Biological control of green and blue mould disease of citrus fruit by yeast

Transcription

1 Indian Phytopath, 49 (4) : (1996) Biological control of green and blue mould disease of citrus fruit by yeast N.K. MEHROTRA, NEETA SHARMA, RATNA GHOSH (NAYEK) and MADHULIKA NIGAM Department of Botany, University of Lucknow, Lucknow ABSTRACT: The yeast antagonist Debaryomyces hansenii was effective in reducing incidence of Penicillium digitatum and P. italicum decay of orange fruits. A water suspension of the yeast cells applied to wounds on tile surface of the fruit prior to inoculation with spore suspensions of pathogens reduced disease by 80-90%. The efficacy of the antagonist in reduction of green and blue mould was affected by the concentration of both the yeast cells and spore suspension. No reduction of disease was exhibited by an autoclaved preparation of yeast cells, or by a yeast culture filtrate. Debaryomyces hansenii did not inhibit the growth of the pathogens on PDA. The antagonist grew readily on injured citrus fruit leachate and multiplied rapidly on wound site. Keywords : Penicillium rot, citrus, biological control, yeast Green and blue mould caused by Penicillium digitatum Sacco and P. italicum Weh, respectively, account for most major post- harvest losses of citrus fruit (Bancroft et al., 1984). Synthetic chemical fungicides, often in combinaiton with physical means, are used traditionally to control the e diseases. It has been observed that fungicide efficacy is frequently decreased by the development of resistant strains of the pathogens. In addition, public awareness and concern regarding pesticide residues in our food has emphasised the need to find alternative means for controlling post- harvest diseases of citrus. Numerous laboratory studies revealed tl1e potential of antagonist against post-harvest diseases (Pusey et al., 1986; Wilson and Pusey, 1985). Bacillus subtilis, as a potential microbial biocontrol agent of post-harvest disease of citrus, was reported by Gutter and Littauer (1953). The efficacy of Received for publication July 5, 1996 this antagonist bacterial strain was due to the release of antibiotics (Singh and Deverall, 1984). The present study was undertaken to find new biological control antagonists of citrus pathogens, particularly those that did not produce antibiotic substance' as part of their mode of action. MATERIALS Isolation AND METHODS of antagonist Potential antagonists were isolated from the surface of healthy citrus fruit by placing each fruit in 500 ml beaker containing 200 ml sterile distilled water. Each beaker was placed on a rotary shaker at 100 rpm for 10 minutes. Wash water (0.1 ml) was then spread on a NYDA medium plates and allowed to incubate at 25 C for 24 hours. Several bacterial and yeast colonies developed. Selected yeast colonies were then streaked 3 times across NYDA plates in order to obtain single cell colony. Single cell colonies were isolated and maintained in pure cultures and stored in refrigerator.

2 [Vol. 49(4) 1996] Screening of antagonist Cultures of individual isolates to be tested were grown on NYDA medium for 3 days. A loop of 3 days old culture was then transferred to flasks containing 50 ml NYDB and incubated at room temperature on a rotary shaker for 24 hours at 200 rpm. After 24 hours, one rnl of each seed flask was then transferred into another flask containing 50 ml NYDB and incubated for 48 hours before testing them on fruits. Fresh healthy fruits were surface sterilized by 95% ethanol and placed on a moist blotting paper in trays. Two conical wounds (3 mm deep and 3 mm wide) were cut on the fruit peel with the help ofa dissecting sterilized needle. 50 ul of the antagonist - NYDB mixture, 10 9 cells nu- I, were then applied to each wound and the droplets were allowedto dry for 1-2 hours at room temperature. In the control set, only sterile broth was applied. The fruits were then inoculated by placing 20 III of a pathogen spore suspension, 10 4 spores ml'. After inoculation, the trays were covered with polythene sheets for moisture retention and incubated at 25 C for four days. The number of infected wounds were counted and wound sites were observed for upto 14 days. Each treatment in each experiment consisted of 3 replicates. For evaluating the effect of water soluble nutrients present in citrus fruit on the efficacy of D. hansenii in the inhibition of Penicillium rots, fresh citrus fruits were sterilized, homogenized and then Indian Phytopathology 351 centrifuged at 9000 rpm for 10 minutes. The supernatent was diluted with water and filter-sterilized for the preparation of pathogen spore suspension used for inoculation. Population changes of Debaryomyces on healthy and inoculated fruit surfaces were evaluated by cutting the inoculated sites (5 x 5 em) and vigorously shaking the tissue in 10 ml of sterile water for I hour. After appropriate dilutions, 10 ml of washing liquid were plated in triplicates on NYDA and the number of colonies counted after 48 hours of incubation. The interaction between antagonist and pathogens was tested by placing 4 mm agar discs of 7 days old culture of the pathogen in a petridish containing 15 ml of NYDB with and without antagonist cells. The effect of the antagonist on the growth of the pathogens in the synthetic medium was evaluated by the determining the dry weight of the cultures after a week of incubation at 25 C (Wishiewski et al., 1988). The effect of yeast antagonist on the growth of the pathogen was compared with that of a known antibiotic producing bacterial antagonist Bacillus subtilis. RESULTS Results of extensive screening trials indicated that Debaryomyces hansenii effectively reduced the infeciton of the two major post-harvest pathogens of citrus fruits (Table 1). Table 1. Inhibition of Penicillium rots of citrus by Debaryomyces hansenii Pathogen Penicillium digitatum Penicillium italicum Db = Debaryomyces. Antagonist Db Control Db Control Incubation time (days) (per cent infection)

