Study of Antagonistic Yeasts Isolated from Some Natural Sources of West Bengal Swapan Kr

Home , Penicillium digitatum, Zygosaccharomyces

Study of antagonistic yeasts isolated from some natural sources of West Bengal Swapan Kr. Ghosh1, Tumpa Santra2 and Amit Chakravarty2 .1Mycopathology Lab., P.G. Department of Botany, Ramakrishna Mission Vivekananda Centenary College, Rahara, Kolkata 700118, India 2Department of Microbiology, Institute of Genetic Engineering, Badu. Kol-128, W.B., India. ABSTRACT

An intensive survey was conducted and 196 samples were collected from different zones of 12 districts of West Bengal. The collected samples were various fruits ( apple , green grapes . brown grapes , date fruits , jamun and fig ) ,juices ( date juice , toddy , cane juice ) and milk. From these samples 588 yeast colonies were isolated .These yeasts were identified by conventional morphological, microscopical and biochemical testing methods. These colonies comprise of 23 genera and 38 species of yeasts. Out of 38 isolated & identified species, 26 are ascomycetous while 12 are basidiomycetous yeasts .Maximum number of species were found in Candida (5) and followed by Pichia(4), Rhodotorula(3) and Saccharomyces (2).The percent of occurrence , Berger – Parker dominance index of each species were calculated. All yeast species isolated were screened by dual culture plate method for their antagonistic property against Penicillium digitatum, causal pathogen of Penicillium rot of Citrus . Out of 38 species of yeasts,15 species were antagonistic to Penicillium digitatum. Candida famata gave maximum percent of radial inhibition ( 70.24 PIRG)followed by Pichia membranifaciens (68.21PIRG) and Rhodotorula mucilaginosa (60.56 PIRG).Therefore , these natural sources of West Bengal are good habitats of various yeast species including antagonistic yeasts and the antagonistic yeasts can be applied as biological control agents against post harvest Penicillium rot disease of Citrus

Key Words : Yeast , Diversity, Antagonism , Penicillium digitatum , Citrus

INTRODUCTION

Yeasts are unicellular eukaryotic fungi which are 2007;Pimenta et al.2008;Kurtzmanet al. generally multiply quickly by budding or fission . 2012).Unfortunately yeast flora or diversity is highly Yeasts are generally distributed into two phyla neglected in India . :a)Ascomycota b)Basidiomycota and in mitosporic The main objectives of this study are to know the group-Deuteromycota. Dimorphic yeasts are also diversity of yeast flora in natural sources of West present which become filamentous under certain Bengal and to find out the antagonistic potential of environmental conditions. Ascomycetous are two yeast and their application as biocontrol agent types: Budding yeasts (Saccharomyces cerevisiae) against Penicillium fruit rot of orange. and fission yeasts (Schizosaccharomyces pombe).According to Kurtzman& Fell (1999), there MATERIALS AND METHODS are about 100 genera and 700 species. Study area: West Bengal is a state (88,750 km2), in Yeast applications are used in many ways -wine and the eastern region of India. It is bordered by the bakery industry, SCP production ,carotenoid countries of Nepal, Bhutan, and Bangladesh, and the production, recombinant vaccine production, in Indian states of Orissa, Jharkhand, Bihar, Sikkim, understanding of cell cycle and so on . Modern trend and Assam. This stare is divided in to two parts – is application of yeasts as biocontrol agents against North Bengal &South Bengal . The Study area is many post harvest diseases of many fruits and South Bengal. It is the gangatic plain .It includes the vegetables (Droby et al. 2003; Coelho et al. districts -24-Parganas(N) , 24 –Parganas (S), Nadia Agric. Biol. J. N. Am., 2013, 4(1): 33-40

