Carrier for microbial inoculants

Transcription

1 Carrier for microbial inoculants The goal in developing sustainable agriculture is to find models which strike an acceptable balance between production and benefit and ecological conservation by the reduced use of chemicals, pesticides or other harmful materials. The use of biological materials such as microbial fertilizers and compost will be of basic importance. Biofertilizers are the products containing agriculturally important beneficial viable microorganisms, which have an ability to mobilize nutritionally important elements from non-usable form to usable form through biological processes. They not only provide nutrients at a much slower rate but also add the much-required biomass to save the soil deterioration. In large sense, term may include all organic resources (manures) for plant growth which are rendered in an available form for plant absorption through microorganisms or microorganisms-plant association or interaction. Thus biofertilizers can make a significant contribution towards the development of strategies for productivity improvement, which do not lead to an exponential rise in the consumption of non-renewable forms of energy. Essentially Such products can be more appropriately called "Microbial inoculants". The first and foremost example of this kind is the Rhizobium legume inoculants which was first marketed in U.S.A. during the early part of this century (Fred et ai., 1932). "Azotobacterin" and "Phosphobacterin" containing cells of Azotobacter chroococcum and Bacillus megaterium var. phosphaticum, respectively, were 143

2 used in the erstwhile U.S.S.R. and east European countries for bacterizations P J!..tr-.., of crops with a view to exploit nitrogen of Azotobacter and/ Bacillus. / Phosphorus has not nature channel for the return of large annual net losses. Hence, the supply is inevitably shrinking and the deposits are limited. Therefore, the release of insoluble phosphates in soils and fixed phosphorus in the clay minerals by microorganisms assumes significance. It has been realized that phosphate solubilizing microorganisms (PSMs) could be effective as biofertilizer in enhancing crop yield in phosphate deficient SOil~( Subba Rao, 1982; GoldStain, 1986; Kucey et ai., 1989). Current interest in research on PSMs is aimed towards enhancing the use of rock phosphate(rp) for direct application in soil. The prime objective in inoculating seeds is to provide a sufficient number of highly effective organisms in the rhizospheres. Longevity of the organism is essential for success. The effective life of an inoculants is therefore a primary concern of the inoculants manufacturer, the seedsman and the farmer. Several investigators have attempted to test the value of seed inoculated with PSM on growth and yield of plants. A significant increase in the grain yield of wheat was obtained under field conditions when RP was applied to soil and seeds were inoculated with Pseudomonas striata (Gaur et ai., 1980). Gaur (1985) also observed significant increase in the yield of chick pea due to seed inoculation by Aspergillus awamori over control i.e. RP alone. Rasal et. al.(1988) noted that the addition of A. awamori with RP increased the availability of phosphorus in soil, thereby improving significantly the nodulation 144

3 of chick pea. Similar results are also reported by Shinde and patilh 985) and Wani et al. (1978). Phosphorus dissolving bacteria and fungi P. striata, Bacillus polymyxa and A. awamori have been developed as inoculants. The preparation is known as 'IARI Microphos culture'. These cultures were tested in multilocational field trials on wheat, paddy, gram, soyabean, lentil and potato which resulted in increase in the yield of the crops (Gaur,1983). The performance of biofertizer is highly unpredictable due to their biological nature and susceptibility to biotic and abiotic stresses. There is a need to develop more effective, competitive and stress tolerant strains to increase nutrient supply from biofertilizers. Apart from these, for production of any microbial inoculant a suitable and cheap carrier material is required. The selflife of carrier-based inoculant and the cheapness of material are the important criteria for the selection of a suitable carrier. The qualities which a good carrier should possess are (i) (ii) (iii) (iv) (v) non-toxic to microorganisms. good absorption qualities easily pulverized and sterilized good adhesive to seeds, and readily available at moderate cost. The survival proliferation of the selected microorganisms was studied in different carriers such as peat, soil, farm yard manure (FYM), cow dung cake 145

4 (CDC) powder, wood charcoal-soil mixture by Gaur and Gaind (1984) and Kundu and Gaur (1981). Sterilized peat powder proved to be the best carrier for survival and multiplication of the phosphate solubilizing bacteria and fungi. Peat is not readily and conveniently available in India. In the present study, soil FYM, CDC, lignite and betonite were examined for their suitability as carrier for survival and proliferation of Geotrichum sp. and Issatchenkia orientalis in preparation of inoculants. Materials and Methods Organisms : Geotrichum sp. and I. orientalis maintained on Pikovskaya's agar slants were used. Culture Media: 1. Pikovskaya's broth 2. Glucose yeast extract (GYE) broth The medium was used to prepare yeast inoculum The compositions of Pikovskaya's broth and GYE are mentioned in chapter 2. All media were sterilized by autoclaving for 15 min at 15 Ibs p.s.i. (121' C). Carriers: Soil, FYM, CDC, Lignite, Bentonite. Reagents: M Sodium bicarbonate % 2, 4-dinitrophenol indicator 3. 2 N Sulfuric acid 146