3 352 indian Phytopathology Table 2. Inhibition of Penicillium rots of citrus by antagonistic Debaryomyces as affected by pretreatments of the antagonistic cells Pretreatment Per cent infection P. digitatum P. italicum None Autoclaved Culture Sterile medium Water control Table 3. Growth rate in culture of different citrus postharvest pathogens in the presence of the antagonist Debaryomyces hansenii Treatment Dry weight of mycelium (mg)' P. digitatum P. italicum Control 58.3 a 48.9 a D. hansenii 17.4 b 18.8 b Control used v 0.7 c 1.4 c _Replenished" 59.4 a 51.2 a Enriched W 8.4 d 49.4 a D. hansenii 69.2 d 47.9 a z-values in the same column (same fungus) followed by different letters are significantly different (P=O.OS)according to Duncan's multiple range test. v-d. hansenii was cultured on the synthetic medium for 48 hours prior to the use of the medium. x-the used medium was replenished with original nutrients. w- The medium was enriched with yeast extract and nutrient broth. Efficacy of the antagonist was affected by the yeast concentration in the wound as well as by the number of spores of the pathogen used for inoculation (Fig. 1). At a high spore concentration (10 6 spores ml') of pathogen, low concentrations of the antagonist did not reduce the disease while [Vol. 49(4) 1996] the highest antagonist concentration used (10 9 cells ml') reduced the per cent infection. The autoc1aved cell suspension and the culture filtrate failed to check the infection (Table 2). The yeast antagonist multiplied rapidly at the wound sites of injured citrus fruit (Fig. 2). The presence of yeast in a synthetic medium inhibited mycelial growth of the pathogens (Table 3). However, the normal mycelial growth of the pathogens was restored when the used medium was sterilized and replenished with nutrients before inoculation. Agar discs of Debaryomyces hansenii failed to inhibit the growth of any of the two pathogens on NYDA while a similar disc of B. subti/is inhibited the growth of the pathogens as was evident by the formation of an inhibition zone around the disc. DISCUSSION Debaryomyces hansenii inhibited two major post-harvest wound pathogens of orange fruit under conditions that favour disease development (Table 1). While the efficacy of the yeast antagonist depended on the relationship between the number of antagonist cells and concentration of the pathogen spores (Fig. 1), the rapid.., " 0 ~ , so..~ ~ loa 10' --ill-,-- P.digftatum -Po italicum ' \06 \0' Ie! 10" 10'0 Antagonislcellslml Fig. 1. Relationship between two citrus rotting nmgi (Penicillium digitatum and Penicillium italicum) and the development of infection on citrus treated with antagonist Debaryomyces hansenii.

