Role of Micrococcus luteus SNSr7 strain NH54PC02 in Sustainable agriculture by behaving as Biocontrol agent

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1 International Journal of Microbiology and Allied Sciences (IJOMAS) ISSN: August 2016, 3(1):1-13 IJOMAS, 2016 Research Article Page: 1-13 Role of Micrococcus luteus SNSr7 strain NH54PC02 in Sustainable agriculture by behaving as Biocontrol agent Nisha Sharma 1 and Baljeet Singh Saharan 1 * 1 Microbial Resource Technology Laboratory, Department of Microbiology, Kurukshetra University, Kurukshetra , India. Abstract *Corresponding Author: Baljeet Singh Saharan Microbial Resource Technology Laboratory, Department of Microbiology, Kurukshetra University, Kurukshetra , India. baljeet.kuk@gmail.com Micrococcus luteus SNSr7 strain NH54PC02 is a phyllospheric bacterium having plant growth promoting activities and isolated from spinach phyllosphere. It is a gram positive micrococcus bacterium that plays an important role in sustainable agriculture by its antimicrobial properties against plant pathogenic microbes. During our research, we observed that Micrococcus luteus SNSr7 had prominent role in HCN and siderophore production. It also showed antifungal activity against Curvularia sp., Alternaria alternata, Rhizoctonia solani, Fusarium oxysporum and Cladosporium sp. Maximum antifungal activity was observed at conc. of 2000µL/mL after 14 and 21 days of incubation of SNSr7 culture in nutrient broth at 37 o C. This inhibition was either due to secondary metabolites (that are toxic for pathogens) or through certain enzymatic activity as it showed cellulase, protease and chitinase enzyme activities. Maximum percentage inhibition was estimated against Alternaria alternata (76.74%) and Curvularia sp. (78%). With the help of all these important properties it can be concluded that Micrococcus luteus SNSr7 strain NH54PC02 might be act as biocontrol agent and thus help in sustainable agriculture. Key words: Antifungal, HCN, Phyllosphere, Seed germination, Siderophore. Introduction Phyllosphere is the entire surface of plant excluding rhizosphere (root area) and including leaf and stem surfaces. The phyllosphere has many features that make it an excellent habitat for microorganisms. Leafs involves fresh environment and microorganisms can be predicted by direct way on leaf surface with the help of microscopic study. Phyllospheric area also includes large variety of microorganisms in comparison to rhizospheric soil area but less number of microorganisms lie on leaf surface as compared to root surface this is because of limited nutrient concentration on leaf surface as compared to soil, also the changes in environmental conditions may also effect the 1

