IMPACT: International Journal of Research in Applied, Natural and Social Sciences (IMPACT: IJRANSS) ISSN (P): 2347–4580; ISSN (E): 2321–8851 Vol. 8, Issue 1, Jan 2020, 13–16 © Impact Journals EFFECT OF SALTS ON PROTEASE AND LIPASE PRODUCTION IN SEED-BORNE FUNGI OF SOYBEAN Kesare Umesh Associate Professor, Department of Botany, Adarsh Senior College, Osmanabad, Maharashtra, India Received: 12 Jan 2020 Accepted: 20 Jan 2020 Published: 31 Jan 2020 ABSTRACT During the process of biodeterioration, seed mycoflora produces enzymes to degrade protein, carbohydrate and oil. These enzymes are called as hydrolytic enzymes. The enzymes which degrade proteins are called protease and enzymes and which degrade oil are lipase. Five different salts at 0.05% concentration were added separately in to the basal medium, and it is observed that Barium chloride shows inhibitory effect on protease production by the fungal species, such as Aspergillus niger, A. glaucus, A. ustus and Trichoderma viride, while there is no activity by the fungi like Curvularia lunata, Fusarium roseum, F. oxysporum and Spicaria violecia. Sodium chloride also reveals similar effect except some fungi like A. niger, A. glaucus, C. lunata and F. oxysporum. Potassium chloride also is an inhibitory factor for protease production by almost all the fungi except Alternaria alternata, A. flavus and A. niger. Barium chloride stimulates the lipase activity by A. alternata and inhibited by A. glaucus, while it was in total inhibition in the other fungi. KEYWORDS: Salts, Protease, Lipase, Fungi INTRODUCTION Seed plays a vital role for the production of a healthy crop. These seeds are also responsible for disease transmission. This takes place either in the field or in ill-storage condition. Neergard (1977) reported that in the presence of seed-borne pathogens, several types of abnormalities like reduction in seed size, seed rotting, discoloration of seeds, seed necrosis, loss in germ inability, toxification and other physiological disorders. Acording to Sandikar (1990) the species of fusarium are found to be significantly destructive and responsible to cause harmful effect on seed health resulting in to seed deterioration and poisoning of seeds. During the process of biodeterioration, fungi prduce enzymes to degrade proteins, carbohydrates and oil. Sharma and Satyanarayana(1980) studied production of protease by some fungi such as Helminthosporium, Glomerella cingulata, Curvularia geniculata, Alternaria pelandui. Umatale(1995),Charya and Reddy(1982) also studied on lipase production in certain oil seeds. Umatale found Aspergillus flavus, A.helianthi, Macrophominaphasiolina and Rhzopus nigricans are more active to produce lipase. MATERIALS AND METHODS Collection of samples and detection of seed mycoflora. For the collection of seed samples, the method described by Neergaard (1973) has been adopted. Accordingly from fields, store houses market places and seed companies. A composite sample of each variety was prepared by mixing the individual samples together. The seed mycoflora was isolated by using standered moist blotter paper method (SMB) and agar plate method (APM) as recommended by International seed testing association (ISTA 1966),De Tempe (1970),Neergaard (1973) and Agarwal (19760. Impact Factor(JCC): 4.6148 – This article can be downloaded from www.impactjournals.us 14 Kesare Umesh Identification of Seed Borne Fungi The fungi occurring on each and every seed in the plates were identified preliminary on the basis of sporulation characters like sexual or asexual spores with the help of stereoscopic binocular microscope. The identification and further confirmation of seed-borne fungi was made by preparing slides of the fungal growth and observing them under compound microscope. The identification was made with the help of manuals as per Nelson,et.al. (1983), Singh, et.al.(1991), Mukadam D.S.