scholarly journals Enhancement of Postharvest Biocontrol Activity of the Yeast Candida oleophila by Overexpression of Lytic Enzymes

2003 ◽  
Author(s):  
Samir Droby ◽  
Michael Wisniewski ◽  
Martin Goldway ◽  
Wojciech Janisiewicz ◽  
Charles Wilson
Keyword(s):  
Mode Of Action ◽  
Biocontrol Agent ◽  
Biocontrol Agents ◽  
Transformation System ◽  
Lytic Enzymes ◽  
Candida Oleophila ◽  
Biocontrol Activity ◽  
Yeast Antagonist

Enhancing the activity of biocontrol agents could be the most important factor in their success in controlling fruit disease and their ultimate acceptance in commercial disease management. Direct manipulation of a biocontrol agent resulting in enhancement of diseases control could be achieved by using recent advances in molecular biology techniques. The objectives of this project were to isolate genes from yeast species that were used as postharvest biocontrol agents against postharvest diseases and to determine their role in biocontrol efficacy. The emphasis was to be placed on the yeast, Candida oleophila, which was jointly discovered and developed in our laboratories, and commercialized as the product, Aspire. The general plan was to develop a transformation system for C . oleophila and either knockout or overexpress particular genes of interest. Additionally, biochemical characterization of the lytic peptides was conducted in the wild-type and transgenic isolates. In addition to developing a better understanding of the mode of action of the yeast biocontrol agents, it was also our intent to demonstrate the feasibility of enhancing biocontrol activity via genetic enhancement of yeast with genes known to code for proteins with antimicrobial activity. Major achievements are: 1) Characterization of extracellular lytic enzymes produced by the yeast biocontrol agent Candida oleophila; 2) Development of a transformation system for Candida oleophila; 3) Cloning and analysis of C.oleophila glucanase gene; 4) Overexpression of and knockout of C. oleophila glucanase gene and evaluating its role in the biocontrol activity of C. oleophila; 5) Characterization of defensin gene and its expression in the yeast Pichiapastoris; 6) Cloning and Analysis of Chitinase and Adhesin Genes; 7) Characterization of the rnase secreted by C . oleophila and its inhibitory activity against P. digitatum. This project has resulted in information that enhanced our understanding of the mode of action of the yeast C . oleophila. This was important step towards enhancing the biocontrol activity of the yeast. Fungal cell wall enzymes produced by the yeast antagonist were characterized. Different substrates were identified to enhance there production in vitro. Exo-b-1, 3 glucanase, chitinase and protease production was stimulated by the presence of cell-wall fragments of Penicillium digitatum in the growing medium, in addition to glucose. A transformation system developed was used to study the role of lytic enzymes in the biocontrol activity of the yeast antagonist and was essential for genetic manipulation of C . oleqphila. After cloning and characterization of the exo-glucanase gene from the yeast, the transformation system was efficiently used to study the role of the enzyme in the biocontrol activity by over-expressing or knocking out the activity of the enzyme. At the last phase of the research (still ongoing) the transformation system is being used to study the role of chitinase gene in the mode of action. Knockout and over expression experiments are underway.

scholarly journals Mode of Action of Yeast Biocontrol Agents of Postharvest Diseases of Fruits

1996 ◽  
Author(s):  
Edo Chalutz ◽  
Michael Wisniewski ◽  
Samir Droby ◽  
Yael Eilam ◽  
Ilan Chet
Keyword(s):  
Spore Germination ◽  
Mode Of Action ◽  
Biocontrol Agent ◽  
Biocontrol Agents ◽  
Postharvest Diseases ◽  
Direct Inhibition ◽  
Calcium Salts ◽  
Biocontrol Activity ◽  
Extracellular Materials ◽  
Very High