2 growth of microorganism. Some microorganism in phyllosphere play favourable role while some have drastic effects e.g. beneficial role of microorganisms is in term of nutrient supply for plants by behaving as plant growth promoting bacteria (PGPB) and also through inhibitory effect against plant pathogenic microorganisms. Micrococcus luteus SNSr7 strain NH54PC02 (M. luteus) is one such type of PGPB isolated from spinach phyllosphere. It also acts as bioinoculant as well as biocontrol agent and play important role in sustainability of agriculture. As there is a need to prevent the plants and crops from pathogenic microorganisms as large amount of crops yield got lost because of these harmful microorganisms and the use of chemical fungicides or chemical fertilizers leads to the loss of wealth as well as are harmful for human and animals or cause environmental threat. Cost of production is also increasing day by day because of use of these costly chemical reagents (chemical fertilizers/fungicides). The earlier research showed that plant diseases by pathogenic microorganisms reduces the plants/crops growth and yields in the range of 10% in developed countries and more than 20% in less developed countries of the world every year [1]. Thus there is an essence to use such kind of microorganisms that help in protecting the crop and their yield from pathogen without causing any harmful effect to environment and also they should be profitable. Biocontrol agents exhibit their role in two ways, one is by effecting phytopathogens as bio-fungicides by producing certain enzymes e.g. cellulase, chitinase and protease etc. and also by second way in which they help to promote the plant growth by producing phytoharmones (IAA), Siderophore, and by enhancing seed germination, beside this they also helps in providing mineral molecules to the plants from soil. Siderophore is an iron relating promoter that are produced by bacteria and some fungi in iron limited area that may help to provide iron molecules to plant for growth from the deep region of soil. It helps in growth of most bacteria as an enzymatic component have important roles in transfer of electron, helps in providing resistance to active oxygen intermediates and RNA synthesis. Beside this it also play role in protection of plants against pathogenic microorganisms as bio-control agent [2]. Micrococcus luteus SNSr7 strain NH54PC02 exhibited their role as biocontrol agent by two ways one is by direct method in which HCN and Siderophore production along with certain enzyme activities (cellulose, protease and chitinase) and thus inhibit the plant pathogen. The indirect method includes inhibition of the plant pathogenic fungi by performing antifungal activity against pathogenic fungi (Alternaria alternata (A. alternata), Cladosporium sp., Fusarium oxysporum (F. oxysporum), Rhizoctonia solani (R. solani) and Curvularia sp.) which were performed due to production of secondary metabolites that had toxic effect on pathogens. A. alternata is an opportunistic pathogenic fungus that causes leaf spot, leaf blight and leaf rot diseases on various plants e.g. Alternaria blight in pigeonpea [3]. Cladosporium is a plant pathogenic fungi causes infection in leaves, root, fruits and seedlings of various plants like pepper, tomato, spinach and basella alba crops (Dhar et al. [4]. F. oxysporum causes infection in vascular system, chlorosis, wilting, necrosis, immature leaf drop, stunting, and dampingoff diseases in various plants e.g. Cucurbits, Banana, Tomato, legumes and sweet potatoes etc. R. solani is a soil borne plant pathogenic fungus causes root rot disease in sugar beet, collar rot diseases, and patches in cereals, damping off in seedling of various plants and belly rot of cucumber etc. Curvularia is also one of such pathogenic fungi causes Curvularia blight and leaf spots on Sorghum plant [5]. Here we introduced the role of M. luteus SNSr7 bacterial strain as biocontrol agent by studying its inhibition effect against all these harmful plant pathogenic fungi thus it can help in promoting the plant yield and growth and help in sustainable agriculture. 2

3 Materials and Method HCN Production Bacterial isolate was screened for the production of HCN by adding 4.4 g glycine/l in nutrient agar medium and bacterial culture was streaked on it. Whattman s filter paper no. 1 was soaked in 2% sodium carbonate (Na2CO3) in 0.5% picric acid solution and placed on the top lid of the plate. After streaking the bacterial culture plates were sealed with the help of parafilm and incubated at ºC for 3-5 days. Development of orange to red colour was the indication of HCN production [6, 7]. Siderophore Production The qualitative assay of siderophore production was performed in Chrome Azurole s (CAS) agar medium. CAS agar plates were prepared and streaked with test organism and incubated at C for 3-5 days. Development of halo zone around the colony was considered as a positive result [8]. Maximum percentage (%) siderophore activity was calculated by incubating the bacterial culture in a minimal basal broth medium at o C from 1-7 days under shaker conditions (100 rpm). The cells were removed by centrifugation after every day at 3000 rpm for 15 minutes (min) and 0.5 ml of the culture supernatant was then mixed with 0.5 ml CAS solution also with 10 μl of shuttling solution (sulfosalicylic acid). The colour was obtained and determined by using the spectrophotometer at the optical density (O.D.) of 630 nm after setting culture for 20 min at room temperature. Necessary blank (minimal medium) and reference solution (minimal medium + CAS dye + shuttle solution) were used during the determination [8]. Percentage siderophore was production was calculated by using formula % Siderophore production = O.D. of reference (Ar) O.D. of bacterial culture (As) O.D. of reference (Ar) Antifungal Assay The isolate was tested for antagonistic activity against a wide range of phyto-pathogens on PDA medium by dual-culture technique. The isolate was grown in nutrient broth medium at 37 o C in a rotary shaker (150 rpm) for 3, 5, 7, 14 and 21 days to check the maximum inhibition effect due to production of secondary metabolites. The culture broth was then centrifuged at rpm for 10 min to obtain the cell-free supernatant. The PDA plates were spreaded with different plant pathogenic fungi and wells (5-7 mm diameter) were made into the PDA plates by using a sterile cork borer, 100 µl of clear bacterial supernatant was also loaded into each well for the detection of antagonistic activity against different pathogenic fungi and allowed to incubate at o C for 3-5 days. Along with this, the inoculum conc. was also used in range of µl/ml by diluting the bacterial inoculum with sterile broth for the analysis of maximum inhibition effect. The antifungal activity of the culture filtrate was evaluated by measuring the diameter of inhibition zones [9]. In-vitro antifungal activity was also observed by inoculating bacterial culture and fungal pathogen in same petri plate in which bacterial isolate was streaked horizontally at the one side of the petri plate producing 2 sectors and at other sector disk of fungal pathogen was kept. Inhibition of fungal growth by the bacterial isolate determined antifungal activity. This test is known as visual agar plate assay or disk agar method [10, 11]. The percentage inhibition was calculated by using formula: % Inhibition in radial growth r1- r2 100 r1 - radial mycelial growth in control r2 - radial mycelial growth in treatment Enzyme production for antifungal activity Certain enzyme activities that showed inhibition of pathogenic fungi either by toxin production or by degradation of cell wall were detected by specific enzyme activity r1 3

