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Pseudomonas as a Prospective Candidate For Minimization in use of Chemical Pesticides or their Gradual Replacement with Biocontrol Agents on Agricultural Fields

Pseudomonas as a Prospective Candidate For Minimization in use of Chemical Pesticides or their... DOI: 10.2478/v10207-012-0015-6 POORNIMA SHARMA Government Model Science College, Pachpedi, Jabalpur, M.P. Mata Gujri Women's College, Marhatal, Jabalpur, M.P. SHARMA, P.: Pseudomonas as a prospective candidate for minimization in use of chemical pesticides or their gradual replacement with biocontrol agents on agricultural fields. Agriculture (Ponohospodárstvo), vol. 58, 2012, no. 4, pp. 138­145. Isolates of Pseudomonas species obtained from rhizosphere, rhizoplane and endophytic habitats of various plants were tested for their antagonistic activity against plant pathogenic Fusarium oxysporum isolated from the roots of a diseased pea plant. Pseudomonas isolate P5 showed best activity against F. oxysporum followed by P4, P22 and P17. Some isolates of Pseudomonas, P24, P25 and P26, grew unrestrictedly and completely covered the Fusarium colony. The antagonistic effect of Pseudomonas on F. oxysporum observed in this research can be attributed to the ability of antibiotic synthesis by Pseudomonas and also the ability to counter the toxic metabolites of Fusarium oxysporum. Key words: plant disease, Fusarium oxysporum, biological control, Pseudomonas Human has developed an agricultural ecosystem which is an artificial ecosystem for the cultivation of plants and animals appropriate for food. This artificial ecosystem has disturbed a steady balance between the host plant and pathogen allowing development of organisms pathogenic to the host crop plant in excess; therefore, the cultivated plants are more vulnerable to disease (Oku 1994). To overcome this problem, new varieties of crops are developed. But, after a period of time these new varieties also become susceptible to the pathogen, as new races of the pathogen develop with time (Oku 1994; Trigiano et al. 2004). Fusarium wilts and root rot diseases have been a problem throughout the world for a long period of time. Padwick (1938), Buxton and Storey (1954), Haware (1971), Clarkson (1978), Manzies and Koch (1990), Keinath (1994) and many other researchers have discussed Fusarium wilt as a major problem in several crops. The problem still exists in the present times. Walsh et al. (2010), Polizzi et al. (2011) and several researchers have reported the spread of Fusarium wilt diseases to new host varieties of crops and in new regions of the world. A number of Fusarium species have been implicated in this problem, majorly F. oxysporum. The control of this organism has been an important confront as the pathogenic forms of Fusarium oxysporum show great genetic variability (Leslie & Summerell 2006). There is an increasing consideration in using physical methods and cultural practices in disease management as alternatives to pesticides for the control of soil-borne pathogens (Katan 2000). But, the physical methods being unfeasible for the general masses of farmers and unsuitable for large areas may not be acceptable in the long run. Crop rotation is a cultural method of disease control which may be effective in some cases. Poornima Sharma, Institutional Address 1: Government Model Science College, Pachpedi, Jabalpur-482001, Madhya Pradesh, India. Institutional Address 2: Mata Gujri Women's College, Civic Center, Marhatal, Jabalpur-482002, Madhya Pradesh, India. E-mail: poornima.sharma.india@gmail.com Chemical treatment has been the most effective and popular method of disease control till date. Fungicides such as methyl bromide, captan and carbendazim have been used effectively against Fusarium oxysporum (Melero-Vara et al. 2005; Mukhtar 2007). But with time, the uncontrolled use of chemicals has posed many environmental problems and risks to the health of humans and other organisms. Therefore, there is a need to minimize the use of hazardous chemicals and gradually increase the use of natural and safe methods of controlling pathogen. Widespread investigation on the biological interactions that occur in the rhizosphere of plants has revealed a number of agents which are able to control plant diseases. The mechanisms involved by these biocontrol agents have been classified as antibiosis, competition, parasitism and induced resistance to plant diseases. The concept of rhizosphere competence of these agents is a major consideration. These micro-organisms called biological control agents are also implicated in plant growth promotion (Whipps 2001). A number of bacteria and fungi have been studied which are known to control pathogenic micro-organisms in the rhizosphere. Among fungi, Trichoderma has been considered as a promising biocontrol agent (Harman et al. 2004; Zeilinger & Omann 2007; Sharma 2011). Bacteria such as Bacillus subtilis, B. cepacia Serratia plymuthica, Pseudomonas putida and P. fluorescens, P. aureofaciens (Duijff et al. 1999; Whipps 2001) have also been evaluated with promising results. These bacterial agents especially Pseudomonas spp. are known to show strong antibiosis to pathogens. They produce antibiotics such as butyrolactones, HCN, kanosamine, viscosinamide and zwittermycin A and many other antibiotics some of which are yet to be characterized (Kim et al. 1999; Whipps 2001). Another mechanism of biological control is the competition for nutrients. In case of Pseudomonas, it is recognized that bacterial iron chelators can impound iron when it is present in limited quantity and make it unavailable to pathogenic fungi (Loper & Henkels 1999). The pathogenic forms of Fusarium oxysporum require available Fe for the germination and penetration of root tips of susceptible host. Both, fluorescent Pseudomonas and F. oxysporum produce specific Fe chelating compounds (siderophores). But, siderophores of F. oxysporum have less affinity for Fe than the siderophores of Pseudomonas. Thus, Pseudomonas can easily deprive F. oxysporum of Fe under conditions of iron scarcity and result in decreased likelihood of root infection (Baker 1986). Parasitism is yet another mechanism involved in biocontrol. Certain bacteria such as Arthrobacter are known to parasitize the fungus Pythium debaryanum and cause complete lysis of fungal host (Mitchell & Hurwitz 1965). Rhizosphere bacteria, specifically, Bacillus and Pseudomonas are believed to induce systemic resistance in plants by enhanced expression of stress related genes. Some of these bacteria are also able to colonize root tissue internally (van Loon 1998; Whipps 2001). The role of rhizospheric bacteria in plant health promotion may be indirect by suppressing pathogens in the rhizosphere of plants or by the supporting growth of mycorrhizal fungi. Plant growth enhancement may also be achieved directly by associative nitrogen fixation and solubilization of nutrients by these bacteria. Thereafter, these nutrients are made available to plants (Whipps 2001). With such an intensive investigation on the attributes of bacterial agents and their promising prospective in agriculture attempts to popularize biological methods of pest control is necessary. This research aims at reinforcing faith in biocontrol agents and a gradual reduction in use of synthetic chemicals in agriculture. MATERIAL AND METHODS The pathogen taken under study was Fusarium oxysporum (isolate FP-02/G) isolated from the diseased pea plant root. The pathogen has been deposited at ARI, Pune (India) under accession no. NFCCI2195. Isolation and screening of microbes to obtain potential antagonists of the pathogen Fungi and bacteria both were screened to isolate antagonists of the pathogen F. oxysporum. A similar line of investigation was followed for both. Amongst fungi, several strains of Trichoderma showed considerable activity against F. oxysporum. Activity of Trichoderma has been discussed in another paper (Sharma 2011). The present paper focuses on bacterial isolates showing biocontrol potential. The screening for bacterial agents was performed in two phases. In the first as well as the second phase screening, healthy pea plants and other plants from var- ious regions of Jabalpur district (India) were arbitrarily selected. The roots of these plants were employed for isolation of microbial agents from rhizosphere, rhizoplane and endophytic habitats with expected potential to control the growth of pathogen under study. All the isolated microbes were screened for their antagonism on the pathogen. Dual culture technique was followed for the first and second phase screening. All isolations in the first phase were done on nutrient agar medium (3.0 g Beef Extract, 5.0 g Peptone, 2.0 g Sodium chloride, 15.0 g Agar) and the inoculated medium plates were incubated at 30°C for 24 hours. Modified methods were adopted for isolation from rhizosphere and rhizoplane (Pandey & Palni 1998; Hasan 2002). a) Isolation of microbes from rhizosphere: Roots of freshly obtained plants were taken and the soil clumps adhered to roots were shed off. The roots were washed in a minimum amount of sterilized distilled water for 10 to 15 seconds. This washing was taken as sample suspension containing rhizosphere microbes; 0.1 ml of suspension was spread on the medium. b) Isolation of microbes from rhizoplane: Roots were washed under tap water for 2­3 minutes and cut into segments of 3­4 centimetre. The segments were washed in sterilized distilled water several times and left in sterilized distilled water for 5 minutes. Thereafter, the segments were placed on the medium. c) Isolation of endophytes: Modification of method by Paul et al. (2007) was adopted. Root segments were washed as mentioned in b) and then treated with 0.05% of HgCl2 for 30 seconds to one minute depending on the kind of root. The segments were rinsed several times in sterilized distilled water. About a half centimetre portion was cut off from either side of each root segment and then the segment was placed on the medium. Test of antagonism by dual culture technique on 90 mm petri-plate (modified methods adopted from Dhingra and Sinclair 1995; Nair and Anith 2009) The isolates obtained by the above method were subjected to dual culture technique to test their activity against the pathogen. Isolates showing zone of inhibition against pathogen were tentatively identified to be Pseudomonas when subjected to the gelatine liquefaction test. Also, they showed growth below 10°C (King et al. 1954; Blazevic et al. 1973). Based on the results obtained in the first screening phase, second screening phase was planned. In the second screening phase, Pseudomonas spp. were specifically isolated on Pseudomonas selective medium (for fluorescein) [HiMedium, M120, Lot-24035] and subjected to the dual culture technique. The test was performed on potato dextrose agar medium (Central Drug House (P) Ltd., JO 0013, Batch 51037). The fungal pathogen was point inoculated and 200 µL broth culture of the expected control agent was aliquoted in a well (7 mm diameter) at a distance of 12 mm opposite to the pathogen (Figure 1). C ­ control agent, Z ­ zone of inhibition X ­ growth towards control agent Y ­ growth in the opposite direction Z is directly proportional to difference (Dfp) between X and Y % Inhibition = (Dfp / Y) × 100 Figure 1. Diagrammatic representation of biocontrol activity Analysis of observations recorded in Pseudomonas-Fusarium dual culture Observations were taken as the pathogen approached the well. The difference (Dfp) in growth of the pathogen towards the well (control agent) from the point of inoculation and in the opposite direction from point of inoculation was associated with the zone of inhibition (Z) formed by the control agent against pathogen. This relation was analysed statistically by MS Excel Data Analysis ToolPak application for Correlation. Identification of Pseudomonas Pseudomonas was cultured in Pseudomonas selective (for fluorescein) broth medium for 48 hours till visibly sufficient density was obtained; thereafter, glycerol (20% v/v) was added to the culture and was stored at about 10°C. Pseudomonas was tentatively identified on the basis of its growth on Pseudomonas selective (for fluorescein) agar medium, gram staining, and gelatine liquefaction test (King et al.1954; Blazevic et al. 1973). from rhizoplane and eleven isolates obtained from rhizosphere of plants showed considerable results. In this research, endophytic isolates did not show comparably good results. Table 1 shows the potential of these twenty-three isolates. Unidentified antibiotics released from Pseudomonas culture aliquot in the well, diffused into the surrounding agar medium and inhibited the growth of F. oxysporum. This was evident by measuring the growth of F. oxysporum towards and away from Pseudomonas culture aliquot and relating the growth RESULTS AND DISCUSSION Forty five isolates of Pseudomonas were tested in both the phases of screening. Twelve isolates obtained T a b l e 1 Analysis of antagonistic activity of Pseudomonas spp. on Fusarium oxysporum % inhibition of growth posed by Pseudomonas sp. on F. oxysporum (Dfp / Y) ×100 44 36 24 24 19 24 18 16 15 13 15 10 9 13 8 9 9 7 7 8 7 3 3 Source plant / habitat rhizoplane (rp) rhizosphere (rs) Abelmoschus esculentus Datura spp. Annona squamosa Brassica oleracea Solanum molongera Solanum lycopersicum Carica papaya Pisum sativum Musa paradisiaca Bougainvillae "Barbara K" