"In-vitro" Evaluation of Antagonistic Potentials of Some Yeast Isolates Against Different Plant Pathogenic Fungi

In-vitro EVALUATION OF ANTAGONISTIC POTENTIALS OF SOME YEAST ISOLATES AGAINST DIFFERENT PLANT PATHOGENIC FUNGI

Arijit Das Prasad Singha 1 , Dharnendra Reang, Mayanglambam Ranjana Devi

Abstract:

An experiment was conducted to evaluate the antagonistic potentials of 38 yeast isolates against different post harvest fungal pathogens. Four different plant pathogenic fungi (Colletotrichum musae, Alternaria solani, Rhizoctonia solani, and Fusarium oxysporum f.sp.ciceri) were tested for their growth and behavior in presence of yeasts in dual culture plates using a modified PDA medium. The potential of antagonism of each yeast isolates were measured by a range of inhibition percentages against the test pathogen in dual plates and according to that measurement, the antagonistic potential was grouped in three categories as- low (<20% inhibition); medium (21%-50%); and high (>50%). According to the measurement high antagonistic potentials were found from Y4 and Y22 against Colletotrichum musae; Y7 against Rhizoctonia solani; Y49 against Alternaria solani but against Fusarium oxysporum f.sp.ciceri, no single yeast isolate was found to show more than 50% inhibition in dual culture plates.

Key words: dual culture technique, antagonistic yeasts, bio-control efficiency and plant pathogenic fungi.

Introduction:

Research on the control of plant diseases by phyllosphere applied biological control agents has produced a wealth of informations on a wide range of fungal and bacterial biocontrol agents and their applications for controlling diseases affecting leaves, flowers and fruits. Research into their mode of action and ecological adaptation has provided critical insights that have increased commercial utilization of phyllosphere applied biocontrol agents has proven to be challangeing because the leaf surface presents a relatively hostile environment for introduced microbes with a relative paucity of available nutrients (Beattie and Lindow,1999;Beattie,2002), wide water availability fluxes (Beattie and Lindow,1995), direct exoposure to ultraviolet radiation(Sundin,2002) and infrared radiation and competition with other phyllosphere colonist. Foliar applied biocontrol agents do not have the avoidance strategy of endophytic growth available to pathogens (Beattie and Lindow,1995) and therefore must primarily utilize a tolerance strategy whereby they colonise and survive on the leaf surface or in protected sites that may or may not be the same as the pathogen they are intended to control. Because of the variable effects of the biological and physical environment, the vast majority of phyllosphere applied biocontrol agents research has been focused on glasshouse or storage environment wherethe physical environment is more predictable and stable (Paulitz and Belanger,2000). During the past 20 years several biocontrol agents have been exploited and widely investigated against different postharvest fungal pathogens, like Aspergillus spp, Botrytis spp, Monilinia spp, Penicilium spp, Rhizopus spp. (Droby et al., 2002). Many of the early studies aimed at the study of the mode of action and evaluation of the efficacy of some potential biocontrol bacteria, such as, Brevibaccilus subtilis, producer of antibiotics (Pusey et al., 1986), however, the application of such bacteria on fruit did not prove to be commercially acceptable. Wilson and Wisniewski(1994) indicated the following characteristics of an ideal antagonist: genetic stability, efficacy at low concentrations and against a wide range of pathogens on various fruit products, simple nutritional requirements, survival in adverse environmental conditions, growth on cheap substrates in fermenters, lack of pathogenicity for the host plant and no production of metabolites potentially toxic to humans, resistance to the most frequently used pesticides and compatibility with other chemical and physical treatments. Yeasts seem to possess a good number of the above mentioned features and during the last few years, research has been made on the selection and study of yeasts (Chalutz and Droby, 1998).

Materials and methods:

Antagonistic potential of various yeast isolates(total 38) were tested through dual culture technique against the pathogens like- Colletotrichum musae, Rhizoctonia solani, Alternaria solani and Fusarium oxysporum f.sp.ciceri.

For this experiment, 6 mm diameter block of the pathogen from pre grown plates were inoculated on Yeast Potato Dextrose Agar medium supplemented with yeast extract at a distance of 2 cm from the periphery of the sterile plate (9 cm diameter). On the other side of the plate a streak of yeast isolate was made at the same distance as that of the pathogen. It should be noted that both the yeast and the antagonist were of the same age when inoculated. A similar plate but without the yeast isolate was also set up as a control for the experiment. The plates containing the paired cultures were then incubated at 28 ± 10C for about 6 days.

Interactions between the pathogen and the yeast isolates were noted when the pathogen had just touched the line where sterile distilled water was inoculated in place of the yeast isolate in the control plate. The pathogen growth was measured and from this observation the percent growth inhibition was calculated as {(Control- Treatment)/Control} ×100. Besides, observation was also made as to whether the yeast isolate was suppressive, competitive or inhibitory towards the test pathogen.