In a previous BARD-supported study, three of the investigators of this research were involved in a study on biological control of postharvest diseases of citrus and deciduous fruits. Several naturally occurring, non-antibiotic producing yeast antagonists were identified. Application of some of these antagonists resulted in very high levels of biocontrol under laboratory conditions but lower efficacy in semi-commercial tests. It was felt that the lack of knowledge on the mode of action of the biocontrol agents was limiting their efficient use. The current study was aimed at narrowing this gap in our knowledge. Two specific objectives were outlined: to study the mechanism by which calcium salts enhance biocontrol activity and to determine the role, if any, of the yeast extracellular materials and/or enzymes which degrade fungal cell walls during the interaction between the antagonists, the pathogen and the host. CaCl2 but not MgCl2, inhibited spore germination, and germ-tube elongation of Botrytis cinerea, Penicillium expansum and P. digitatum in culture. It also inhibited the pectinolytic activity of the pathogens. Biocontrol of apple decay by isolate 182 of Candida oleophila, an effective biocontrol agent, was enhanced by the addition of CaCl2 whereas there was no effect on the biocontrol activity of isolate 247 of this yeast. Similarly, CaCl2 enhanced efficacy of the US-7 isolate of Pichia guilliermondii in reducing infection of P. digitatum in citrus fruit. CaCl2 by itself also reduced the infection of peel wounds and stimulated ethylene production by grapefruit peel. This antagonist exhibited a very high ability to maintain cytosolic Ca2+ homeostasis when exposed to high CaCl2 concentrations. It is postulated, therefore, that enhanced biocontrol activity by calcium is the result of direct inhibition of the pathogen by calcium ions on spore germination and metabolism and indirectly due to the ability of the biocontrol agent to maintain normal metabolism in the presence of high levels of calcium. The extracellular materials produced by P. guilliermondii in culture and on the fruit inhibited, at low concentrations, the pathogen in culture and reduced percent infection of the fruit. The direct inhibition of the pathogen by these materials may thus be involved in the mode of action of the antagonist. This study contributed to our knowledge on the action of calcium salts and the yeast antagonist extracellular materials on biocontrol activity and will contribute to a more efficient use of this technology in the control of postharvest diseases of fruits.

Characterization of extracellular lytic enzymes produced by the yeast biocontrol agent Candida oleophila

2004 ◽  
Vol 45 (3) ◽  
pp. 140-148 ◽  
Author(s):  
Meirav Bar-Shimon ◽  
Hila Yehuda ◽  
Lea Cohen ◽  
Batia Weiss ◽  
Alexsandra Kobeshnikov ◽  
...  
Keyword(s):  
Biocontrol Agent ◽  
Lytic Enzymes ◽  
Candida Oleophila

scholarly journals Development of molecular tools for the yeast Papiliotrema terrestris LS28 and identification of Yap1 as a transcription factor involved in biocontrol activity

2021 ◽  
Author(s):  
Raffaello Castoria ◽  
Cecilia Miccoli ◽  
Giuseppe Barone ◽  
Davide Palmieri ◽  
Filippo De Curtis ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Biocontrol Agent ◽  
Integrated Control ◽  
Biocontrol Agents ◽  
Alternative Methods ◽  
Penicillium Expansum ◽  
Postharvest Disease ◽  
Molecular Tools ◽  
Postharvest Diseases ◽  
Biocontrol Activity

Fungal attacks on stored vegetable and fruits are responsible for losses of products. There is an active research field to develop alternative strategies for postharvest disease management, and the use of biocontrol agents represents a promising approach. Understanding the molecular bases of the biocontrol activity of these agents is crucial to potentiate their effectiveness. The yeast Papiliotrema terrestris is a biocontrol agent against postharvest pathogens. Phenotypic studies suggest that it exerts its antagonistic activity through competition for nutrients and space, which relies on its resistance to oxidative and other cellular stresses. In this study, we developed tools for genetic manipulation in P. terrestris to perform targeted gene replacement and functional complementation of the transcription factors Yap1 and Rim101. In vitro phenotypic analyses revealed a conserved role of Yap1 and Rim101 in broad resistance to oxidative stress and alkaline pH sensing, respectively. In vivo analyses revealed that P. terrestris yap1Δ and rim101Δ mutants display decreased ability to colonize wounded fruit as compared to the parental WT strain; the yap1Δ mutant also displays reduced biocontrol activity against the postharvest pathogens Penicillium expansum and Monilia fructigena, indicating an important role for resistance to oxidative stress in timely wound colonization and biocontrol activity of P. terrestris. In conclusion, the availability of molecular tools developed in the present study provides a foundation to elucidate the genetic mechanisms underlying biocontrol activity of P. terrestris, with the goal of enhancing this activity for the practical use of P. terrestris LS28 in pest management programs based on biological and integrated control. IMPORTANCE The use of fungicides represents the most effective and widely used strategy for controlling postharvest diseases. However, their extensive use has raised several concerns, such as the emergence of plant pathogens’ resistance as well as the health risks associated with the persistence of chemical residues in fruits, vegetables, and in the environment. These factors have brought attention to alternative methods for controlling postharvest diseases, such as the utilization of biocontrol agents. In the present study we developed genomics and genetics resources to investigate at molecular level the mechanisms involved in the biocontrol activity of Papiliotrema terrestris, a basidiomycete yeast that is an effective biocontrol agent against widespread fungal pathogens, including Penicillium expansum, the etiological agent of blue mold disease of pome fruits. A deeper understanding of how postharvest biocontrol agents operate is the basic requirement to promote the utilization of biological (and integrated) control for the reduction of chemical fungicides.

