Attachment microbes antagonistic against Botrytis cinerea—biological control and scanning electron microscope studies in vivo

Bacteria and yeasts, selected by an attachment assay for their ability to adhere to Botrytis cinerea hyphae or conidia, were evaluated for biocontrol potential against B. cinerea on excised tomato stems. Eight of the 12 bacteria and seven of the eight yeast isolates conferred 90% to 100% biocontrol activity when antagonist populations were applied at three to 80 times the pathogen inoculum density. Biocontrol was maintained at similar levels when biocontrol agent (BCA) application was delayed up to 48 h after pathogen challenge. Scanning electron microscopy showed extensive colonisation of B. cinerea mycelium or conidia by many of these isolates and also evidence of pathogen degradation. The biocontrol efficacy and potential for the assay are discussed with respect to cell-to-cell adhesion as a vehicle to deliver antagonistic mechanisms to highly specific pathogen sites.     

Botrytis species: relentless necrotrophic thugs or endophytes gone rogue?

Plant pathology has a long‐standing tradition of classifying microbes as pathogens, endophytes or saprophytes. Lifestyles of pathogens are categorized as biotrophic, necrotrophic or hemibiotrophic. Botrytis species are considered by many to be archetypal examples of necrotrophic fungi, with B. cinerea being the most extensively studied species because of its broad host range and economic impact. In this review, we discuss recent work which illustrates that B. cinerea is capable of colonizing plants internally, presumably as an endophyte, without causing any disease or stress symptoms. The extent of the facultative endophytic behaviour of B. cinerea and its relevance in the ecology and disease epidemiology may be vastly underestimated. Moreover, we discuss the recent discovery of a novel Botrytis species, B. deweyae, which normally grows as an endophyte in ornamental daylilies (Hemerocallis), but displays facultative pathogenic behaviour, and is increasingly causing economic damage. We propose that the emergence of endophytes ‘gone rogue’ as novel diseases may be related to increased inbreeding of hybrid lines and reduced genetic diversity. These observations lead us to argue that the sometimes inflexible classification of pathogenic microbes by their lifestyles requires serious reconsideration. There is much more variety to the interactions of Botrytis with its hosts than the eye (or the plant pathologist) can see, and this may be true for other microbes interacting with plants.     

Yeasts and filamentous fungi carried by the gynes of leaf-cutting ants

Insect-associated microbes exhibit a wide range of interactions with their hosts. One example of such interactions is the insect-driven dispersal of microorganisms, which plays an essential role in the ecology of several microbes. To study dispersal of microorganisms by leaf-cutting ants (Formicidae: Attini), we applied culture-dependent methods to identify the filamentous fungi and yeasts found in two different body parts of leaf-cutting ant gynes: the exoskeleton and the infrabuccal pocket. The gynes use the latter structure to store a pellet of the ants’ symbiotic fungus during nest founding. Many filamentous fungi (n = 142) and yeasts (n = 19) were isolated from the gynes’ exoskeleton. In contrast, only seven filamentous fungi and three yeasts isolates were recovered from the infrabuccal pellets, suggesting an efficient mechanism utilized by the gynes to prevent contamination of the symbiotic fungus inoculum. The genus Cladosporium prevailed (78%) among filamentous fungi whereas Aureobasidium, Candida and Cryptococcus prevailed among yeasts associated with gynes. Interestingly, Escovopsis, a specialized fungal pathogen of the leaf-cutting ant-fungus symbiosis, was not isolated from the body parts or from infrabuccal pellets of any gynes sampled. Our results suggest that gynes of the leaf-cutter ants Atta laevigata and A. capiguara do not vertically transmit any particular species of yeasts or filamentous fungi during the foundation of a new nest. Instead, fungi found in association with gynes have a cosmopolitan distribution, suggesting they are probably acquired from the environment and passively dispersed during nest foundation. The possible role of these fungi for the attine ant–microbial symbiosis is discussed.     

Microbial incidence in upper respiratory tracts of workers in the paper industry.