5 4. 4 N Sodium carbonate 5. Standard potassium dihydrogen phosphate solution. 6. Sulphomolybdic acid solution 7. Chlorostannous acid solution The preparation of all these reagents is given in chapter no. 2 Physical and chemical properties of carriers: 1. ph : 50.0 gm of each carrier was added separately to 100 ml distilled water and shaken mechanically for 1 h. The ph of the resulted solution was read using 'Eli co' ph meter. 2. Water holding capacity: The water holding capacity of the carriers was measured by passing 100 ml distilled water through weighed amount of carrier. From the amount of water obtained after passage, the water holding capacity was calculated using standard formula. 3. Available phosphorus : Available phosphorus of the carriers was determined by method of Jackson (1973) as described in chapter Organic matter: Organic matter in different carriers was determined by method of Walkley and Black (1934) as described in chapter 2. Preparation of an inoculant: For the preparation of carrier based inoculant, cell suspensions of 24h old Geotrichum sp. and I. orienta/is were inoculated, separately, in 500 ml sterilized Pikovskaya's broth in a fiask of 1 litre capacity. The inoculated fiasks 147

6 were incubated at 28 ± 0.2 C for 48h in static condition. The carriers were oven dried at 80 C for 24 h. Ground and passed through a 60mes sieve. 100 gm of each carrier was dispensed in to a flask of appropriate size, moistened to 40% of their water holding capacity and plugged with cotton. The flasks were autoclaved at 151bs ps.i. (121 C) for 2 h consecutively fo~~~day~,,\.r'\ ~f",,1 \ Individual carrier was inoculated ascetically with 5 ml inoculum. An inoc~m in each flask was thoroughly mixed to distribute the culture uniformly. The initial count of the inoculated carrier in each flask was measured by serial dilution plate count technique(collins, 1967) using Pikovskaya's agar medium and count was expressed on the basis of wet weight of carrier. The flasks were inoculated at 28 ± 0.2 C for 60 d and sampled at the intervals of 10, 20, 30, 45 and 60 d for counting cell survivals by the serial dilution plate count technique. The colonies showing solubilization zone around them were considered as of phosphate solubilizers. Results and discussion Table 14.1 indicates the physicochemical properties of different carrier rn t'ocm'jt t~ materials tested. All the materials originally have ph,4.0 to 9.6. The water holding capacity was highest with Bentonite, as 430%, followed by CDC, FYM, soil and lignite. The maximum organic matter was noted with CDC as 9.9% while maximum available phosphorus (P) was in FYM. Bentonite "",.the ;'0 only source where~ the organic matter and available P were nil. Table 14.2 shows the survival count of Geotrichum sp. in different carrier materials with days of incubation. Geotrichum sp. survived and multiplied in all 148

7 carriers up to 60 d and survival count.:..,;.j.cjirectly proportional to the days of incubation. The survival and multiplication of the organism was highly..s'lfpoh(.,t by FYM and CDC, this may be due to high amount of organic matter present in the compounds. Less number',. of survivors was observed in Bentonite, while lignite and soil supported little increment in the survival number. CDC and FYM proved better up to 45 d but FYM proved better over CDC up to 60 d. Samaha (1998) observed that CDC was best carrier for Oebaryomyces hansenii. The initial number of I. orientalis per gram of carrier and its survival at periodic intervals up to 60 d are shown int<lble The survival and proliferation of I. orientalis were high in soil, FYM and CDC. This also may be due to high organic matter contents of substances. The viable count of soil and FYM was same on the 60 th d. Survival count of bentonite was low compared to soil and FYM. Compared to soil, the number of organisms was more in CDC upto 30 th d but incredibly increased in soil than in CDC on 60 th d. The study provei that soil and FYM o.\v..e. the best carrier> for survival, proliferation and maintenance of phosphate solubilizing yeasts, Geotrichum sp and I. orientalis. Soil, as best carrier was reported by Dave(1999) for Pseudomonas fluorescens, by Samaha (1998) for Aspergillus niger and by Narsian(1995) for A. aculeatus. Kundu and Gaur(" 981) reported that the soil cooked be serve as good carrier for spore former B. polymyxa and A. awamori. Kundu and Gaur (1984) noted CDC and FYM as good carrier materials. 149

8 Tomar (1997) reported that FYM along with phosphate solubilizing bacteria, P. striata in field conditions increased the yield level of blackgarm ( Phaseolus mungol) by saving 25% fertilizer. From the study we can deduce that FYM is a good carrier material for both the organisms and also it can increase organic matter contained in soil. 150

9 Table 14.1 : Physical and chemical properties of different carriers. Carriers ph WHC OM Available P (% ) (%) (ppm) Soil CDC FYM Bentonite Lignite

10 Table 14.2 Survival count of Geotrichum sp. in different carrier materials. Days of incubation Carriers S 60 X 10 6 cfu/g X 10 7 cfu/g X 10' cfu Ig X 10' u cfu/g X 10' cfu/g X 10" cfulg Soil CDC FYM Lignite Bentonite

11 Table 14.3: Survival count of Issatchenkia orienta/is in different carrier materials. Days of incubation Carriers X10'cfulg X10' cfu /g X10'u cfu /g X10'" cfu /g X10" cfu/g X10 14 cfull! Soil CDC FYM Bentonite Lignite