Microbiological
Communication
Biosci. Biotech. Res. Comm. 9(2):
In vitro antifungal potential of rhizospheric isolates against Fusarium oxysporum causing Fusarium wilt of
LS Raut1* and VS Hamde2
1Department of Microbiology, Sant Tukaram Arts and Science College, Parbhani - 431401, Maharashtra, India
2Department of Microbiology, Yogeshwari Mahavidyalaya, Ambajogai - 431517, Maharashtra, India
ABSTRACT
Fusarium wilt is one of the drastic diseases of
KEY WORDS:
ARTICLE INFORMATION:
*Corresponding Author: lasraut76@gmail.com Received 10th June, 2016
Accepted after revision 25th June, 2016 BBRC Print ISSN:
Thomson Reuters ISI SCI Indexed Journal NAAS Journal Score : 3.48
© A Society of Science and Nature Publication, 2016. All rights309 reserved.
Online Contents Available at: http//www.bbrc.in/
LS Raut and VS Hamde
INTRODUCTION
Bt- cotton is one of the most important cash crops cul- tivated in India. In north zone of India, Maharashtra is state first rank in cultivation area but trails on third rank for production (current cotton scenario,
14)due to different environmental factors and attack of pests. Fusarium wilt is one of major destructive and yield reducing diseases of
Present control strategies to control the Fusarium wilt disease is by using chemical pesticides. Chemical control measures create imbalances in the microbial community, which may be unfavorable to the activity of beneficial organisms and could lead to the development of resist- ant strains of pathogen and pollution of environment. Since, Fusarium wilt is increasingly destructive in cotton production, and to avoid the hazardous effect of chem- icals new ways of alternative controlling the diseases need to be searched. The production of siderophores in rhizosphere fungi was higher than those isolated from the contaminated soil. Siderophore production by Fun- gal M10 strain was studied using CAS blue agar and the
ganism and diffused through the medium producing a colour change from blue to orange. Microbial sidero-
phores production increases virulence power of micro- organisms and serve as good biocontrol agents, (Talebi et al., 2011, Hussein and Joo, 2012, Vinale et al., 2013 and Balado et al. 2015).
Microbiological control of plant pathogen by rhizos- pheric microorganisms offers an attractive alternative over chemical control. There are evidences that microor- ganism can be served as good microbial control candi- date to suppress the diseases. U.S. Environmental Protec- tion Agency registered eight species of microorganisms for commercial use against soil borne plant pathogens in the United States (Cook et al., 1996). Researchers from different region reported microorganisms as biocontrol agents such as Pseudomonas sp. (Chernin et al., 2011 Sandheep et al., 2012; ), Bacillus sp. (Saha et al., 2012; Lamsal et al., 2012), Trichoderma sp. ( Ahith and Laksh- midevi, 2010; Otadoh et al., 2011; Sandheep et al., 2012) Serratia sp (Chernin et al., 2011) etc. Several research- ers working on biological control of Fusarium wilt of different crops but there is less literature available on microbiological control of Fusarium wilt of Bt- cot- ton. Biological Control of Fusarium Wilt of Cotton was studied by using endophytic bacteria (Chen et al. 1995),
Trichoderma sp (Sivan and Chet, 2008 and Mali and Ramaiah., 2015).