,Howrah , Hooghly, Burdwan , Birbhum, Midnapore et.al(2000);Kurtzman et al. (2012) and Rose & (W), Midnapore (E), Bankura , Purulia and Harisson (1993). Murshidabad(Fig 1) .The main seasons are summer, Morphological & Microscopical investigation: The rainy season, a short autumn, and winter. colonies were observed and described on MA and MYGPA medium The isolates were also grown in MA &MYPGA broth for determination of their cultural

characteristics (pellicle ,sedimentation or ring formation).In certain cases, the isolates were grown on sterile slices of carrot for induction of ascospore formation . Biochemical investigations: For carbon and nitrogen assimilation ,the basal medium of Barnett,et al.(2000) was used . the results were determined after the3th, 7th, ,14th ,21th and 28th day . The ability of some carbohydrates for anaerobic assimilation (fermentation) was determined by using Durhan glass tubes after 3 weeks . The quantity of the tested carbohydrates was 2% . For Diazonium blue –B (DBB) test, a ten day old 0 Fig.1 Map of the West Bengal citing study districts culture on MYPGA was held at 55 for three hours and then flooded with ice –cold DBB reagent . The Study samples: The collected materials were reagent was prepared by dissolving diazonium blue various fruits ( Apple , green grapes . brown grapes , salt (Sigma) in cold 0.5M-tris –HCL buffer pH 7.0 at date fruits , jamun and fig ) , juices ( date juice , toddy 1mg /ml .The reagent was kept ice –cold and used , cane juice ) and milks from different traders of within few minutes of preparation . twelve districts] of West Bengal , India Other additional tests such as starch formation ,urea Isolation &Purification of yeasts:Fruit/juice/milk hydrolysis , cycloheximide (0.01% or 0.1%), were samples from different districts of West Bengal ,India performed. were collected in separate sterile biodegradable Isolation and purification of pathogen from polythene bags and brought in laboratory. Known diseased fruit: The rotten oranges(Fig.2) were collected weight of each sample was washed with known in sterilized polythene bags and carried to laboratory from volume of sterilized distilled water by shaking on a different markets . Five gram of rotten fruit was aseptically rotary shaker. The washings/fruit juices were 10 fold cut out and smashed and mixed in 50 ml sterile distilled serially diluted and plated on MA medium ( MA; malt water under Laminar air flow . The solution and diluents extract, 2g;agar2g ;Distilled water,100ml). The Petri were streaked on to PDA Petri dishes containing penicillin dishes were incubated at 25o C for 3 days .(Beech (1.6ug /ml )and incubated at 28 o C temperature for 3 days and Devanport, 1971;Ghosh,2011) .After three days , each colony of the fungus was transferred to PDA slant by inoculating needle and Single cell isolate of each type of yeasts was incubated at 280 C for one week and stored at 4oC in obtained by streaking loop full of cells on MA medium refrigerator ( Hee.2003; Dingra& Sinclair 1985 ). and transferring well isolated colonies to MA. The isolates were maintained on Malt-Yeast –Glucose- Peptone –Agar medium (Dry malt extract ,3g.;dry years ,3g.Peptone,5g.,D- glucose 10g, agar ,20g.

Distilled water, 1L) at 28oC with monthly subculturing. Identification of Yeasts: Identification of each isolate of yeast up to species level was carried on the basis of standard morphological, and physiological /biochemical tests presented by Barnett Fig.2 Disease symptom

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Identification of the pathogen:The pathogen was Where , R1=Radial growth of P. digitatum in control identified by its growth pattern and microscopical plate. characteristic (Fig.3&Fig 4)consulting with published R2= Radial growth of P. digitatum in dual Keys of Penicillium (Nagamani, et al., culture plate interacting with yeast. 2006;Domsch,et al . ,1980). PIRG=Per cent of Inhibition of Radial growth

of P.digitatum.

Each treatemt was carried out 5 replications and plates were arranged in randomized Block Design. Data analysis:The percent of occurrence of each species was determined by total number of yeast

