Production and Optimization of Rhodotorula Glutinis for Biocontrolling of Strawberry Blight

ISSN: 0975-8585 Research Journal of Pharmaceutical, Biological and Chemical Sciences Production and Optimization of Rhodotorula glutinis for biocontrolling of Strawberry Blight. Haggag Wafaa M1, Abd-El-Kreem F1, SM Singer2, and Mohamed DEH Aly1. 1Plant Pathology Dept. National Research Centre, Dokki, Egypt. 2Vegetable Research Dept., National Research Centre, Dokki, Egypt. ABSTRACT Biocontrol agents represent an alternative or supplement to chemicals for disease control. Rhodotorula glutinis was evaluated for its activity in reducing blight and gray mould diseases of strawberry caused by Botrytis cinerea and Phomopsis obscurans in vitro and in vivo. Optimization of biomass and metabolite production were determined using some cultural conditions like different incubation time in hours, pH, carbon sources , concentrations and nitrogen source. Yeast was produced hydrolysis enzymes and siderophores (Rhodotorulic acid ) when grown in a medium with excess fructose as carbon source and soybean as nitrogen source . The stabilization technique was have evaluated using liquid formulations . Yeast showed high preserve viability of when stored at 4oC for six months. Preharvest application of liquid formulation of certain microbial isolate provided an effective control of leaves blight as well as fruit gray mould disease pre and protect fruit during storage than the tested fungicide at the recommended levels. Keywords: Antifungal , Botrytis cinerea , Phomopsis obscurans , Optimization , Rhodotorula glutinis , strawberry blight and yeast *Corresponding author Page No. 599 5(6) RJPBCS 2014 November - December ISSN: 0975-8585 INTRODUCTION Strawberry (Fragaria ananassa L.) production in Egypt is very minor compared to the principal fruits. Leaf blight, caused by the fungus Phomopsis obscurans, is an important summertime disease because so few varieties have adequate resistance to it. (Haggag, Wafaa, 2009). Grey mould diseases caused by Botrytis cinerea are major flower, fruit, and foliar in Egypt (Haggag, Wafaa, 2008) pathogens of field-grown strawberries and can affect almost every variety in Egypt. Postharvest losses of fruit and vegetables can reach very high values, representing more than 25% of the total production in industrialized countries and more than 50% in developing countries . Exploring biological fungicides from microbial metabolites is an important research direction of fungicides and has been received more attention (Haggag, Wafaa, 2013). Yeasts of the genus Rhodotorula (particularly Rhodotorula glutinis) have been reported as good biocontrol agent against Penicillium expansum, in apples and pears (and against Botrytis cinerea in strawberry (Helbig 2001) when preharvest and/or postharvest applications are made. The production of hydrolysis enzymes, rhodotorulic acid, a siderophore, by the yeast Rhodotorula glutinis contributes greatly to biocontrol of blue mold on apples and the addition of some nutrients, e.g. proline, and manipulating pH stimulated production of the siderophore (Calvente et al., 2001 ) and carotenoids ( Saenge et al., 2011). The important characteristics of yeast cell wall are referred to as mannans, mannan conjugates, high water solubility; broadspectrum of biological activity, low toxicity, stability and anti-mutagenic effects via different modes of action (Dae et al., 2001 and Haggag, Wafaa, 2013). Beta-carotene is an excellent antioxidant (Bhosale, and Gadre, 2001). Enhancing biocontrol using these manipulations may be possible if they do not benefit the growth of the pathogen and outweigh the benefits of higher siderophore production by the antagonist. In order to obtain semi-commercial amounts of the microbial agent, it is necessary to scale-up the production process, at least to the level of pilot plant, using a low cost culture medium. To our knowledge, there are no papers that have addressed the scale-up aspects of any Rhodotorula fermentation process. The levels of fermentation are related to fermentation time, ventilation, temperature, initial pH etc. Biocontrol strain fermentation is greatly influenced by the combination of media components and culture conditions in laboratory or industrial fermentation. Fermentation time is a very important factor, which affect the yield and quality of metabolites. The microbial agent should be formulated as a product having long storage stability, of at least 6 months (Janisiewicz and Korsten 2002). In this study, we tried to optimize fermentation condition of yeast Rhodotorula glutinis and determine the optimal combination of the medium composition , culture conditions optimization and fermentation , formulation and shelf life to improve the antifungal productivity, enhance the control effect of blight diseases caused by