(1997) and Mukadam et.al. (2006). Production of Protease Production of protease(s) was made by growing the fungi on liquid medium containing glucose 10g, gelatin 10g, dipotassium hydrogen phosphate 1.0g, MgSO 7H O -500mg and distilled water-1000ml. pH of the medium was adjusted 4. 2 at 5.5. Twenty five ml of medium was poured in 100ml Erlenmeyer conical flasks and autoclaved as 151bs pressure for 20 minutes. The flasks on cooling were inoculated separately with 10ml standard spore/mycelial suspension of test fungi prepared from 7 days old cultures grown on PDA slants. The flasks were incubated for 6 days at 25±1oC with diurnal periodicity of light. On 7th day, flasks were harvested by filtering the contents through Whatman’s filter No.1. The filtrates were collected in the pre-sterlised bottles and termed as crude enzyme preparation. Assay Method (Cup-Plate Method) Determination of protease(s) activity was done with the help of cup-plate method, adopted by Hislop et. al., (1982) and Rajamani (1990). A basal medium was prepared by adding 2% (w/v) agar and one percent (w/v) gelatin. pH of the medium was adjusted at 5.6 with Mcllavaine’s buffer. Then it was sterilized at 15 lbs pressure for 15 minutes. About 15 ml of the medium was poured in presterilized petriplates under aseptic condition. On solidification 6mm diameter cups/cavities were made in the centre of each of the agar plate with a sterilized cork borer (No.4). The cups/cavities were filled carefully with about 0.5ml of culture filtrate (crude enzyme preparation). The plates were incubated at 25oC for 24 hours. Then the plates were flooded with 15 percent mercuric chloride in 7N HCl. After 10 minutes of standing, a clear transparent zone indicated the hydrolysis of gelating by extracellular proteolytic enzymes, whereas the rest of the region of the petriplates become opaque due to the coagulatin (protein) by mercuric chloride. Diameter of the clear zone was used as measure (mm) of protease activity, while non appearance of clear zone considered absence of protease (s) in the culture filtrates. Production of Lipase Lipase activity was studied by growing the fungi on liquid medium at pH5.6 containing oil-10g, KNO -2.5g, KH PO - 3 2 4- 1.0g, MgSO – 0.5g and distilled water 1000ml. 25ml of the medium was poured in 100ml conical flasks and autoclaved at 2 15 1bs pressure for 30 minutes, then on cooling the flasks were inoculated separately with 1.0ml spore suspension of the fungi which were incubated for 6 days at 25± 1oC with diurnal periodicity of light. On 7th day of the flasks were harvested by filtering the contents through Whatman filter paper no.1. The filtrates were collected in presterilized culture filtrate bottles and termed as crude lipase. Assay Method (Cup-Plate Method) Determination of lipase activity was done with the help of cup-plate method. The medium contains Difco peptone-10g, NaCl-5g, Cac1 .2H O-1.0g, agar 2 percent and 10ml lipid substrate serbitan mono laurate (Tween-20) (Pre-sterilized) was 2 2 added to it. The pH of the medium was adjusted to 6.00. The medium was poured in each Petri plate. On solidifying the NAAS Rating: 3.00 – Articles can be sent to [email protected] Effect of Salts on Protease and Lipase Production in Seed-Borne Fungi of Soybean 15 medium with the help of a cork borer (No.4) was made in the centre and was filled with 0.1ml culture filtrate. The plates were incubated at 28oC. After 24 hours, a clear circular zone was measured (mm) as lipase activity. Similar procedure was followed for the culture filtrate in the central cavity instead of the active enzymes. RESULTS AND DISCUSSIONS Five different salts at 0.05% concentration were added separately in to the basal medium and their effect on protease production was studied and the results are shown in Table 1. Barium chloride reveals inhibitory effect on protease production by the fungal