The incidence of microbes in the nasal cavities of workers in three paper and board mills was investigated. A total of 234 persons exposed to microbial aerosols and splashes from paper machine wires and debarker drums formed the exposed group. The control group consisted of 294 workers from the dry working areas: the winding and packing sections. Chi-square analysis was used to test the differences in the frequency of microbial incidence and various symptoms between the exposed and control groups. The nasal cavities of many workers, particularly workers in the debarkers, proved to be contaminated by Klebsiella pneumoniae, other coliforms, yeasts, and molds; usually only one microbe was involved, but sometimes two or several species were found. Nasal bacteria and yeasts were largely derived from the mill and debarker air; the microbes in the air came mainly from process waters. Lack of association of nasopharyngeal symptoms with either exposure to aerosols or nasal microbial contamination was interpreted as an indication of host defenses that were adequate to protect workers from harmful microbial colonization in paper mill environments.     

A new improved strategy for the selection of cold-adapted antagonist yeasts to control postharvest pear diseases

Postharvest diseases cause considerable losses of harvested fruits during transportation and storage. Many yeast species have been reported as good antagonists against postharvest pear pathogens. In this work, we used a novel selection strategy that involves the isolation of yeasts from washing fluids, showing biocontrol activity against a regional Penicillium expansum strain (primary screening), originally obtained from fruit wounds after long time storage at ?1/0°C. About 26 isolates representative of the 11 yeast species identified in the 27 selected washing waters were chosen to be evaluated in a secondary screening against a regional Botrytis cinerea strain on pear wounds. Among yeasts tested, 38% showed complete control of P. expansum, but only 15% reduced the decay incidence of B. cinerea to 60–80% at ?1/0°C. These results reveal that some of the yeasts found can be biological alternatives to fungicides in the control of P. expansum and B. cinerea infections. Based on the data obtained, our strategy seems to be much more effective than the previously reported methods in obtaining successful biocontrol agents.     

The signalling mucin Msb2 regulates surface sensing and host penetration via BMP1 MAP kinase signalling in Botrytis cinerea

Botrytis cinerea is a necrotrophic fungus that infects a wide range of fruit, vegetable and flower crops. Penetration of the host cuticle occurs via infection structures that are formed in response to appropriate plant surface signals. The differentiation of these structures requires a highly conserved mitogen‐activated protein (MAP) kinase cascade including the MAP kinase BMP1. In yeast and several plant‐pathogenic fungi, the signalling mucin Msb2 has been shown to be involved in surface recognition and MAP kinase activation. In this study, a B. cinerea msb2 mutant was generated and characterized. The mutant showed normal growth, sporulation, sclerotia formation and stress resistance. In the absence of nutrients, abnormal germination with multiple germ tubes was observed. In the presence of sugars, normal germination occurred, but msb2 germlings were almost unable to form appressoria or infection cushions on hard surfaces. Nevertheless, the msb2 mutant showed only a moderate delay in lesion formation on different host plants, and formed expanding lesions similar to the wild‐type. Although the wild‐type showed increasing BMP1 phosphorylation during the first hours of germination on hard surfaces, the phosphorylation levels in the msb2 mutant were strongly reduced. Several genes encoding secreted proteins were found to be co‐regulated by BMP1 and Msb2 during germination. Taken together, B. cinerea Msb2 is likely to represent a hard surface sensor of germlings and hyphae that triggers infection structure formation via the activation of the BMP1 MAP kinase pathway.     

Microorganisms in Daqu: a starter culture of Chinese Maotai-flavor liquor

Maotai-flavor liquor, a famous traditional Chinese drink, is distilled from fermented sorghum in the southern province of China. Moreover, it is of interest as one of the few examples of liquor distilled from the product of a fermentation using a wild microflora starter. Daqu is the starter of this fermentation process. Daqu is a mixture of two components: milled wheat and a complex microbial community. The composition and the effects of this microbial group are largely unknown. In this study, we have analysed the constituents of the microbial community and the development of microorganisms in the industrial Daqu preparation and ripening process. More than 200 colonies were isolated and characterized. The isolates were discriminated by phenotypic and conventional biochemical taxonomic methods. The results revealed the presence of bacteria, moulds and yeasts. Bacteria consist of Bacillus, Acetobacter, Lactobacillus, and Clostridium, among which Bacillus strains were found to be predominant. Moulds consisted of Aspergillus, Mucor, Rhizopus, Monascus and Trichoderma, and Aspergillus strains were found to be predominant in the six different biotypes. Yeasts comprised Saccharomyces, Hansenula, Candida, Pichia, and Torulaspora. The most frequently isolated yeasts belonged to the genus Saccharomyces. The microbial diversity shift showed that the microbial genera changed with increasing ripening time. Knowledge of the microbial diversity in Daqu provides a basis for microflora management and understanding of the role of microbes in the Daqu production process, and the contribution of Daqu performed as a starter culture to Maotai-flavor liquor.     

