. uvicola at post veraison revealed all varieties to be susceptible over a range of temperatures (20-35 degrees C). Based on laboratory studies, there was no infection of berries at a relative humidity (RH) < 50%, and infection diminished at 87% RH. Infection did occur if the berries were first incubated at 100% RH for 24 hours, and then transferred to an environment of lower humidity.
Grapes have a complex microbial ecology including filamentous fungi, yeasts and bacteria with different physiological characteristics and effects upon wine production. Some species are only found in grapes, such as parasitic fungi and environmental bacteria, while others have the ability to survive and grow in wines, constituting the wine microbial consortium. This consortium covers yeast species, lactic acid bacteria and acetic acid bacteria. The proportion of these microorganisms depends on the grape ripening stage and on the availability of nutrients. Grape berries are susceptible to fungal parasites until véraison after which the microbiota of truly intact berries is similar to that of plant leaves, which is dominated by basidiomycetous yeasts (e.g. Cryptococcus spp., Rhodotorula spp. Sporobolomyces spp.) and the yeast-like fungus Aureobasidium pullulans. The cuticle of visually intact berries may bear microfissures and softens with ripening, increasing nutrient availability and explaining the possible dominance by the oxidative or weakly fermentative ascomycetous populations (e.g. Candida spp., Hanseniaspora spp., Metschnikowia spp., Pichia spp.) approaching harvest time. When grape skin is clearly damaged, the availability of high sugar concentrations on the berry surface favours the increase of ascomycetes with higher fermentative activity like Pichia spp. and Zygoascus hellenicus, including dangerous wine spoilage yeasts (e.g. Zygosaccharomyces spp., Torulaspora spp.), and of acetic acid bacteria (e.g. Gluconobacter spp., Acetobacter spp.). The sugar fermenting species Saccharomyces cerevisiae is rarely found on unblemished berries, being favoured by grape damage. Lactic acid bacteria are minor partners of grape microbiota and while being the typical agent of malolactic fermentation, Oenococcus oeni has been seldom isolated from grapes in the vineyard. Environmental ubiquitous bacteria of the genus Enterobacter spp., Enterococcus spp., Bacillus spp., Burkholderia spp., Serratia spp., Staphylococcus spp., among others, have been isolated from grapes but do not have the ability to grow in wines. Saprophytic moulds, like Botrytis cinerea, causing grey rot, or Aspergillus spp., possibly producing ochratoxin, are only active in the vineyard, although their metabolites may affect wine quality during grape processing. The impact of damaged grapes in yeast ecology has been underestimated mostly because of inaccurate grape sampling. Injured berries hidden in apparently sound bunches explain the recovery of a higher number of species when whole bunches are picked. Grape health status is the main factor affecting the microbial ecology of grapes, increasing both microbial numbers and species diversity. Therefore, the influence of abiotic (e.g. climate, rain, hail), biotic (e.g. insects, birds, phytopathogenic and saprophytic moulds) and viticultural (e.g. fungicides) factors is dependent on their primary damaging effect.
Keywords: Vitis vinifera, Greeneria uvicola, grape, epidemiology, bitter rot. Thesis (M.S.)--North Carolina State University. Includes bibliographical references (p. 39-45). Includes vita.
Aureobasidium pullulans is the source of the commercially valuable polysaccharide pullulan and the enzyme xylanase. Isolates are typically off-white to pale pink or black on solid media, while some tropical isolates have been described as 'color variants' with bright pigments of red, yellow or purple. We sequenced 5 loci (internal transcribed spacer, intergenic spacer 1, translation elongation factor-1 alpha, beta tubulin, and RNA polymerase II) from 45 new isolates from Thailand. Based on the phylogenetic analyses, isolates were classified into 12 clades. Each clade showed different colors on different culture media including two clades with 'color variants' and some clades exhibited high levels of pullulan production or xylanase activity. Colony characteristics do not correlate perfectly with DNA sequence phylogeny or the physiological characters, but DNA sequence differences rapidly identify isolates with genetic novelty.
ABSTRACT The yeast Candida oleophila, the base of the commercial product Aspire, is recommended for the control of postharvest decay in citrus and pome fruit. Its modes of action include nutrient competition, site exclusion, and direct mycoparasitism. In the present study, we showed that application of Candida oleophila to surface wounds or to intact 'Marsh Seedless' grapefruit elicited systemic resistance against Penicillium digitatum, the main postharvest pathogen of citrus fruit. The induction of pathogen resistance in fruit was already pronounced 24 h after elicitation; it was distance, concentration, and time dependent and restricted to the peel tissue closely surrounding the yeast application site. The induction of pathogen resistance required viable yeast cells at concentrations of 10(8) to 10(9) cells ml(-1). Nonviable autoclaved or boiled yeast cells or lower yeast concentrations were ineffective in enhancing fruit disease resistance. Application of Candida oleophila cell suspensions to grapefruit peel tissue increased ethylene biosynthesis, phenylalanine ammonia lyase activity, and phytoalexin accumulation, and increased chitinase and beta-1,3-endoglucanase protein levels, indicated by western immunoblotting analysis. Scanning electron microscope observations revealed that spore germination and germ tube growth of Penicillium digitatum were markedly inhibited in wounds made near the yeast-treated sites. Overall, this study provides evidence that induced resistance against postharvest decay of citrus fruit should be considered an important component of the multiple modes of action of the yeast Candida oleophila.
The performance of denaturing gradient gel electrophoresis (DGGE) for analysing yeasts associated with wine grapes was compared with cultural isolation on malt extract agar (MEA). After optimisation of PCR and electrophoretic conditions, the lower limit of yeast detection by PCR-DGGE was 10(2) cfuml(-1), although this value was affected by culture age and the relative populations of the species in mixed culture. In mixed yeast populations, PCR-DGGE detected species present at 10-100-fold less than other species but not when the ratio exceeded 100-fold. Aureobasidium pullulans was the main species isolated from immature, mature, and both damaged and undamaged grapes. It was not detected by PCR-DGGE when present at populations less than 10(3) cfug(-1). When approaching maturity, damaged grapes gave a predominance of Metschnikowia and Hanseniaspora species (10(5)-10(7) cfug(-1)), all detectable using PCR-DGGE. However, various species of Rhodotorula, Rhodosporidium and Cryptococcus were not detected by this method, even when populations were as high as 10(4) cfug(-1). PCR -DGGE was less sensitive than culture on MEA for determining the yeast ecology of grapes and could not reliably detect species present at populations less than 10(4) cfug(-1). However, this method detected a greater diversity of species than agar plating.
Studies on the american rot of grapes due to
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Complex mechanism of action involved in the biocontrol activity of yeasts antagonists against postharvest diseases of fruits and vegetables
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