scholarly journals Penggunaan Bakteri Kitinolitik sebagai Pengendali Hayati Colletotrichum capsici pada Tanaman Cabai

2018 ◽  
Vol 14 (4) ◽  
pp. 120
Author(s):  
Dian Syahfitri ◽  
Nisa Rachmania Mubarik ◽  
Lisdar A Manaf
Keyword(s):  
Biocontrol Agent ◽  
Bacterial Isolate ◽  
Disease Incidence ◽  
Antagonistic Activity ◽  
Biocontrol Agents ◽  
Bacterial Isolates ◽  
Chitinolytic Bacteria ◽  
Colletotrichum Capsici ◽  
Carrier Materials

Use of Chitinolytic Bacteria as Biological Control of Colletotrichum capsici on Chili PlantsColletotrichum capsici is known as the causal agent of anthracnose disease in chili plant and may cause reduction of crop yield. Chitinolytic bacteria, namely Serratia marcescens KAHN 15.12, Bacillus thuringiensis SAHA 12.12, and BAE 36 were reported to have antagonistic activity against C. capsici. Therefore, a study was conducted to determine the potential of chitinolytic bacteria on controlling C. capsici on chili plants in greenhouse experiment. Three bacterial isolates used as biocontrol agent was formulated by using talcum as carrier materials. The methodologies consisted of characterization of bacterial isolates, formulation of biocontrol agent, viability test of bacterial isolate, efficacy of biocontrol agents in the laboratory and in the greenhouse.  Disease severity in the laboratory reached 64% when chili treated with isolate formulation of BAE 36.  In the greenhouse, BAE 36 isolate formulation and consortium formulation were able to suppress infection of C. capsici; each was indicated by disease incidence of 25% and 50%, respectively. These results indicated that chitinolytic bacterial formulations could be potencial as biocontrol agents of C. capsici.

scholarly journals Role of Reactive Oxygen Species (ROS) in Tritrophic Interactions in Postharvest Biocontrol Systems

2012 ◽  
Author(s):  
Samir Droby ◽  
Michael Wisniewski ◽  
Ron Porat ◽  
Dumitru Macarisin
Keyword(s):  
Oxidative Stress ◽  
Mitogen Activated Protein Kinase ◽  
Yeast Cells ◽  
Penicillium Expansum ◽  
Antioxidant Genes ◽  
Candida Oleophila ◽  
Biocontrol Activity ◽  
Mitogen Activated Protein ◽  
Apple Fruits