Indicator microbes correlate with pathogenic bacteria, yeasts and helminthes in sand at a subtropical recreational beach site

Aims: Research into the relationship between pathogens, faecal indicator microbes and environmental factors in beach sand has been limited, yet vital to the understanding of the microbial relationship between sand and the water column and to the improvement of criteria for better human health protection at beaches. The objectives of this study were to evaluate the presence and distribution of pathogens in various zones of beach sand (subtidal, intertidal and supratidal) and to assess their relationship with environmental parameters and indicator microbes at a non‐point source subtropical marine beach. Methods and Results: In this exploratory study in subtropical Miami (Florida, USA), beach sand samples were collected and analysed over the course of 6 days for several pathogens, microbial source tracking markers and indicator microbes. An inverse correlation between moisture content and most indicator microbes was found. Significant associations were identified between some indicator microbes and pathogens (such as nematode larvae and yeasts in the genus Candida), which are from classes of microbes that are rarely evaluated in the context of recreational beach use. Conclusions: Results indicate that indicator microbes may predict the presence of some of the pathogens, in particular helminthes, yeasts and the bacterial pathogen Staphylococcus aureus including methicillin‐resistant forms. Indicator microbes may thus be useful for monitoring beach sand and water quality at non‐point source beaches. Significance and Impact of the Study: The presence of both indicator microbes and pathogens in beach sand provides one possible explanation for human health effects reported at non‐point sources beaches.     

A lytic enzyme cocktail from <Emphasis Type="Italic">Streptomyces</Emphasis> sp. B578 for the control of lactic and acetic acid bacteria in wine

Beside yeasts, lactic acid bacteria (LAB) are the most abundant microbes in must during vinification. Whereas Oenococcos oeni is commercially used as a starter culture for the biological acid reduction in wines, other species are responsible for different types of wine spoilage. Members of the genera Pediococcus, Weissella, Leuconostoc, and Lactobacillus are producers of exopolysaccharide slimes, biogenic amines, acetic acid, and other off-flavors. In order to control microbial growth, different procedures such as heating of must and addition of sulfite or lysozyme from egg white are generally applied. Yet, because of health risks, the application of sulfite should be reduced and lysozyme is not effective against all LAB. In this study, we describe exoenzymes from a Streptomyces sp. strain B578 lysing nearly all wine-relevant strains of LAB and Gram-negative acetic acid bacteria. The lytic enzymes were active under wine-making conditions, such as the presence of sulfite and ethanol, low temperatures, and low pH values. The analysis of the exoenzyme composition revealed a synergistic action of different cell wall hydrolases. In conclusion, the lytic cocktail of Streptomyces sp. B578 is a promising tool for the control of wine-spoiling bacteria. This article is dedicated to Prof. Dr. Ferdinand Radler on the occasion of his 80th birthday.     

Dual Antagonism of Aldehydes and Epiphytic Bacteria from Strawberry Leaf Surfaces against the Pathogenic Fungus Botrytis cinerea in vitro

Dual culture experiments were conducted in vitro to evaluate the potential combined biological effect of epiphytic bacteria and plant volatiles formed during fatty acids degradation on the pathogenic fungus Botrytis cinerea. The aliphatic aldehydes hexanal, (E)-2-hexenal, (Z)-3-hexenal and (E)-2-nonenal showed an enhancing effect on the antagonistic interaction between the epiphytic bacteria Pseudomonas lurida, Pseudomonas rhizosphaerae, Pseudomonas parafulva, and Bacillus megaterium against the pathogenic fungus. The unsaturated aldehydes were found to be the most potent with the minimum effective concentration being 1 ppm. Increasing volatile concentrations led to the inhibition of Botrytis cinerea growth with concomitant increase of colony diameters of epiphytic bacteria. Especially (E)-2-nonenal showed a stronger inhibitory effect on different strains of the plant pathogenic fungus Botrytis cinerea than on the epiphytic bacteria. These results suggest that co-application of antagonistic bacteria with natural plant volatiles can enhance the effectiveness of the biocontrol agents against B. cinerea.     