4 confirmation based agar medium as shown below. Cellulase activity This activity was evaluated by streaking bacterial culture on carboxymethyl cellulose (CMC) agar media. The culture was then incubated at o C for hours (h). The clear zone around the colony indicated the positive cellulase activity [12]. Protease enzyme activity The bacterial culture was streaked on skim milk agar media and incubated at o C for h. The clear zone around the colony showed positive protease enzyme activity [13]. Chitinase enzyme activity The chitinase enzyme activity was confirmed by streaking the bacterial culture on chitin agar media. The clear zone was formed around the colony after incubation at o C for 5-7 days that was confirmed by flooding the plates with 0.1% (w/v) congo red dye for 15 min and then washed with distilled water [14, 15]. Isolation of antimicrobial compound The bacterial strain was grown in nutrient broth medium and incubated at 37 o C for 7 days. The filtrate was separated by centrifuging the inoculum containing broth at 10,000 rpm for 10 min. and then with the ethyl acetate in separating funnel (ratio of 1:1) in the form of (v/v) followed by continues shaking for 30 min. This process was repeated and ethyl acetate was allowed to evaporate. Ethyl acetate:chloroform:aceticacid (5:4:1) was used as solvent and crude extract was spotted on silica gel thin layer plates (TLC) for up to cm. The process was performed under closed glass chamber, for the complete saturation of air when TLC plates were dipped in it for the process. The chromatogram was visualized under UV light at the range of 365nm. The Rf values of the compounds were calculated using the following formula: Distance travelled by the compound 4 Rf = Distance travelled by the solvent front Results HCN production HCN production ability of M. luteus SNSr7 was confirmed by conversion of whatt man s filter no. 1 paper from yellowish (that was due to picric acid) to orange colour (Figure 1) due to reaction with the picric acid leads to the production of hydrogen cyanide gas that converts the yellow colour of picric acid containing filter paper into orange colour. Results of Siderophore production Halo zone was formed around the colony in CAS media plates confirmed that siderophore production activity was present in M. luteus SNSr7 (Figure 2.1) and zone formation was estimated by well diffusion method (Figure 2.2) that showed CAS halo zone size in range of 27 mm of size. Quantitatively % siderophore was estimated by taking O.D. at 630 nm along with reference (blank+ CAS dye) and blank only with the incubation gap from 1-10 days by using UV/Vis spectrophotometer (Figure 2.3). With the help of this formula it was estimated that M. luteus SNSr7 has capability of 88.38% of siderophore production after incubation of 9 days at 37 o C having O.D. value 0.23 (Figure 2.4) while O.D. of reference was recorded as 1.98 and further specific testing of siderophore showed hydroxymate type of siderophore production by SNSr7. Enzyme assay for biocontrol role of M. luteus SNSr7 M. luteus SNSr7 showed positive response for certain enzyme activities that play important role in antagonistic activity against plant pathogenic fungi either by inhibition of protein synthesis (transcriton) or by degradation of fungal cell wall. Cellulase, protease and chitinase enzyme activity are present in SNSr7 which were confirmed by using specific media. As cellulase enzyme activity was confirmed by the formation of clear zone around the colony in CMC agar