Botrytis cinerea and Phomopsis obscurans under field and storage conditions in two harvest seasons. MATERIALS AND METHODS Culture Conditions and Medium Yeast Rhodotorula glutinis was isolated from Strawberry plants and stored in the laboratory (Haggag Wafaa et al. 2005) . Isolate was grown on YPD medium containing 2% (w/v) glucose, 2% (w/v) peptone and 1% (w/v) yeast extract. B. cinerea and P. obscurans were isolated from diseased Strawberry grown in Boheria governorate. Botrytis cinerea and Phomopsis on PDA. They were stored at 4°C until required. These isolates were maintained at –20°C as spore suspensions in 20% glycerol and were activated in potato dextrose agar for 7 days. In vitro antagonist assays Petri plates (90 mm diameter), preinoculated 48 h before – to compensate the velocity growth differences – with 1 cm2 plug of a fresh culture of B. cinerea and P. obscurans (grown on PDA), were inoculated with a yeast suspension containing 1 · 108 CFU ml)1. After the yeast was inoculated, the plates were incubated for 48–72 h at 29 ± 1_C. In vitro antagonistic assays were conducted in two manners: (a) The fungal pathogen and the antagonistic yeast were inoculated at the opposite extremes of the 90-mm diameter Petri plate with PDA medium. (b) The yeast was plated in order to get a circumference growth and the fungus was inoculated in the centre of the plate. The control plate was only inoculated with the fungal pathogen either in the centre or at the extreme of the Petri plate. After incubation for 24–72 h, plates were evaluated. Page No. 600 5(6) RJPBCS 2014 November - December ISSN: 0975-8585 Antifungal Activity Assay Antifungal activity was measured by assaying the mycelium growth inhibition rate of B. cinerea and P. obscurans. Briefly, 1mL sterile fermentation filtrate mixed with 9 mL PDA was replaced in a dish or without sterile fermentation filtrate as control. After the medium solidifying we inoculated B. cinerea and P. obscurans mycelium discs which as incubated for 3d on the dish. The dishes were incubated in the dark at 28°C in incubator for 72 h . The percentage of inhibition was calculated from the following equation: Inhibition (%) = [(growth diameter in the control sample – growth diameter in the sample with treated yeast) × 100]/growth diameter in the control sample. The experiment was repeated twice in triplicates. Measurements of R. glutinis cell growth protein and biopolymers production Cell growth, protein and biopolymers produce by R. glutinis were measured in culture filtrates after four and seven days of incubation. Cells growth were monitored by measuring the difference in protein contents between culture broth and culture supernatant. Cells growth were determined by measuring the optical density at 610 nm. Protein content in the supernatant was determined at 595 nm by the method of Bradford (1976) using bovine serum albumin as a standard. Enzymes activities Activities of hydrolysis enzymes were assessed in culture filtrates of the tested yeast isolates over 7 days using a Labsystems Uniskan II microtiter plate spectrophotometer. Exo-glucanase activity was detonated using glucose oxidase-O-dianisidine reaction (Sigma Chemicals, glucose determination kit 510-A) which specifically measures glucose produced from laminarin hydrolysis. ß-1,3-Glucanase activity was assayed by incubating 0.2 ml of culture filtrate in 50 mM potassium acetate buffer (pH 5.5) with 50 µl of enzyme solution appropriately diluted in the same buffer. Reaction mixtures were incubated at 37°C for 30 min and were stopped by boiling for 5 min. One unit of ß-1,3-glucanase activity was defined as the amount of enzyme that releases 1 µmol of reducing sugar equivalents (expressed as glucose) per min under the standard assay conditions. The activity of exo-chitinase, was measured as the release of N-acetylglucoseamine from chitin and one unit (U) of enzyme activity was defined as the amount of enzyme that release 1 µmol of reducing groups/ min/ ml of the filtrate (Bradford, 1976). Siderophores assay The amount of siderophores (Rhodotorulic acid ) excreted into the culture medium was determined by spectrophotometer. Concentration was calculated using absorption maximum and the molar absorption coefficient (max; 400 nm) according to the method of ( Meyer and Abdallah, 1978). Batch Fermentation Incubation period The optimization of composition of incubation period, and cultural conditions was carried out based on stepwise modification of the governing parameters for antifungal production. The cultures were transferred to seed broth (200 mL of Medium) contained in a 500 mL Erlenmeyer flask and incubated at 30°C on a rotary shaker (175 rpm) for 6-8 hours. A 500 mL Erlenmeyer flask containing 200 mL of the same seed medium was incubated as specified above.

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