Bacterial diversity in the rhizosphere of maize and the surrounding carbonate‐rich bulk soil

Maize represents one of the main cultivar for food and energy and crop yields are influenced by soil physicochemical and climatic conditions. To study how maize plants influence soil microbes we have examined microbial communities that colonize maize plants grown in carbonate‐rich soil (pH 8.5) using culture‐independent, PCR‐based methods. We observed a low proportion of unclassified bacteria in this soil whether it was planted or unplanted. Our results indicate that a higher complexity of the bacterial community is present in bulk soil with microbes from nine phyla, while in the rhizosphere microbes from only six phyla were found. The predominant microbes in bulk soil were bacteria of the phyla Acidobacteria, Bacteroidetes and Proteobacteria, while Gammaproteobacteria of the genera Pseudomonas and Lysobacter were the predominant in the rhizosphere. As Gammaproteobacteria respond chemotactically to exudates and are efficient in the utilization of plants exudate products, microbial communities associated to the rhizosphere seem to be plant‐driven. It should be noted that Gammaproteobacteria made available inorganic nutrients to the plants favouring plant growth and then the benefit of the interaction is common.     

Impact of <Emphasis Type="Italic">Quillaja saponaria</Emphasis> saponins on grapevine ecosystem organisms

The control of grapevine pathogens is a rising concern in Vitis vinifera culture. The current international trend is toward banning chemicals that are highly toxic to the environment and human workers, and adopting tighter regulations. We evaluated the impact of saponins on three kinds of organisms found in grapevine culture. The ectoparasitic nematode Xiphinema index, the parasitic fungus Botrytis cinerea and various yeast strains representative of the must fermentation population were incubated on synthetic media supplemented with variable concentrations of Quillaja saponaria saponins. Saponins induced reduction in the growth of B. cinerea and showed nematicide effects on X. index. The control of X. index and Botrytis cinerea is discussed in the context of the potential use of these chemicals as environmentally-friendly grapevine treatments. With Saccharomyces cerevisiae and other yeasts, saponins showed higher toxicity against S. cerevisiae strains isolated from wine or palm wine whereas laboratory strains or strains isolated from oak exhibited better resistance. This indicates that Q. saponaria saponins effects against yeast microflora should be assessed in the field before they can be considered an environmentally-safe new molecule against B. cinerea and X. index.     

Ecology of Microbial Saprophytes and Pathogens in Tissue Culture and Field-Grown Plants: Reasons for Contamination Problems In Vitro

This review compares published surveys of microbial populations in plant tissue and cell cultures with the microbial saprophytes and pathogens found on field grown plants and microbial populations in the laboratory environment. From this comparison and the measured reduction in contamination after improvements in working practices in the laboratory, conclusions can be drawn about the importance of the explant and the laboratory as sources of contamination.

Mechanisms of pathogenicity in vitro are described to explain why bacteria, fungi, and yeasts that are not pathogenic to plants in the field become pathogens in plant tissue cultures. Conversely, plant metabolism and its effect on the tissue culture environment are described to explain why prokaryotes, viruses, and viroids that cause disease in the field can stay latent in vitro.

Detection methods for latent contaminants in plant tissue cultures are summarized, and the strategies and methods for prevention or treatment of contamination are discussed.     

Effects of microbial floras on the distributions of five domestic Drosophila species across fruit resources

Summary The distributions of five Drosophila species and four components of the microflora have been compared across a total of 48 traps baited with four different fruit and vegetable substrates in two domestic compost heaps in Canberra (Australia). Large and consistent differences are found, both among the Drosophila and among the microbial classes, in their distributions across traps baited with different substrates. Moreover the distribution of each Drosophila species shows a unique set of strong associations with the microbial distributions. Thus the distributions of both D. simulans and D. melanogaster are found to be strongly negatively correlated with the abundance of bacteria while D. simulans is also strongly positively correlated with the titre of fermenter yeasts. D. immigrans is strongly positively correlated both with bacteria and with non-fermenter yeasts. D. hydei is positively correlated with nonfermentery yeasts and D. busckii is negatively correlated with fermenter yeasts. Moulds are the only microbial class not consistently associated with the distribution of any of the Drosophila species. The correlations with the other microbial classes are sufficient to explain the majority of the abundance differences of the Drosophila species among the trap types. It is therefore proposed that the clear partitioning of the fruit resources by the Drosophila is due to their differing primary interactions with the microflora.     