5 Figure 1: HCN production by M. luteus SNSr7 in the form of deep orange colour formation on whatt man s filter paper in comparison to control Figure 2.1: M. luteus SNSr7 along with SNSr8 and SNKp25 bacterial strain showed clear halo zone around colony as compared with control Figure 2.2: Positive siderophore production shown by formation of halo zone around well filled with M. luteus SNSr7 media and zone was easily visible when treated with gram s iodine (Figure 3.1a) while protease activity was confirmed by formation of zone in skim milk agar media and treatment with HCl solution (Figure 3.1b). Chitinase is also one of important enzymatic activity of bacteria that help to kill 5 the pathogenic microorganism by degrading their cell wall (fungal cell wall is made up of chitin). This enzyme activity is also shown by M. luteus SNSr7 bacterial strain as clear zone was formed in chitin agar medium when treated with congo red solution (0.2%) and washed with 1M NaCl solution (Figure 3.1c).

6 Figure 2.3: Yellow to orange colour showed positive response for siderophore production by M. luteus SNSr7 with blue coloured reference Figure 2.4: Maximum siderophore production by M. luteus SNSr7 at 37 o C after 9 days of incubation using CAS assay Fig. 3.1a Fig. 3.1b Figure 3.1a: Cellulase enzyme activity of M. luteus SNSr7 was confirmed by formation of clear zone in CMC agar media when treated with gram s iodine Figure 3.1b: Protease enzyme activity performed by SNSr7 by forming clear zone in Skim agar medium in comparison to control Fig. 3.1c Chitinase activity present in M. luteus SNSr7 was estimated by formation of clear zone around the colony when flooded with congo red dye solution as shown in in comparison to uninoculated control 6

7 Figure 4.1: Inhibition of Rhizoctonia solani by M. luteus SNSr7 strain NH54PC02 compared with control Figure 4.2: Inhibition of Cladosporium sp. by M. luteus SNSr7 shown in comparison to untreated control Figure 4.3: Inhibition performed by M. luteus SNSr7 against A. alternata in comparison to untreated control Figure 4.4: Inhibition activity by M. luteus SNSr7 against Curvularia sp. was shown in and compared with control 7

8 Figure 4.5: Antifungal activity shown by M. luteus SNSr7 against F. oxysporum as shown and compared with untreated control Figure 4.6: M. luteus SNSr7 strain NH54PC02 inhibition activity against R. solani as shown positive effect in by disk method in comparison to control Figure 5: Inhibition effect of different conc. of M. luteus SNSr7 strain NH54PC02 inoculum against five plant pathogenic fungi 8

9 Fig. 6.1 Fig. 6.2 Figure 6.1: TLC results showing movement of unknown antimicrobial compound (b) along with the mobile phase (ethylacetate:chloroform:acetic acid) visualized by using iodine vapours by keeping under UV light and with KMNo4 also compared with chloramphenicol antibiotic (c in fig) Figure 6.2: TLC results of unknown antimicrobial compound using (Benzene:Petroleum ether: Ethyl acetate) and visualized by using Iodine vapours B Figure 6.3: Solubility of unknown compounds of SNSr7 bacterial strain designated as B along with other compounds moving along with mobile phase (Ethylacetate:Hexane :Petroleum ether) according to its solubility under UV light Results of Antimicrobial Activity M. luteus SNSr7 bacterial strain has the ability to inhibit the growth of five plant pathogenic fungi named as R. solani (Figure 4.1), Cladosporium sp. (Figure 4.2), A. alternata (Figure 4.3), Curvularia sp. (Figure 4.4) and zone inhibition was observed. Inhibition effect was also performed by using disk method (Figure 4.5; Figure 4.6) to check is there any difference in inhibition effect in both of these methods. Inhibition effect was estimated along with different concentration ( µl/ml) of bacterial inoculum that was allowed to inoculate in well against these plant pathogenic fungi and it was found that SNSr7 maximally inhibited A. alternata and Curvularia sp. and R. solani at the concentration of 1000 µl/ml and 2000 µl/ml and inhibition zone was measured in mm along with standard deviation (SD). At the conc. of 2000 µl/ml, maximum inhibition zone was observed for A. alternata, Curvularia sp., and then R. solani, Cladosporium sp. and F. oxysporum 9

