Studies on soil fungistasis: Effect of certain ... - DSpace@NEHU

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356 R. R, MISHRA AND K. K. PANDEY gation or by long air drying and that it is lessened when microbial activities are reduced and can be restored in sterilized soil inoculated with natural soil, specific organisms and/or plant materials s 9 The phenomenon of fungistasis, however, is not yet clearly understood in relation to different physical and biological factors like different degree of sterilization, incubation period, spore age, addition of fungal species and their metabolites to the soil. In this paper an effort has been made to investigate the role of the soil parameters on the soil fungistasis of two cultivated fields. MATERIALS AND METHODS Soil samples for the study of soil fungistasis were collected from two cultivated fields cropped with Ca]anus ca]an and Lathyrus sativus. The soil samples collected at different stages of the plants growth were air dried, finely powdered and then used for the experiments detailed below. Soil samples collected were kept separately in an electric oven at 40, 50, 60, 70, 80, 100 and 120~ for 6 hrs and the sterilization of soil samples was done in autoclave for different time intervals i.e. 5, 10, 15 and 20 mrs at 15 lb/sq, inch pressure. The soil samples thus treated were tested for fungistasis by method of Jackson 5. Mycoflora of the above soil samples was also estimated by dilution plate method using Martin's medium. In another set of the experiment the soil samples collected from the fields of the two crops after powdering and sieving were packed in the petriplates. The plates were incubated at 25 :t: 1 ~ for 1, 4, 7 and 15 days and thereafter fungistasis was assessed by a method suggested by J ackson5. Throughout the course of study the moisture status of the soil samples was maintained at 25 percent, by adding sterilized distilled water. This part of the experiment consisted of the evaluation of fungistasis in relation to spore age The spores of Rhizopus nigricans, Aspergillus flavus, .Penicillium oxalicum, Verticillium albo-atrum, Curvularia lunata, Helminthosporium sativum and Fusarium oxysporum harvested from 7, 10, 15, 20, 25, 30, 45, 60 and 90 days-old culture medium (PDA) were used for the study of fungistasis in relation to spore age. The test fungi were maintained in triplicate for each type of spore age. After the desired age the spores were removed and spore suspension was prepared in sterilized double distilled water, centrifuged at 5000 rpm, diluted with sterilized double distilled water so that each microscopic field contained about 50 spores. After preincubation (6-8 h) one drop of the above suspension was pipetted out separately on agar blocks with a sterilized pipette. Inoculated plates were again incubated at 25 4- 2~ for 24 h and thereafter the agar blocks were gently removed from the surface of the soil. The surface of the block which was in contact
STUDIES ON SOIL FUNGISTASIS 357 with the so il was then sliced off with the help of cover glass and kept on slide. All the blocks removed at a time were killed and stained with lactophenol cotton blue and were examined under microscope for the germination of fungal spores. The percentage inhibition was calculated on the basis of 1000 spore number counted. In this part of the investigation the effect of soil amendment with seven days old cultures of Rhizopus nigricans, Mucor hiemalis, Aspergillus niger, A. [lavus, A. sydowi, A. terreus, Penicillium oxalicum, Curvularia lunata, A crophialophora ]usispora, A lternaria lumicola, Helminthospor~um sativum and Fusarium oxysporum on fugistasis was studied. Ten circular agar blocks of uniform size (1 cm) of the above fungi grown on PDA medium were mixed separately to 50 g of sterilized soil. The plates were then incubated at 25:1: I~ for 15 days and thereafter, fungistasis was determined. For an other set of this experiments the fungi grown similarly were mixed together in different combinations, viz A. niger + A. ]lavus, A. /lavus + A. tenuis, A. niger -r H. sativum, A. tenuis + F. oxysporum and A. tenuis + H. sativum. In this case five blocks of each constituent measuring 1 cm in diameter were mixed and incubated as described above and the fungistasis was assessed. The fungistasis of sterilized soil was also determined for reference. The effect of culture filtrate of M. hiemalis, A. niger, A. /lavus, A. sydowi, P. oxalicum, A. tenuis, H. sativum and F. oxysporum on the fungistasis was studied in this experiment. The fungi were grown separately in 250 ml flasks containing 100 ml sterilized liquid Czapek's medium in an incubator (25 1 ~ for 15 days, The flasks were intermittently shaken during the incubation period and thereafter the medium was filtered through Whatman filter paper no. 1 and finally through millipore filter (0.22 ~). The clear solution thus obtained was designated as culture filtrate. The culture filtrate of above fungi was mixed with sterilized soil at the rate of 1 ml per 10 g of soil and the fungistasis was assessed. In all the experiments the fungistasis was assessed by the method suggested by J a c ks o n 5 and the test organisms used were Rhizopus n,gricans, Aspergillus [lavus, Penicillium oxalicum, Verticillium albo~atrum, Curvularia lunata, Helminthosporium sativum and Fusarium oxysporum. The fungistasis has been expressed in terms of percentage inhibition which has been calculated on the basis of 1000 spore number counted. RESULTS E/[ect o/sterilization on/ungistasis (i) Oven sterilization. A perusal of the table 1 indicates that as the temperature increases the fungistatic value decreased. The fungistasis was very much reduced at 80~ At 100~ and above the fungistasis was lost. The soil samples of L. sativus field exhibited higher fungistatic value than the samples from C. caian field. The organisms tested
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