colony divided by number of colony of each speciesand the result is multiplied by hundred. The Berger –Parker Dominance index (D) was caculated or measured ( Harrison, et al.2004)by the Fig.3 P. digitatum on PDA formula :D=n/N, where nindicated the No. of individuals in a species and N represented as total No. of individual.The Berger – Parker dominance was calculated in species level. The species with D>0.1 were considered as Dominant , the speices with lying between 0.1 &0.05 were called General and the species with D <0.05 wereconsidered as Rare . RESULTS AND DISCUSION The data presented in the Table 1 showed that from 196 samples of different natural sources, 588 yeast colonies were isolated. These yeasts were identified Fig.4 Penicillus of P.digitatum by conventional morphological, microscopical and biochemical testing methods. These colonies Screening of yeasts as antagonists against comprise of 23 genera and 38species of yeasts. Penicillium digitatum: Dual culture assay: Five Maximum number of species were found in Candida mm diameter of mycelial colony from the margin of (5) and followed by Pichia(4), Rhodotorula(3) and actively growing colony of P. digitatum was placed on Saccharomyces (2) .Out of 38 isolated & identified one end of Petri plate and that of yeast was species, 26 are ascomycetous while 12 are incubated simultaneously at opposite ends of a Petri basidiomycetous yeasts (Table1). The data of table 1 dish containing 25 ml of sterilized PDA medium . The indicated the distribution or habitats of isolated & plates containing the paired culture were incubated at 0 0 identified yeasts. Some yeasts were isolated from 28 ± 1 C for 9 days in a B.O.D. incubator .The five restricted fruits’ surface/fruit juice/milk and some PDA plates inoculating only with 5mm diameter of were isolated from maximum fruits’ surface & juices mycelial colony from the margin of actively growing 0 0 .The Candida famata , Pichia membranifaciens , colony of P. digitatum were incubated at 28 ± 1 C Rhodotorula mucilaginosa, Saccharomyces for 9 days .These were control. The degrees of cerevisiae and Debaryomyces hansenii were present antagonism were calculated by the formula: in maximum and same percent of occurrence i.e. 5.1.The relative occurrence of these five yeasts were R1- R2 ‘General’ where as other 33 species of yeasts were ‘Rare’ in occurrence by calculating the Berger – PIRG= ------x100 Parker dominance index. Filobasidium uniguttulatum and Lipomyces yarrowii were present in minimum i.e.

R1 at below 1.00 percent of occurrence(Table2). Phaffia rhodozyma has been isolated from grape fruit. This yeast has great important in biotechnology for its’ carotenoid (astaxanthin).Ghosh and

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Fig.5 Control (Left ), P. digitatum (P.d.)X R.mucilaginosa Fig.7P.digitatum X P. membranifaciens (P.m.)

Samaddar (1987) isolated different types of yeasts from different sources like palm juice, toddy, molasses, grape and cane juice. Saccharomyces cerevisiae, Schizosaccharomyces pombe, Saccharomyces kluveri, Rhodotorula sp, Candida sp were isolated and identified by them. Identification of yeasts up to species level was carried on the basis of standard morphological and physiological/ biochemical tests proposed for each group by Barnett et al; ( 2000) and Kartzman and Fell ( 1999). Isolation and taxonomic study of yeast strains from Bulgarian dairy products were reported by Savova (2002).They identified Debaryomyces hansenii, Candida famata, Rhodotorula muiilaginosa, Kluveromyces lactis, Kluveromyces maximus etc. Isolation and Fig.6 P. digitatum X C. famata identification of yeast flora from the soil was reported by Mushtaq et al.( 2004). Ghosh (2011) isolated and identified five genera and seven species of yeast from fruit surface of Syzygium cumini Table 1 Isolated, identified species of yeasts and their distribution in different natural sources

S.No. Yeast sps Phylum/ Sources/Samples Group(Stage) 1 Aureobasidium pollulens Ascomycota(Anamorph) Apple , green grapes . brown grapes , date fruits . 2 Bullera pyricola Basidiomycota(Anamorph) Green grapes , date fruits 3 Bensingtonia phyllada Basidiomycota(Anamorph) Apple , green grapes , brown grapes 4 Candida famata Ascomycota(Anamorph) Apple , green grapes . brown grapes , date fruits , jamun, toddy 5 C. rugosa Ascomycota(Anamorph) Apple, fig fruit , toddy 6 C. spherical Ascomycota(Anamorph) Toddy , cane juice 7 C krusei Ascomycota(Anamorph) Apple, fig , toddy 8 C. succiphila Ascomycota(Anamorph) Green grape 9 Cryptococcus albidus Basidiomycota(Anamorph) Apple 10 Cryptococcus victoriae Basidiomycota(Anamorph) Fig 11 Debaryomyces yamadae Ascomycota(Telomorph) Date fruits , jamun and fig 12 Debaryomyces hansenii Ascomycota(Telomorph) Brown grape, jamun and fig

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13 Filobasidium uniguttulatum Basidiomycota(Telomorph) Green grape 14 Galactomyces citri-auranth Ascomycota(Telomorph) Apple 15 Hensenula anomala Ascomycota(Telomorph) Date fruit& date juice 16 Henseniaspora uvarum Ascomycota(Telomorph) Apple 17 Kadamaea anthophila Ascomycota(Telomorph) Green grape 18 Kloeckera apiculata Ascomycota(Anamorph) Green grape & milk 19 Kluveromyces lactis Ascomycota(Telomorph) Milk 20 K. thermotolerans Ascomycota(Telomorph) Milk 21 Lipomyces yarrowii Ascomycota(Telomorph) Milk 22 Mycotorula rogosa Ascomycota(Anamorph) Apple ,brown grape 23 Phaffia rhodozyma Basidiomycota(Anamorph) Green grape 24 Pichia jadini Ascomycota(Telomorph) Green grape 25 P .lachancei Ascomycota(Telomorph) Jamun 26 P. euphorbiae Ascomycota(Telomorph) Date fruits , brown grapes 27 P .membranifaciens Ascomycota(Telomorph) Apple , green grape,milk 28 Rhodotorula mucilaginosa Basidiomycota(Anamorph) Toddy ,cane juice, jamun, green &brown grapes 29 R .toruloides Basidiomycota(Anamorph) Apple , green grapes, milk 30 R. minua Basidiomycota(Anamorph) Date fruit& fig 31 Saccharomyces cerevisiae Ascomycota(Telomorph) Date juice , toddy ,cane juice, green grape, apple 32 S. oviformis Ascomycota(Telomorph) Date juice , toddy ,apple 32 Saccharomycodes ludwigii Ascomycota (Telomorph) Date juice , toddy , cane juice , milk, grapes 34 Sporidobolus ruinensiae Basidiomycota (Telomorph) Green grape &fig 35 Trichosporon belgelli Basidiomycota (Anamorph) Apple 36 T. mucoides Basidiomycota (Anamorph) Apple 37 Zygosaccharomyces lentus Ascomycota (Telomorph) Green grapes . brown grape 38 Z. bailli Ascomycota(Telomorph) Green grape

All yeast species isolated were screened by dual lentus gave minimum inhibition (5.45 and 7.00 PIRG culture plate method for their antagonistic property respectively) .Potential use of yeasts as biocontrol against Penicillium digitatum, causal pathogen of agents of soil born fungal plant pathogens and as Penicillium rot of Citrus .The data presented in the plant growth promoters were recent investigated by table3 showed that out of 38 species of yeasts , 15 EL- Tarabily and Sivasiyhamparam ( 2006) .Wide species are antagonistic to Penicillium digitatum variety of yeasts have been used extensively for .Candida famata ( Fig. 6 ) gave maximum percent of biological control of post harvest diseases of fruits & radial inhibition (70.24 PIRG)followed by Pichia vegetables (Punja,1997).The yeast Torulapsis membranifaciens (68.21 PIRG)( Fig. 7 ), Rhodotorula candida (Candida famata) effectively controls mucilaginosa (60.56 PIRG)( Fig. 5),Debaryomyces Penicillium digitatum infection on Citrus fruits ( Arras hansensii (55.40 PIRG ), Saccharomyces cerevisiae ( et al, 1999). Candida saitoana &C. oleophila control 45.23PIRG) C. spherical (42.22PIRG ).(Table post harvest diseases of apple &Citrus fruits (El- 3).Kluveromyces lactis and Zygosaccharomyces Ghaouth et al. 2000).

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Table 2. Isolated and identified yeasts ,their number of colony and their percent of occurrence ,Berger – Parker dominance index and Relative dominance.