To elucidate the role of ROS in the tri-trophic interactions in postharvest biocontrol systems a detailed molecular and biochemical investigation was undertaken. The application of the yeast biocontrol agent Metschnikowia fructicola, microarray analysis was performed on grapefruit surface wounds using an Affymetrix Citrus GeneChip. the data indicated that 1007 putative unigenes showed significant expression changes following wounding and yeast application relative to wounded controls. The expression of the genes encoding Respiratory burst oxidase (Rbo), mitogen-activated protein kinase (MAPK) and mitogen-activated protein kinase kinase (MAPKK), G-proteins, chitinase (CHI), phenylalanine ammonia-lyase (PAL), chalcone synthase (CHS) and 4-coumarate-CoA ligase (4CL). In contrast, three genes, peroxidase (POD), superoxide dismutase (SOD) and catalase (CAT), were down-regulated in grapefruit peel tissue treated with yeast cells. The yeast antagonists, Metschnikowia fructicola (strain 277) and Candida oleophila (strain 182) generate relatively high levels of super oxide anion (O2−) following its interaction with wounded fruit surface. Using laser scanning confocal microscopy we observed that the application of M. fructicola and C. oleophila into citrus and apple fruit wounds correlated with an increase in H2O2 accumulation in host tissue. The present data, together with our earlier discovery of the importance of H₂O₂ production in the defense response of citrus flavedo to postharvest pathogens, indicate that the yeast-induced oxidative response in fruit exocarp may be associated with the ability of specific yeast species to serve as biocontrol agents for the management of postharvest diseases. Effect of ROS on yeast cells was also studied. Pretreatment of the yeast, Candida oleophila, with 5 mM H₂O₂ for 30 min (sublethal) increased yeast tolerance to subsequent lethal levels of oxidative stress (50 mM H₂O₂), high temperature (40 °C), and low pH (pH 4). Suppression subtractive hybridization analysis was used to identify genes expressed in yeast in response to sublethal oxidative stress. Transcript levels were confirmed using semi quantitative reverse transcription-PCR. Seven antioxidant genes were up regulated. Pretreatment of the yeast antagonist Candida oleophila with glycine betaine (GB) increases oxidative stress tolerance in the microenvironment of apple wounds. ROS production is greater when yeast antagonists used as biocontrol agents are applied in the wounds. Compared to untreated control yeast cells, GB-treated cells recovered from the oxidative stress environment of apple wounds exhibited less accumulation of ROS and lower levels of oxidative damage to cellular proteins and lipids. Additionally, GB-treated yeast exhibited greater biocontrol activity against Penicillium expansum and Botrytis cinerea, and faster growth in wounds of apple fruits compared to untreated yeast. The expression of major antioxidant genes, including peroxisomal catalase, peroxiredoxin TSA1, and glutathione peroxidase was elevated in the yeast by GB treatment. A mild heat shock (HS) pretreatment (30 min at 40 1C) improved the tolerance of M. fructicola to subsequent high temperature (45 1C, 20–30 min) and oxidative stress (0.4 mol-¹) hydrogen peroxide, 20–60 min). HS-treated yeast cells showed less accumulation of reactive oxygen species (ROS) than non-treated cells in response to both stresses. Additionally, HS-treated yeast exhibited significantly greater (P≥0.0001) biocontrol activity against Penicillium expansum and a significantly faster (Po0.0001) growth rate in wounds of apple fruits stored at 25 1C compared with the performance of untreated yeast cells. Transcription of a trehalose-6-phosphate synthase gene (TPS1) was up regulated in response to HS and trehalose content also increased. 

Digital x-ray mapping of nanometer-size supported bimetallic catalysts

1988 ◽  
Vol 46 ◽  
pp. 530-531
Author(s):  
L. T. Germinario
Keyword(s):  
Background Radiation ◽  
Metal Cluster ◽  
Single Element ◽  
Beam Diameter ◽  
X Rays ◽  
X Ray ◽  
Major Interest ◽  
Induced Changes

Understanding the role of metal cluster composition in determining catalytic selectivity and activity is of major interest in heterogeneous catalysis. The electron microscope is well established as a powerful tool for ultrastructural and compositional characterization of support and catalyst. Because the spatial resolution of x-ray microanalysis is defined by the smallest beam diameter into which the required number of electrons can be focused, the dedicated STEM with FEG is the instrument of choice. The main sources of errors in energy dispersive x-ray analysis (EDS) are: (1) beam-induced changes in specimen composition, (2) specimen drift, (3) instrumental factors which produce background radiation, and (4) basic statistical limitations which result in the detection of a finite number of x-ray photons. Digital beam techniques have been described for supported single-element metal clusters with spatial resolutions of about 10 nm. However, the detection of spurious characteristic x-rays away from catalyst particles produced images requiring several image processing steps.

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