10 Table 1: Effect of different conc. of M. luteus SNSr7 bacterial inoculum on phytopathogenic fungi in the form of inhibition zone size (mm) Conc. of SNSr7 inoculum (µl/ml) R. solani (mm)±sd F. oxysporum (mm)±sd A. alternata (mm)±sd Cladosporium sp. (mm)±sd Curvularia sp. (mm)±sd ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±0.32 Table 2: Effect of incubation time period of broth culture of M. luteus SNSr7 strain NH54PC02 against four plant pathogenic fungi and their inhibition zone (mm) Sr. Time of R. solani F. A. Cladosporium Curvularia no. incubation (mm) ±SD oxysporum alternata (mm) ±SD (mm) in broth (mm)±sd (mm) ±SD ±SD (days) ± ±1 0.97± ±0.25 1± ± ± ± ± ± ± ± ± ± ± ± ±0.36 Table 3: Percentage inhibition by SNSr7 against all five plant pathogenic fungi by fungal disk method Sr. No. Name of fungi r1 (mm) (Control) r2 (mm) (Treated with M. luteus SNSr7) 1 R. solani F. oxysporum A. alternata Cladosporium sp Curvularias p % Inhibition (antifungal activity) respectively (Table 1; Figure 5). Antifungal activity is due to production of some secondary metabolites that inhibit the growth of pathogenic fungi. Thus, to observe this effect of antifungal toxic compound production by SNSr7, we inoculated the bacterial strain in nutrient broth and incubated at 37 o C with the time incubation of 3, 5, 7, 14, 21 days and we found that maximum inhibition was at the incubation of SNSr7 for14 and 21 days in nutrient broth was also evaluated against A. alternate, Curvularia sp., and then against R. solani while less inhibition 10

11 zone was found against F. oxysporum and Cladosporium sp. respectively (Table 2). We also analysed the percentage inhibition by M. luteus SNSr7 and maximum percentage inhibition was against Curvularia sp., A. alternata, and then against Cladosporium sp. while in comparison to these less % inhibition was observed against F. oxysporum and R. solani using fugal disk method (Table 3). TLC results TLC analysis showed that the extracted antimicrobial compound using ethylacetate after incubation for 21 days in nutrient broth maximum solubility in solvent ratios of ethylacetate: chloroform: acetic acid (50:40:10) in TLC. The rf value was obtained as total length of solvent front was 6.4 cm and 5.7 cm surface was covered by SNSr7 in TLC by moving with mobile phase and thus rf value was 0.89 while the rf value of chlorophenicol was estimated around That showed the unknown compound is approximately resembled with this antibiotic either with its structural or functional groups. The solubility movement zone of unknown compound was detected by using iodine vapours and KMNo4 (Figure 6.1) and by keeping the spotted TLC plates with these anitimicrobial compound of SNSr7 in mobile phase containing Benzene:Petroleum ether:ethyl acetate (5:2:3) and it was observed that this compound showed zone near the top of the solvent end in plates (Figure 6.2) so the rf value of compound B was calculated as But less solubility of this unknown antimicrobial compound was detected when move along with mobile phase of solvent containing ethylacetate:hexane:petroleum ether in the ratio of (5:3:2). The rf value was observed less in this mobile face as compared to in first i.e It also visualized by keeping the TLC plates under UV light for clear band detection (Figure 6.3). Thus, due to maximum solubility of our unknown compound in second mobile phase solvent (Benzene:Petroleum ether:ethyl acetate) so, it might be the compound having acidic or ketonic group along with hydrocarbon ring (complex group of carbon chain) along with this it also showed its maximum stability at temperature even above from o C. Discussion This work on M. luteus SNSr7 strain NH54PC02 investigated that this strain has ability to act as biocontrol agent by mean of production of certain compounds like siderophore, HCN, inhibitory enzymes like chitinase, protease, cellulase and also by production of secondary metabolites that may help to prevent plants from pathogenic fungi and can be helpful in improvement of crops production by this. In our recent work it was investigated that Hydrogen cyanide (HCN) gas production ability was shown by M. luteus SNSr7 as it plays defensive role against phytopathogens and supported by Ahmad et al. [16] and Attar et al. [17]. They also showed the effect of PGPR against pathogen due to these activities. It was also observed that qualitatively siderophore production was in range of 27 mm size of halo zone which was favoured by Deb et al. [18]. Who stated that maximum of 16 mm of CAS halo zone was shown by isolate KD7. Our bacterial strain showed good effect of siderophore production than Deb et al. [18] as clearly seen from the zone size. Our findings showed certain enzymetic activities like cellulase, protease and chitinase are also present in M. luteus SNSr7 that are helpful in performing antagonistic activity against pathogenic microorganism (phytopathogenic fungi) and is supported by earlier literature report in which peroxidase and chitinase etc. enzymes role in antagonistic activity against phytopathogens have been discussed Maksimov et al. [19]; Suryadi et al. [15]. They also reported the antimicrobial activity has been shown with the help of extracellular enzymes (chitinase and glucanse) and help to kill the pathogen by degrading their cell wall thus these similar properties of extracellular enzyme activity was also performed by SNSr7. Similarly, Kuddus and Ahmad [14] also reported that antimicrobial activity due to chitinase enzyme. Along with this as secondary metabolites play their inhibition role in antifungal activity thus, 11

12 during our research we also observed that M. luteus SNSr7 bacterial strain showed positive response toward plant pathogenic fungi including F. oxysporum, Cladosporium sp., A. alternata and R. solani as well as against Curvularia sp. SNSr7 showed maximum inhibition against A. alternata and R. solani at the conc. of µl/ml and was supported by Karuppiah and Rajaram [20]. They reported that Antiphytopathogenic fungal activity of Bacillus isolates were increased with the increase in conc. of culture suspension from µl/l against Pencillium sp., Cercospora and F. oxysporum. It was also sustained by Ahemad and Khan [21] who reported that effective inhibition effect of Pseudomonas putida strain PS9 against pathogenic fungi was at the conc. range of ( μg/ml with a two times dilution. It was evaluated that % inhibition activity of M. luteus SNSr7 strain NH54PC02 was maximum against these pathogenic fungi is in the range of 76.74% for A. alternata, 75.56% for Cladosporium, in case of Curvularia and less against F. oxysporum. Similar to our findings, Grobelak et al. [22] also reported that maximum % inhibition of bacterial strain was 71% against A. alternata and 20% for F. oxysporum however we observed maximum % inhibition by M. luteus SNSr7 against these plant pathogenic fungi which suggests it role as beneficial antagonistic effect against plant pathogenic fungi. On the basis of our outcomes, we can suggest that M. luteus SNSr7 strain NH54PC02 is an important bio-inoculant and can be used as biocontrol agent. References 1. Glick BR Biocontrol mechanisms. In: Beneficial plantbacterial interactions. Springer International Publishing Pedraza RO Siderophores Production by Azospirillum: Biological importance, assessing methods and biocontrol activity. In: Handbook for Azospirillum. pp Springer International Publishing. 3. Sharma M, Ghosh R, Pande S Occurrence of Alternaria alternata causing Alternaria blight in pigeonpea in India. Advance in Bioscience and Biotechnology. 4: Dhar D, Ghoshal M, Mitra DAK Effect of Fusarium sp. and Cladosporium sp. infection on Basella alba leaves. Journal of Chemical Biological and Physical Science. 5(2): Dhar D, Ghoshal M, Mitra DAK Effect of Fusarium sp. and Cladosporium sp. infection on Basella alba leaves. Journal of Chemical Biological and Physical Science, 5(2): Lorck H Production of hydrocyanic acid by bacteria. Physiology of Plant. 1: Alstrom S, Burns RG, Cyanide production by Rhizobacteria as a possible mechanism of plant growth inhibition. Biology and Fertility of Soil. 7: Schwyn, B, Neilands JB Universal chemical assay for the detection and determination of siderophores. Anals of Biochemistry. 160: Sowndhararajan K, Marimuthu S, Manian S Biocontrol potential of phylloplane bacterium Ochrobactrum anthropi BMO-111 against blister blight disease of tea. Journal of Applied Microbiology. 114: Kamil Z, Saleh M, Moustafa S Isolation and identification of rhizosphere soil chitinolytic bacteria and their potential in antifungal 12

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