S.No. Yeast sps Number of Occurrence ( %) Berger- Parker Relative dominance colony dominance index(D) 1 Aureobasidium pollulens 7 1.1 0.0119 R 2 Bullera pyricola 9 1.5 0.0153 R 3 Bensingtonia phyllada 8 1.3 0.0136 R 4 Candida famata 30 5.1 0.0510 G 5 C. rugosa 15 2.5 0.0255 R 6 C. spherical 13 2.2 0.0221 R 7 C krusei 12 2.0 0.0204 R 8 C. succiphila 27 4.5 0.0459 R 9 Cryptococcus albidus 15 2.7 0.0255 R 10 Cryptococcus victoriae 15 2.5 0.0255 R 11 Debaryomyces yamadae 23 3.9 0.0391 R 12 Debaryomyces hansenii 31 5.2 0.0527 G 13 Filobasidium uniguttulatum 4 0.6 0.0068 R 14 Galactomyces citri-auranth 6 1.0 0.0102 R 15 Hensenula anomala 8 1.3 0.0136 R 16 Henseniaspora uvarum 11 1.8 0.0187 R 17 Kadamaea anthophila 14 2.3 0.0238 R 18 Kloeckera apiculata 15 2.5 0.0255 R 19 Kluveromyces lactis 14 2.3 0.0238 R 20 K. thermotolerans 19 3.2 0.0323 R 21 Lipomyces yarrowii 4 0.6 0.0068 R 22 Mycotorula rogosa 9 1.5 0.0153 R 23 Phaffia rhodozyma 7 1.1 0.0119 R 24 Pichia jadini 19 3.2 0.0323 R 25 P. lachancei 22 3.7 0.0374 R 26 P. euphorbiae 18 3.0 0.0306 R 27 P . membranifaciens 30 5.1 0.0510 G 28 Rhodotorula mucilaginosa 30 5.1 0.0510 G 29 R .toruloides 26 4.4 0.0442 R 30 R. minua 15 2.5 0.0255 R 31 Saccharomyces cerevisiae 32 5.4 0.0544 G 32 S. oviformis 21 3.5 0.0357 R 32 Saccharomycodes ludwigii 13 2.2 0.0221 R 34 Sporidobolus ruinensiae 11 1.8 0.0187 R 35 Trichosporon belgelli 7 1.1 0.0119 R 36 T. mucoides 8 1.3 0.0136 R 37 Zygosaccharomyces lentus 11 1.8 0.0187 R 38 Z. bailli 10 1.7 0.0170 R Total 588

The yeast elicited production of phytoalexins, P. expensum based on nutrition and space scopoletin and scoparine and did not produce toxic competition ( Coelho,2007).The expression of an substances against the pathogens Penicillium antifungal peptide ( cecropin- A based peptide) in digitatum and Botrytis cinerea. It suggested that the Saccharomyces cerevisiae, inhibited the germination yeast may reduce fungal infection by activating host's of Colletotrichum coccodes ( causal organism of defense mechanisms (Arras& Arru, 1999). Fifteen tomato fruit decay) was reported (Jones antagonistic yeasts showed inhibitory activity against &Prusky,2005 ) .Capdeville et al.(2007) worked on

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the interaction between C. magnus and C. CONCLUSION gloeosporioides on papaya fruits under electron Therefore , these various fruits ( Apple , green microscope and reported that the yeast colonized grapes . brown grapes , date fruits , Jamun and Fig ) wound surface of fruits much faster than the ,juices ( Date juices , Toddy , Cane juice )and milk of pathogen, over competing the latter for space and West Bengal are good habitats of various yeast nutrition .Moreover ,C magnus produced a flocculent species including antagonistic yeasts and the matrix , which affected hypal integrity of the antagonistic species can be applied as biological pathogen. Rhodotorula sp produces rhodotorulic control agents against post harvest Penicillium rot acid which inhibits the growth of the pathogen .In this disease of Citrus. experiments the mechanism of inhibition of growth of P. digitatum may be due to antifungal substances ACKNOWLEDGEMENT secreted by yeasts . The first author is very grateful to the Principal , This work including the diversity of yeast flora in R.K.M.V.C.College , Rahara, providing Lab. facilities different natural sources of West Bengal and and UGC , New Delhi for providing financial support. screening of antagonistic yeasts against P. digitatum REFERENCES is new in its kind in India. Arras, G, Dessi, R., Sanna, P. and Arru, S. (1999).Inhibitory Table 3.Screening of yeasts for their antagonistic activity of yeasts isolated from fruits against property. Penicillium digitatum. Acta.Horticulturae 48( 5):37-45.

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