Results and Discussions:

Four different plant pathogenic fungi (Colletotrichum musae, Alternaria solani, Rhizoctonia solani, and Fusarium oxysporum f.sp.ciceri) were tested for their growth and behavior in presence of yeasts in dual culture plates using a modified PDA medium (with 5.0gm yeast extract/lit.).

1. Against Colletotrichum musae

Out of the 38 yeast isolates obtained from different phylloplanes as well as some local yeast formulations, date palm wines and decomposing fruits, five yeast isolates showed better inhibitory effects than rest of 33 isolates on C musae in dual culture plates six days after inoculation among which three yeasts were isolated from phylloplanes (Y22, Y26 and Y44) and two (Y2 and Y4) from fermenting solution (wines). Y4 and Y2 though isolated from wines obtained from plants from where the yeasts were grown as contaminant of sugar rich plant extract. Radial growth inhibition was maximum by Y22 to the extent of 71.01% over control followed by Y4 which showed an inhibition upto 53.17%. Y44, Y2 and Y26 showed inhibitory activity to the extent of 42%, 42% and 39.13% respectively after 6 days of inoculation but after that C musae grew on the other side of the yeast colonies. The results are presented in the Table-1.1.

Table 1.1: Growth and behavior of C musae against yeast isolates in pure culture:

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Dual culture plates of yeasts and C musae were maintained for few more days to allow the pathogen cover the whole plate and also allowed to sporulate so as to observe the sporulation behavior of the fungus. Full growth on the control plate by C musae i.e the pathogen grew to reach the other end of the plate from the point of inoculation, was observed seven days after inoculation giving a total radial growth of 7 cm with 6 cm radial growth having sufficient sporulation indicated by pink colored slimy exudates and confirmed by microscopy and remain same upto 10 days of inoculation. In majority of the cases of interaction between the yeast and C musae in dual culture plates; the anthracnose fungus grew slowly and sparsely near to the yeast colony as a streak but in some cases e.g. Y22, Y44, Y51, ML1 and a few others, after crossing the yeast colony from underneath, started to grow profusely and reached the margin of the Petri plates as in case of the control plates but with a much reduced zone of sporulation as compared to that of the control plate. The radial growth of C musae was totally restricted after 6 days in case of Y2, Y4, Y22, Y26, Y44, Y52 with no sporulation or very few mm of sporulating zone which was obtained in case of Y4 and Y52. Although in case of Y10 there was no radial growth inhibition (0%) of C musae upto 6 days but growth suppression was indicated by the ceasation of growth after 6 days. At 10 days after inoculation the zone of sporulation in most of the yeast isolates was almost equal to that of the control plate but in comparison (except Y22, Y44, Y51 and ML1), the sparse growth was much less from 6 days of inoculation due to rapid hyphal dissolution of the test pathogen.Microscopic observation of the inhibited C musea hyphae from inhibition zone revealed swelling and development of irregular septation as compared to that of the normal hypahe.

Primary screening for the selection of antagonistic organism against a particular pathogen is normally done by dual culture method using a medium that supports sufficient growth and activity of both the organisms. This practice has been widely adapted in the laboratory. During the present investigation it was found that the potato dextrose medium (PDA) was suitable enough for growth of the test fungus C musae but was not suitable enough for sufficient growth of the isolated yeasts. To select a suitable medium for both type of organisms a modified PDA medium was developed using 1% yeast extract into the medium. Yeast extract provided several undefined growth factors to the yeasts for profuse growth and this fact is equally applicable in other filamentous fungi also when tested along with the yeasts in dual culture plates.

The present study on the in vitro growth and sporulation inhibition by various yeast isolated from phyllosphere indicate the possibility of using or further developing yeasts as bio-control agent against postharvest disease of banana pathogen C. musae and also against other fungal pathogen.

2. Against Rhizoctonia solani

The test pathogen Rhizoctonia solani is an important sclerotial soil borne fungus causing various diseases of plants like- Sheath blight of rice. After incubation of dual culture plates at 28 ± 10C, observations were made at 6 days and 10 days after inoculation respectively. The results were shown in the Table 1.2. Maximum inhibition was shown by Y7 isolate (from palm wine) both at 6 days and 10 days after inoculation in dual culture plate and the inhibition percentage was 55.22% (Plate6) followed by Y33(46.28%) and Y22(43.28%) at 6 days after inoculation. The isolatesY3, Y9, ML2 and Y44 also showed moderate inhibition at 6 days after inoculation but inhibitory effect did not persist upto 10 days of inoculation.

Table 1.2: Growth and behavior of R solani against yeast isolates in pure culture:

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