Soil serves as excellent culture medium for all types of microorganisms due to the abundant availability of nutrient and favorable environmental condition. Rhizo- sphere of plants has been frequently exploited as bril- liant source for searching microbial control agents. It has been suggested that microorganisms isolated from the rhizosphere of a particular crop may be better adapted to that crops rhizospheric environment and may provide better control of diseases than the other plant rhizos- phere species (Cook, 1993). The present study was aimed to i) Isolation of phytopathogen responsible for caus- ing Fusarium wilt of
MATERIAL AND METHODS
ISOLATION OF PHYTOPATHOGEN
Fusarium wilt infected Bt- cotton plants were collected from different fields of Beed, Hingoli and Vasantrao Naik Agricultural University, Parbhani district in sterile polyethylene bags and brought to research laboratory, Department microbiology Yogeshwari Mahavidyalaya, Ambajogai. Infected stems and branches of
ISOLATION OF RHIZOSPHERIC BACTERIA
Rhizospheric soil samples from healthy Bt- cotton grow- ing fields of different districts of Marathwada region (Beed, Latur, Osmanabad, Aurangabad, Jalna, Nanded, Hingoli and Parbhani) were collected in sterile polyethyl-
ene bags and brought to research laboratory. 1g of each rhizospheric soil sample was mixed with 100 ml sterile distilled water and shaken well for 2 min, and then the content of flask was allowed to settle. Different dilutions
IN VITRO SCREENING FOR MICROBIAL CONTROL AGENT
114 rhizobacteria isolates were screened for antifungal activity against Fusarium oxysporum f.sp. vasinfectum by using dual culture technique on King B agar plates (Gull and Hafeez, 2012). 5 mm diameter mycelial disc was punched from margin of actively growing mycelium and placed at the center of 9 cm Petri plate and rhizo- bacterial isolates were inoculated 3 cm apart from the center. Three rhizospheric isolates were placed in a plate along with phytopathogen at the center. Control plate was kept without inoculation of rhizobacteria isolates and the all the plates were incubated at 28 0C for 6 days. The antifungal activity was determined by measuring the inhibition of mycelial growth of Fusarium oxyspo- rum f. sp. vasinfectum.
MECHANISM OF MICROBIAL CONTROL AGENT
To characterize the mechanism of microbial control agent, the efficient rhizospheric isolates were tested for the production of volatile metabolites, diffusible metab- olites and siderophore.
DETECTION OF VOLATILE METABOLITES
Volatile metabolites detection of efficient rhizospheric isolates was done by using double plate method (Dennis and Webster, 1971). The
mmPetri plate separately. Both the inoculated plates were placed facing each other and sealed with cello- phane adhesive tape. Control was kept without inocula- tion of rhizospheric isolate. Procedure repeated for each efficient rhizospheric isolates in triplicates. All the plates were incubated at 28 0C for 6 days. The production of volatile metabolites was then determined by inhibition of Fusarium oxysporum f.sp. vasinfectum and percent- age of radial growth inhibition was calculated by using the formula (Whipps, 1987).
LS Raut and VS Hamde
Where, R1 is radial growth of the pathogen alone (a control value) and R2 is radial growth pathogen in the direction of the antagonist pathogen + antagonist (an inhibition value).
DETECTION OF DIFFUSIBLE METABOLITES
Diffusible antifungal metabolites were detected by well diffusion assay (Schlumbaum et al., 1986). Rhizobacte- rial isolates showing antifungal activity against Fusar- ium oxysporum f.sp. vasinfectum during screening were grown in King B broth at room temperature on rotary shaker at 150 rpm for 48 h to obtain cell free culture filtrate. Kings B agar plates were prepared and after solidification with the help of sterile cork borer three wells 3cm apart from the center 90 mm diameter were punched on a plate. These wells were labeled accord- ing to the rhizobacteria isolates cell free culture filtrate to be loaded. 5 mm plugs from leading edge of 3 day old culture Fusarium oxysporum f.sp. vasinfectum were punched and kept at the center of the plate. Different rhizobacterial isolate broths were filtered by using Mil- lipore syringe filter 0.22 μ
QUALITATIVE DETECTION OF SIDEROPHORE
Siderophore production was determined by using modi- fied Chrome Azurol S (CAS) assay (Milagres et al., 1999). Initially all the glassware’s are rinsed with distilled water and dried. 60.5 mg of CAS was weighed accurately and dissolved in 50 ml of distilled water and to this add 10 ml of iron solution (1 mM FeCl3.6H2O, in 10 mM HCl).
72.9mg of Hexa decyl tri
LS Raut and VS Hamde
culture medium was inoculated with 24 h old rhizos- pheric isolate near the borderline of the two medium. The same procedure was repeated for each rhizospheric isolate.
RESULTS AND DISCUSSION
ISOLATION OF PHYTOPATHOGEN
Fusarium wilt phytopathogen was isolated from infected
Gull and Hafeez, 2012). The phytopathogen was transferred on fresh PDA and Kings B agar plates and also on maintained on slants for further use.
ISOLATION OF RHIZOSPHERIC BACTERIA
114 rhizobacterial isolates from different
IN VITRO SCREENING FOR MICROBIAL CONTROL AGENT
All the114 rhizospheric isolates were screened for in vitro antifungal activity against Fusarium oxysporum f.sp. vasinfectum by dual culture technique Gull and Hafeez, 2012). Out of 114, 13 rhizobacterial isolates inhibited mycelial growth in dual culture. These isolates showed significant differences in mycelial growth inhibition. Radial growth of phytopathogen in test and control was measured and recorded (Table 1 and Fig 2). Rhizospheric isolates showed significant inhibition RLS19 (68.89 %), RLS52 (62.22 %), RLS53 (60.00 %), RLS72 (57.78 %) and RLS101 (57.78 %) of Fusarium oxysporum f.sp. vasin-
fectum in dual culture technique. The highest antifungal activity was shown by rhizospheric isolate RLS19 (68.89 %) and followed by RLS52 (62.22 %), RLS53 (60.00 %).
Similar results were also recorded by other investi- gators, rhizospheric Pseudomonas isolate, isolated from rhizospheric soil of tomato evaluated against Fusarium oxysporum f.sp. lycopersici by dual culture technique and maximum zone inhibition was 22 mm (Asha et al., 2011). Rhizospheric isolates, Pseudomonas sp. and Bacil- lus sp. were evaluated for biocontrol potential against Fusarium oxysporum f.sp. ciceris by dual culture tech- nique, where Bacillus subtilis B28 showed 51.16 % inhi- bition (Karimi et al., 2012). Endophytic bacteria were antagonistic against F. oxysporum, isolates EB1 and EB2 inhibits radial growth 42.60 % and 41.00 % respectively in dual culture test (Edward et al., 2013).Soil isolates
When results of previous researchers (Xu and Kim, 2014; Asha et al., 2011; Karimi et al., 2012; Edward et al., 2013; Xu and Kim, 2014; Srivastava et al., 2015) compared to our findings, our finding found better than the others, where rhizospheric isolates RLS19 (68.89 %), RLS52 (62.22 %), RLS53 (60.00%) RLS72 (57.78 %), and RLS101 (57.78 %) found better in inhibiting the Fusarium sp. Based on screening results in dual culture technique, eight isolates namely (RLS18, RLS19, RLS52, RLS53, RLS58, RLS72, RLS76 and RLS102) with good inhibition activities of Fusarium oxysporum f.sp. vasin- fectum were selected for further study.
MECHANISM OF MICROBIAL CONTROL AGENT
While finding the mechanism of the microbial control agent, the rhizospheric isolates were tested for the pro- duction of volatile metabolite, diffusible metabolite and siderophore production.
DETECTION OF VOLATILE METABOLITES
After 6 days incubation it was observed that three rhizospheric isolates were able to produce volatile metabolites and inhibit the radial growth of Fusarium oxysporum f. sp. vasinfectum (Fig. 3 and Fig. 4). High- est Inhibition of phytopathogen by producing volatile metabolites was shown by rhizospheric isolate RLS101 (50 %) followed by RLS52 (20 %) and RLS79 (10 %).