Detection of Botrytis cinerea by loop-mediated isothermal amplification

Aims: To develop a sensitive, rapid and simple method for detection of Botrytis cinerea based on loop‐mediated isothermal amplification (LAMP) that would be suitable for use outside a conventional laboratory setting. Methods and Results: A LAMP assay was designed based on the intergenic spacer of the B. cinerea nuclear ribosomal DNA (rDNA). The resulting assay was characterized in terms of sensitivity and specificity using DNA extracted from cultures. The assay consistently amplified 65 pg B. cinerea DNA. No cross‐reactivity was observed with a range of other fungal pathogens, with the exception of the closely related species Botrytis pelargonii. Use of a novel real‐time LAMP platform (the OptiGene Genie I) allowed detection of B. cinerea in infected rose petals, with amplification occurring in <15 min. Conclusions: The LAMP assay that was developed is suitable for rapid detection of B. cinerea in infected plant material. Significance and Impact of the Study: The LAMP method combines the sensitivity and specificity of nucleic acid‐based methods with simplified equipment and a reduced reaction time. These features make the method potentially suitable for on‐site use, where the results of testing could help to inform decisions regarding the storage and processing of commodities affected by B. cinerea, such as cut flowers, fruit and vegetables.     

Screening of bacteria for the suppression of Botrytis cinerea and Rhizoctonia solani on lettuce (Lactuca sativa) using leaf disc bioassays

Two leaf disc bioassays were developed for screening bacteria as putative biological control agents of Botrytis cinerea and Rhizoctonia solani on lettuce. Aerobic spore and non‐spore forming bacteria were isolated from the phylloplane, rhizoplane and rhizosphere of symptom‐free lettuce plants grown in the presence and absence of chitin or composted bark soil amendments. Bacteria, previously isolated from other plants, were also included in the primary screen initially against B. cinerea. One hundred and twenty‐seven of 700 isolates reduced botrytis rotting of lettuce leaves by more than 50% in the primary screen. Following a secondary screen against B. cinerea, the lead 50 isolates were also tested for suppression of R. solani infection. Four isolates significantly reduced both botrytis and rhizoctonia leaf rotting. Eleven and five isolates gave control of botrytis and rhizoctonia, respectively, equal to that given by the standard fungicides Rovral WP (iprodione) and Basilex (tolclofos methyl). The two most effective isolates against B. cinerea and R. solani were both identified as Bacillus subtilis. Use of soil amendments did not increase the proportion of efficacious isolates recovered. Effective isolates were originally recovered from roots of oilseed rape and lettuce leaves. In general, it was found that bacteria which controlled one disease effectively did not control the second disease nearly as well. The bioassay protocols developed in this study were used successfully in screening a large number of bacterial isolates in a short time.     

Microbiological quality of pork meat from local Hong Kong markets

Over a 3-week period, samples of fresh, chopped, pork meat were taken every morning and afternoon from 50 meat stalls. Microbiological examination revealed that the samples had (c.f.u./g): total microbes, 1×10³ to 2.14×10⁶; mean probable numbers of coliforms and Escherichia coli, 1.51×10³ to 1.15×10⁴; and yeasts and moulds, 0 to 1.28×10⁴. Salmonella, found in 32 samples from 21 stalls, were serotyped as B (three samples), C1 (four) or E (25). No Campylobacter were found. Because microbial growth and/or contamination of the meat occurred during the day, samples taken in the afternoon had greater total counts (P<0.05) and contained detectable numbers of Salmonella more frequently (42% versus 22%) than those taken in the morning.The author is with the Department of Biology and Chemistry, City Polytechnic of Hong Kong, 83 Tat Chee Ave, Kowloon, Hong Kong     

Biocontrol of Postharvest Grey Mould on Tomato by Yeasts

The fungal pathogen Botrytis cinerea causes severe rots on tomato fruit during storage and shelf life. Biological control of postharvest diseases of fruit may be an effective alternative to chemical control. Yeasts are particularly suitable for postharvest use, proving to be highly effective in reducing the incidence of fungal pathogens. Yeast fungi isolated from the surface of solanaceous plants were evaluated for their activity in reducing the postharvest decay of tomato caused by B. cinerea. Of 300 isolates, 14 strains of Rhodotorula rubra and Candida pelliculosa were found to be strongly antagonistic to the pathogen in vitro and were selected for further storage experiment. The antagonists were evaluated for their effect on the biological control of postharvest grey mould. Artificially wounded fruits were treated by means of a novel technique: small sterile discs of filter paper imbibed separately in suspensions of each yeast and the pathogen were superposed onto each wound. After 1‐week, 11 isolates were significantly effective in reducing the diameter of lesions by more than 60% compared to the control treated with B. cinerea alone. Total protection was obtained with the strain 231 of R. rubra on fruits challenged with pathogen spores. To our knowledge, R. rubra and C. pelliculosa have not been described as biocontrol agents against grey mould caused by B. cinerea. Our data demonstrate that the application of antagonistic yeasts represents a promising and environmentally friendly alternative to fungicide treatments to control postharvest grey mould of tomato.     

Yeasts in malting,with special emphasis on <Emphasis Type="Italic">Wickerhamomyces anomalus</Emphasis> (synonym <Emphasis Type="Italic">Pichia anomala</Emphasis>)

Malted barley is a major raw material of beer, as well as distilled spirits and several food products. The production of malt (malting) exploits the biochemical reactions of a natural process, grain germination. In addition to germinating grain, the malting process includes another metabolically active component: a diverse microbial community that includes various types of bacteria and fungi. Therefore, malting can be considered as a complex ecosystem involving two metabolically active groups. Yeasts and yeast-like fungi are an important part of this ecosystem, but previously the significance of yeasts in malting has been largely underestimated. Characterization and identification of yeasts in industrial processes revealed 25 ascomycetous yeasts belonging to 10 genera, and 18 basidiomycetous yeasts belonging to 7 genera. In addition, two ascomycetous yeast-like fungi belonging to the genera Aureobasidium and Exophiala were commonly detected. Yeasts and yeast-like fungi produced extracellular hydrolytic enzymes with a potentially positive contribution to the malt enzyme spectrum. Several ascomycetous yeast strains showed strong antagonistic activity against field and storage moulds, Wickerhamomyces anomalus (synonym Pichia anomala) being the most effective species. Malting studies revealed that W. anomalus VTT C-04565 effectively restricted Fusarium growth and hydrophobin production during malting and prevented beer gushing. In order to broaden the antimicrobial spectrum and to improve malt brewhouse performance, W. anomalus could be combined with other starter cultures such as Lactobacillus plantarum. Well-characterized microbial mixtures consisting of barley and malt-derived microbes open up several possibilities to improve malt properties and to ensure the safety of the malting process.     

Benefits of Soleris® over the Conventional Method for Enumeration of Microbial Load in Salacia Herbal Extract

Stems and roots of Salacia genus plants have been used as a specific remedy for early-stage diabetes, and one of the four sulphonium sulphates, salacinol is the compound responsible for the anti-diabetic activity. Salacia is prone to microbial contamination and insect infestation; hence, methods to estimate the microbial load in such plants will enhance its nutritional value. This paper highlights the novel use of Soleris^® to quantify microbes of all types, namely bacteria, yeasts, molds, and coliforms in herbal extracts. The microbial analysis results obtained with Soleris^® test vial have been compared with the conventional method, and the results indicate that Soleris^® is equally efficient as the conventional method and in fact displays several advantages over the traditional method. The Soleris^® method is a real time monitoring system that is highly sensitive, user-friendly, and environmentally friendly since it generates very little biomedical waste and saves a large amount of time. The data presented here demonstrate that for highly contaminated samples, results are available within 24 h. For yeasts and molds, the Soleris^® method produces results in 48 h, thus offering considerable time savings compared to other commonly used methods.Key words: Salacia, salacinol, microbial load, bacteria, yeasts, herbals