species, such as Aspergillus niger, A. glaucus, A. ustus and Trichoderma viride, while there is no activity by the fungi like Curvularia lunata, Fusarium roseum, F. oxysporum and Spicaria violecia. Sodium chloride also reveals similar effect except some fungi like A. niger, A. glaucus, C. lunata and F. oxysporum. Potassium chloride is also an inhibitory factor for protease production by almost all the fungi except Alternaria alternata, A. flavus and A. niger. No production of protease was seen by S. violecia. While calcium chloride completely inhibits the production of protease in all the ten fungi, Magnesium chloride also inhibits protease production except A. alternata and F. oxysporum. Barium chloride stimulates the lipase activity by A. alternata and inhibited by A. glaucus, while it was total inhibition in the other fungi. Sodium chloride reveals stimulatory effect in lipase production in most of the fungi except A. flavus, A. ustus, Curvularia lunata and Trichoderma viride. Potassium chloride also reveals stimulatory effect in most of the fungi, except C. lunata, while it was totally inhibited in A. flavus, A. ustus, Spicaria violecia and Trichoderma viride. Calcium chloride reveals inhibitory effect on lipase production by the Aspergillus niger and A. glaucus, and it is totally inhibited in most of the fungi except it is stimulatory by Alternaria alternata. Magnesium chloride reveals stimulatory effect on lipase production by most of the fungi except A. niger, which is inhibited with this salt and other produce totally inhibited lipase, such as A. glaucus, Curvularia lunata, Fusarium roseum, F. oxysporum and S. violecia. Table 1: Effect of Salts on Protease and Lipase Production in Seed-Borne Fungi Fungi Salts AAL ASF ASN ASG ASU CUL FUR FUO SPV TRIV (0.05% conc.) Activity Zone (mm) Protease Production Barium chloride 18 21 16 16 20 - - - - 13 Sodium chloride 14 18 18 21 16 21 18 20 16 14 Potassium chloride 20 20 19 14 20 14 16 14 - 11 Calcium chloride 13 16 17 13 15 14 18 16 14 12 Magnesium chloride 18 17 16 16 18 14 20 21 16 17 Control 18 20 18 19 21 20 22 20 19 20 Lipase Production Barium chloride 12 - - 12 - - - - - - Sodium chloride 15 12 18 17 11 15 17 18 15 16 Potassium chloride 14 - 14 14 - 12 15 12 - - Calcium chloride 13 - 13 12 - - - - - - Magnesium chloride 11 16 11 - 15 - - - - 18 Control 11 16 14 14 15 17 11 11 11 18 Aal - Alternaria alternata Cul - Curvularia lunata Asf - Aspergillu flavus Fur - Fusarium roseum Asn - Aspergillus niger Fuo - Fusarium oxysporum Asg - Aspergillus glaucus Spv - Spicaria violecia Asu - Aspergillus ustus Triv - Trichoderma viride Impact Factor(JCC): 4.6148 – This article can be downloaded from www.impactjournals.us 16 Kesare Umesh CONCLUSIONS Five different salts at 0.05% concentration were added separately in to the basal medium and their effect on protease production was studied. Barium chloride reveals inhibitory effect on protease production by the fungal species, such as Aspergillus niger, A. glaucus, A. ustus and Trichoderma viride, while there is no activity by the fungi like Curvularia lunata, Fusarium roseum, F. oxysporum and Spicaria violecia. Barium chloride stimulates the lipase activity by A. alternata and inhibited by A. glaucus, while it was in total inhibition in the other fungi. REFERENCES 1. Agarwal, V. K., 1976. Technique for the detection of seed-borne fungi. Seed research.4:24–31. 2. Venkatesagowda, B., Ponugupaty, E., Barbosa, A. M., & Dekker, R. F. (2012). Diversity of plant oil seed- associated fungi isolated from seven oil-bearing seeds and their potential for the production of lipolytic enzymes. World Journal of Microbiology and Biotechnology, 28(1), 71–80. 3. Charya, M. A. S. and S. M. Reddy, 1982. Production of proteolytic enzymes by three seed-borne fungi. Acta botanica Indica. 10:296–299. 4. De,Tempe, J., 1970. 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