LS Raut and VS Hamde
FIGURE 1: Isolation of Fusarium wilt causing phytopathogen from infected
(c) isolated Phytopathogen
FIGURE 2: In vitro screening of antifungal activity of rhizos- pheric isolates against Fusarium oxysporum f.sp. vasinfectum (a) Control (b) RLS19 (c) RLS107 (d) RLS58 (e) RLS72 (f) RLS53 and
(g) RLS52
Other isolates were unable produce volatile metabolites and there was no inhibition of Fusarium oxysporum f.sp. vasinfectum growth.Previous researcher found that Pseudomonas aueroginosa P12 isolate inhibits 26.30 % radial growth of Fusarium oxysporum f. sp. ciceris by producing volatile metabolites (Karimi et al., 2012). This
FIGURE 3: Detection of vola- tile metabolite production of rhizospheric isolate RLS102 against Fusarium oxysporum f.sp. vasinfectum (a) Control
(b) Test
FIGURE 4: Production of volatile antifungal metabolites of rhizos- pheric isolates against Fusarium oxysporum f.sp. vasinfectum
FIGURE 5: Detection of diffusible metabolites production of rhizospheric isolate by agar well dif- fusion method against Fusarium oxysporum f.sp. vasinfectum (a) Control (b) RLS18 (c) RLS76 (d) RL19
LS Raut and VS Hamde
FIGURE 6: Siderophore production of rhizospheric isolates by modified CAS assay (a) Control (b) RLS18 (c) RLS58
(d) RLS53
finding suggests that volatile metabolite production by rhizospheric isolates is one of the mechanism by which phytopathogen can be controlled. Here in present study, rhizospheric isolate RLS101 found better in controlling Fusarium oxysporum f.sp. vasinfectum by producing volatile metabolites than Pseudomonas aueroginosa P12 (Karimi et al., 2012).
DETECTION OF DIFFUSIBLE METABOLITES
Diffusible antifungal metabolites were studied by well diffusion assay (Schlumbaum et al., 1986). Eight effi- cient rhizospheric isolates tested showed inhibitory
effect on Fusarium oxysporum f.sp. vasinfectum by producing diffusible metabolites (Fig. 5 and Table 2). Rhizospheric isolate RLS19 (57.50 %) showed highest inhibition of phytopathogen by producing diffusible metabolites followed by RLS52 (55.00 %) and RLS 18 (52.05 %). Bacillus subtilis B28 isolate also inhibiting Fusarium oxysporum f.sp. ciceris (78.30 %) by produc- ing diffusible metabolites (Karimi et al., 2012).
QUALITATIVE DETECTION OF
SIDEROPHORE
After 5 day incubation, eight isolates produced sidero- phore on modified CAS plate. Siderophore production was recorded in the form of grades (Fig.6 and Table 3). The highest siderophore production was recorded in the form of change in colour of the medium from blue to purple or orange (Fig. 6). Highest siderophore produc- ing rhizospheric isolates were RLS18, RLS53 and RLS58. Siderophore production was recorded in the form of change in colour from blue to purple or orange (Milagres et al., 1999). CAS agar plate assay indicated that all the rhizobacterial isolates have siderophore production abil- ity, RLS18, RLS53 and RLS58 found superior compared to other rhizospheric isolates. Similar result was reported by other researchers (Chaiharn et al., 2009). The qualita- tive production of siderophore by B. cereus with orange halos during the exponential growth and Sporulation phases by modified CAS plate assay (Lalloo et al., 2010).
Production of siderophores by different fungal spe- cies isolated from heavy metal contaminated and
Table 1: In vitro screening of antifungal activity of rhizospheric isolates against Fusarium oxysporum f.sp. vasinfectum
LS Raut and VS Hamde
Table 2: Production of diffusible antifungal metabolites by rhizospheric isolates against Fusarium oxysporum f.sp. vasinfectum
Table 3: Siderophore production of rhizospheric isolates by modified CAS assay
+= less siderophore production ++=medium siderophore production +++= high siderophore production
uncontaminated soils was studied by using Chrome azurol sulfonate (CAS) was used for both quantitative and qualitative evaluation of siderophores production. The production of siderophores in rhizosphere fungi was higher than those isolated from the contaminated soil (Hussein and Joo, 2012). Siderophore production by Fungal M10 strain was studied using CAS blue agar and the
CONCLUSION
Our results suggest that the rhizospheric isolate RLS19, RLS52 RLS53 and RLS72 inhibits the mycelial growth
of Fusarium oxysporum f.sp. vasinfectum by producing volatile metabolites diffusible metabolites and sidero- phore. The cumulative effect of these secondary metab- olites resulted in inhibition of mycelial growth of the pathogen causing Fusarium wilt of
ACKNOWLEDGEMENTS
The authors wish to thank Dr. B. R. Chavan Principal, Yogeshwari Mahavidyalaya, Ambajogai for providing facilities to conduct the research.
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