4 [Vol. 49(4) 1996] multiplicaiton of yeast cells at wound site (Fig. 2) andthe fact that a small number of spores usually sufficeto cause decay, suggest that the antagonist may retain its efficacy also under natural condition. D. hansenii lost its antagonistic activity when the cells were killed, and also the culture filtrate didnot show any activity (Table 2). No inhibitory activitywas observed aga inst the fungal pathogen onpda plates. Under similar condition, B. subtilis, anantagonist formed clear mycelial inhibition zone around its colony which was due to secretion of antibioticmaterial (Gueldner et al., 1988). Thus, themode of action of ": is yeast in antagonising the citrusfruit pathogens IS not through the production of antibiotic. Several lines of evidence suggest that competitionfor nutrients at the wound site could be the naininechanism by which D. hansenii inhibits P. ligitatum and P. italicum, e.g. (a) the antagonism could.be partially reversed by the addition of nutrientsto the wounds during inoculation. (b) the culturingof antagonist cell with a pathogen on a syntheticmedium resulted in marked reduction in thethe growth rate of pathogen only under limited nutritionalcondition (Table 3). The growth of D. hansenii in the medium did not result in any residualinhibitory effect on the pathogen which could notbe restored by mere addition of nutrients to the Indian Phytopathology 353 used medium. These data, when considered together with observation of the lack of inhibition on the nutrient rich PDA medium and the rapid growth of the antagonist at the wound site strongly suggest that competition for nutrients is one major mode of action of D. hansenii in the inhibition of citrus pathogens. Competition for nutrients as the mode of action for post-harvest diseases of fruit has been suggested by Wisnewski et al. (1988). Thus, with the lack of evidence of antibiotic production. or any direct interaction between the yeast and pathogens, it 'antagonizes and the rapid depletion of nutrients at the wound site, resulting in disease inhibition suggest that competition for nutrients is likely the major mode of action by which the yeast antagonizes artificially inoculated pathogens on citrus fruits. ACKNOWLEDGEMENT Financial assistance through the DBT research project is gratefully acknowledged. REFERENCES Bancroft, M.N., Gardner, P.D., Eckert, J.W. and Baritelle, J.L. (1984). Comparison of decay control strategies in California lemon packing houses. Plant Dis., 68: 24. Blakeman, J.P. and Fokkema, N.J. (1982). Potential for biological control of plant diseases on the phyi\oplane. Ann. Rev. Phytopathology, 2.0: Chalutz, E. and Wilson, c.i, (1990). Post-harvest biocontrol of green and blue mold and sour rot of citrus fruit by Debaryomyces hansenii. Plant Dis., 74: , 10' b Incubation flj.e (h) " fig. 2. Growth of Debaryomyces hansenii on citrus fruit leachate. Elad, E. and Chet, I. (1987). Possible role of competition for nutrients in biocontrol of Pythium damping-off by bacteria. Phytopathology, 77: Gueldner, R.C., Reilly, c.c., Pusey, L.P., Costello, L.E., Arrendale, R.F., Cox, R.H., Hemmelsbach, D.S., Crumley, F.G. and Cutler, H.G. (1988). Isolation and identification of iturins as antifungal peptides in biological control of peach brown rot

5 Publ 354 Indian Phytopathology with Bacillus subtilis. J. Agr. Food Chem., 36: Gulter, Y. and Littauer, F. (1953). Antagonistic action of Bacilius subtilis against citrus fruit pathogens. Bull. Res. Counc. /sr., 3: Pusey, P.L. and Wilson, c.l. (1986). Compatibility of Bacillus subtilis for post-harvest control of peach brown rot with commercial fruit waxes, dichloran, and cold-storage conditions. Plant Dis., 70: 587. Singh, V. and Deverall, B.1. (1984). Bacillus subtilis as a control agent against fungal pathogens of citrus fruit. Trans. Br. mycol. Soc., [Vol. 49(4) 1996] Wilson, C.L., Franklin, J.D. and Pusey, P. Lawrence (1987). Biological control of Rhizopus rot of peach with Enterobacter cloacae. Phytopathology, 77: Wilson, CiL. and Pusey, P.L. (1985). Potential for biological control of post-harvest plant diseases. Plant Dis., 69: Wisn,iewski, M., Wilfion, C.L., Chalutz, E. and Hershberger, W. (11988). Biological control of postharvest diseases of fruit: Inhibition of Botrytis rot on apple by an antagonistic yeast. Proc. Electron Microse Soc. Amer., 46: \. s o ni ar ad