5856 Advances5856-5859, in Life Sciences Advances in Life Sciences 5(16), Print : ISSN 2278-3849, 20165(16), 2016
Isolation and Characterization of Pseudomonas fluorescens and Bacillus subtilis and their in vitro Evaluation S. KAPALI, R. M. GADE*, A. V. SHITOLE AND S. ASWATHI Department of Plant Pathology, Dr. Panjabrao Deshmukh Krishi Vidyapeeth, Akola, Maharashtra email:
[email protected]
ABSTRACT A total of eight rhizobacterial isolates obtained from rhizosphere of chickpea, pigeonpea, cotton and soybean. All the cultural and biochemical studies confirmed them to be P. fluorescens and B. subtilis. The isolates showed positive response for catalase, citrate, oxidase, IAA, siderophore, HCN and ammonia, while negative for indole and methyl red and voges proskauer. Cultures on NA were also found endospore positive. In vitro antagonism showed that F. oxysporum f. sp. ciceri significantly inhibited by Bacillus subtilis (Bs1 60.37%) and P. fluorescens (Pf1 45.18%). Pseudomonas fluorescens (Pf1) and Bacillus subtilis (Bs4) isolates achieved maximum inhibition of radial growth of R. bataticola by (53.83%) and (49.83%) respectively under in vitro study. Effective isolates of Pseudomonas fluorescens (Pf4, Pf2) and Bacillus subtilis (Bs1) achived maximum inhibition of radial growth of S. rolfsii. Key words
Pseudomonas fluorescens, Bacillus subtilis, wilt, root rot, collar rot.
Plant growth promoting rhizobacteria (PGPR) are associated with most plant species and commonly found in many environments. Rhizobacteria are rhizosphere competent bacteria that aggressively colonize plant roots. Bacillus and Pseudomonas were among the first bacterial isolates to show promising biocontrol characteristics and promoters of plant health and development and are known to survive in the rhizosphere (Santoyo et al., 2012), with the competence ability to tolerate a reasonable range of abiotic factors including temperature, pH and moisture. Pseudomonas fluorescens and Bacillus subtilis had been reported to manage several diseases caused by soil borne pathogens. The present study, aimed at isolation and characterization of antagonistic bacteria against wilt and root rot causing fungal pathogens from the rhizosphere of various crops.
MATERIAL AND METHODS Isolation of Rhizobacteria The rhizobacteria were isolated from different soil samples collected from rhizosphere of chickpea, pigeon pea, cotton and soybean. All the microbial isolates were isolated on their respective media; Bacillus sp. on Nutrient agar and Pseudomonas sp. on King’s B agar (King et al., 1954). One gram of soil (10-6 to 10-8) was aseptically added. Twenty ml of sterilized melted and cooled medium was added and poured in each Petri plate and were incubated at 28±20C for 24 h. The individual colonies were picked up with sterilized
loop and transferred on King’s B and NA medium. The plates were incubated at 28±20C for 24 h. The single colonies developed were transferred on King’s B medium slants and the pure cultures so obtained were stored in refrigerator at 40C.
Isolation of the fungus R. bataticola was isolated from root rot infected gram seedlings. Pathogenicity of isolate was assessed on gram (JG-11) seedlings under greenhouse conditions (22-26°C and 60-70 % RH). Collor rot pathogen, Sclerotinia rolfsii was isolated from diseased gram plant. Pathogenicity of isolate was assessed on gram (ICCV-2 variety) seedlings under greenhouse conditions (22-26°C and 60-70 % RH). F. oxysporum f. sp. ciceri was isolated from wilt infected gram seedlings. Pathogenicity of isolate was assessed on gram (JG-62) seedlings under greenhouse conditions (22-26°C and 60-70 % RH). The respective fungal pathogen cultures were maintained on Potato Dextrose Agar (PDA) medium at room temperature by adopting subsequent sub culturing at periodical and regular intervals. Seven days old culture was used for further studies.
Morphological studies The pure isolates were further cultured on new plates for colony morphology. The colonies morphological characters like margins, shape, raised and pigmentation were observed (Shahzaman, 2014).
Biochemical studies Biochemical test viz., Gram’s reaction, endospore staining catalase production, oxidase, indole production, citrate utilization, methyl red and voges-proskauer test were carried out for conformation of P. fluorescens and Bacillus subtilis.
Siderophore production Siderophore production was detected according to method of Schwyn and Neilands, 1987. The bacterial cultures were streaked on the King’s B medium with streaked onto the King’s B medium amended with the CAS indicator. Presence of siderophore production was indicated by orange halos around the colony due to chelation of iron which bound to CAS dye.
Oxidase test Take an inoculating loop or toothpick. Then touch and spread a well isolated colony on an oxidase disk (Disk contains N, Ndimethyl- p-phenylenediamine oxalate and ánaphthpol). The reaction was observed within 2 minutes at
KAPAL et al., Isolation and Characterization of Pseudomonas fluorescens and Bacillus subtilis and their in vitro Evaluation
5857
Table 1. Morphological and biochemical characterization of rhizobacterial isolates Characters
Pseudomonas fluorescens
Bacillus subtilis
Gram reaction
-ve
+ve
Cell shape
Rod
Rod
Pigmentation
Cream,
Whitish
greenish
to off-whitish
yellowish, whitish Endospore
-ve
+ve
Colony morphology
Smooth margin, flat to raised
Circular, lobate to serrated
Citrate utilization
+ve
+ve
Catalase production
+ve
+ve
Indole production
-ve
-ve
Oxidase
+ve
+ve
Methyl red
-ve
-ve
Voges-proskauer
-ve
-ve
IAA production
+ve
-ve
Siderophore production
+ve
+ve
HCN production
+ve
-ve
NH3
+ve
+ve
+ ve - Positive, - ve - Negative
antagonistic ability against F. oxysporum f.sp. ciceri, R. bataticola and S. rolfsii which causes wilt, root and collar rot in chickpea respectively. The bacterial isolates were screened by dual culture test as followed by Sreedevi and Devi (2012). In Petri plates containing 20 ml PDA (without antibiotics) and a loopful of fresh bacterial culture were streaked in a two side leaving 1cm from the margin, and then 5 mm disc of the fungal cultures were placed at the center and Petri plates were incubated at 280C for 5-7 days. The per cent growth inhibition of the test fungus with each bacterial isolates was calculated. Three replicates plates were maintained for each isolates. Plates streaked with sterilized water in place of bacterial isolates were kept as control. The percent growth inhibition of the test fungus was expressed in comparison to the control plates for each isolates
25-30ºC. Deep purple blue indicate positive reaction.
Methyl red (MR) test A tube of GPPW (5 ml) was inoculated with pure culture of the test organism. It was incubated at 28°C for 48 hour, after this, 5 drops of the MR reagent was added directly to the broth.
Catalase production A drop of 3% H2O2 was taken on glass slide and a small amount of bacterial culture was mixed with platinum inoculation loop. Rapid and sustained production of gas bubbles or effervescence constituted positive test.
Dual culture test Bacterial isolates were further tested for their
Table 2. Efficacy of Pseudomonas fluorescens against F. oxysporum f.sp. ciceri, Rhizoctonia bataticola and Sclerotium rolfsii Isolates
Mycelial growth
Percent growth inhibition (%)
F. oxysporum f.sp. ciceri
R. bataticola
S. rolfsii
F. oxusporum f.sp. ciceri
R. bataicola
S. rolfsii
Pf1
49.33
41.55
81.33
45.18(42.23)*
53.83(47.19) *
9.63(18.07) *
Pf2
52.33
49.77
47.22
41.85(40.31)
44.70(41.96)
47.53(43.59)
Pf3
66.66
60.00
66.33
25.93(30.61)
33.33(35.26)
26.30(30.85)
Pf4
54.10
73.00
35.66
39.89(39.17)
18.88(25.75)
60.37(50.99)
Control
90
90
S. E. (m)±
0.20
0.15
0.11
0.16
0.19
0.19
C.D. (p=0.01)
0.89
0.65
0.49
0.68
0.82
0.80
(* Values in the parenthesis are arc sine transformed)
5858
Advances in Life Sciences 5(16), 2016
Table 3. Efficacy of Bacillus subtilis against F. oxysporum f.sp. ciceri, Rhizoctonia bataticola and Sclerotium rolfsii Isolates
Mycelial growth
Percent growth inhibition (%)
F. oxysporum f.sp. ciceri
R. bataticola
S. rolfsii
F. oxusporum f.sp. ciceri
R. bataicola
S. rolfsii
Pf1
28.33
41.55
81.33
45.18(42.23)*
53.83(47.19) *
9.63(18.07) *
Pf2
70.35
49.77
47.22
41.85(40.31)
44.70(41.96)
47.53(43.59)
Pf3
78.87
60.00
66.33
25.93(30.61)
33.33(35.26)
26.30(30.85)
Pf4
75.15
73.00
35.66
39.89(39.17)
18.88(25.75)
60.37(50.99)
Control
90
90
S. E. (m)±
0.18
0.15
0.11
0.16
0.19
0.19
C.D. (p=0.01)
0.79
0.65
0.49
0.68
0.82
0.80
RESULTS AND DISCUSSION Soil samples were collected from rhizospheric soil of different field crop (chickpea, pigeon pea and cotton). Among these four isolates of Pseudomonas fluorescens and four of Bacilus subtiis were isolated. The isolates of rhizobacteria obtained were evaluated in detail for their morphological and biochemical characteristics as given in Bergey’s manual of systematic bacteriology. All the isolates of Pseudomonas fluorescens showed gram –ve, transparent, smooth and small colonies with diffusible white and yellow green pigment on King’s B medium. All of these were gram negative rods. The isolates were oxidase, catalase and citrate positive and indole, Voges-proskauers, methyl red negative (Table 1). They were also able to produce siderophore, HCN and NH3. Four of the isolates were found to be grampositive, rod shaped bacteria. Four isolates showed profuse growth on NA medium with typical colony morphology which was predominantly off-white to creamish in color and were indole, methylred, vogesproskaur, HCN and IAA negetive and oxidase, catalase, citrate positive. Culture on NA were also found endospore positive (Shobha and Kumudini, 2012). A few of them were positive for siderophore and NH3 (Shahzaman, 2014) (Table. 1). On the basis of these tests, the isolates were tentatively placed into three genera, Bacillus (4) and Pseudomonas (4). Antagonistic potential of 8 of the rhizospheric isolates (Pf1, Pf2, Pf3, Pf4, Bs1, Bs2, Bs3 and Bs4), was tested against F. oxysporum f. sp. ciceri, R. bataticola and S. rolfsii in dual culture under in vitro conditions. The growth of the fungus was lesser as compared to the control plate. Among them Pf1 performed best which gave 45.18% percent inhibition of radial growth followed by Pf2 (41.85%), Pf4 (39.89%) to F. oxysporum f. sp. ciceri, whereas, least inhibition of radial growth was recorded with Pf3 (25.93%) (Table 2). These observations are in accordance with the findings of Khan and Gangopadhyay (2012). Data presented in Table 2 showed that Maximum growth inhibition was observed with Pf1 (53.83 %) followed by Pf2 (44.70 %) to R. bataticola (Belkar and Gade 2012). Data (Table 2) revealed that all the antagonists were found to be significantly effective over control in inhibiting the mycelial growth of S. rolfsii. Maximum growth inhibition was observed with Pf4 60.37% with least mycelial growth 35.66 mm (Patel et al., 2011). Similarly among four isolates of B. subtilis evaluated
against the pathogens in dual culture test, all the isolates reduced the colony growth of F. oxysporum f. sp. ciceri, R. bataticola and S. rolfsii, among them, Bs1 found performed best which gave 68.52% and 45.26% inhibition of mycelial growth to F. oxysporum f. sp. ciceri and S. rolfsii, respectively (Rajput et al., 2012). The data presented in Table 3 Indicate that all isolates were effective to inhibit growth of R. bataticola, Bs4 was found significantly superior to inhibit the mycelial growth of the pathogen with 49.83% growth inhibition. These results are close with the corroboration of Kumar et al. (2011). The difference in percent inhibition of mycelial growth indicates the difference in their antagonistic potential for the test pathogen by production of diverse antimicrobial secondary metabolites (Meena et al., 2012; Singh et al., 2013).The percent inhibition of these rhizobacteria may positively be correlated with extracellular activities of protease, Chitinase, â-1,3glucanase and siderophore (Kandolia and Vakharia, 2013). Suppression of Rhizoctonia bataticola by Pseudomonas fluorecens in agar plate might be due to the production of siderophores (Leong, 1986; Laha et al., 1992) or may be due to volatile antifungal compounds (Abou-aly et al., 2015). Inhibition of the pathogen may be because of antibiosis activity of Bacillus subtilis.
LITERATURE CITED Abou-Aly, H. E., Neweigy, N. A., Zaghlou, R. A., El-Sayed, S. A. and Bahloul, A. M. 2015. Evaluation of some biocontrol agents against soil pathogenic fungi. Res. J. Pharmal. Bio. and Chem. Sci., 6(1): 440. Belkar, Y. K. and Gade, R. M. 2012. Biochemical characterization and growth promotion activities of Pseudomonas fluorescens. J. Pl. Dis. Sci., 7(2): 170 - 174. Buchanan, R. E. and Gibbson, N. E. 1974. Bergey’s Manual of Determinative Bacteriology, 8. Kandoliya, U. K and Vakharia, D. N. 2013. Antagonistic effect of Pseudomonas fluorescens against Fusarium oxysporum f.sp. ciceri causing wilt in chickpea. Agril. Res. Commu. Cen., 36(6): 569-575. Khan, M. A. and Gangopadhay, S. 2012. Effect of soil inhabiting anatgonostic microflora against Fusarium oxysporum f. sp. ciceri, incitant of wilt chickpea. J. Mycol. Pl. Pathol., 42(3): 341-346. King, E. O., Ward, M. K. and Raney, D. E. 1954. Two simple media for the demonstration of pyocenin and fluorescin. J. Lab. Clin. Med., 44: 301-307.
KAPAL et al., Isolation and Characterization of Pseudomonas fluorescens and Bacillus subtilis and their in vitro Evaluation Kumar, S., Sharma, S., Pathak, D. V. and Beniwal, J. 2011. Integrated management of Jatropha root rot caused by Rhizoctonia bataticola. J. Tropical Forest Sci., 23(1): 35-41. Laha, G.S., Singh, R. P. and Verma, J. P. 1992. Biocontrol of Rhizoctonia solani in cotton by Fluorescent pseudomonads. Indian Phytopath., 45(4):412-415. Leong, J., 1986. Siderophore, their biochemistry and role in the biocontrol of plant pathogens. Anna. Rev. Phytopath., 24: 187209. Meena, Y. K., Singh, Y. and Kharayat, B. S. 2012. Biological control of anthracnose of Sorghum caused by Colletotrichum graminicola. Internat. J. Pl. Protec., 5(2): 333-338. Patel, D. B., Singh, H. B., Shroff, S. and Sahu, J. 2011. Antagonistic efficiency of Pseudomonas strains against soil borne disease of chickpea crop under in vitro and in vivo. Elixir Agriculture, 30: 1774-1777. Rajput, V. A., Konde, S. A. and Thakur, M. R. 2010. Evaluation of Bioagents against chickpea wilt complex. J. Soil and Crops, 20(1):155-158.
5859
Santoyo, G., Mosqueda, M. d. C. O. and Govindappa, M. 2012. Mechanisms of biocontrol and plant growth-promoting activity in soil bacterial species of Bacillus and Pseudomonas: a review. Biol. Sci. Tech., 22(8): 855-872. Schwyn, B. and Neilands, J. B. 1987. Universal chemical assay for detection and determination of siderophores. Anal. Biochem., 16: 47-56. Shahzaman, S., 2014. Preliminary screening for fungal root pathogen suppression by plant growth promoting rhizobacteria in Chickpea (Cicer arietinum L.). J. of Agril. Sci., 4(6): 295-298. Shobha, G. and Kumudini, B.S. 2012. Antagonistic effect of the newly isolated PGPR, Bacillus spp. on Fusarium oxysporum. Int. J. App. Sci. Eng. Res., 1(3): 74-77. Singh, S. P., Singh, H. B. and Singh, D.K. 2013. Trichoderma harzianum and Pseudomonas sp. mediated management of Sclerotium rolfsii rot in tomato(Lycopersicon esculentum Mill.). The Bioscan. 3: 801-804. SreedevI, B. and Devi, M. C. 2012. Mechanisms of biological control of root rot of groundnut caused by Macrophomina phaseolina using Pseudomonas fluorescens. Indian Phytopath., 65(4): 360365.
Received on 11-08-2016
Accepted on 16-08-2016
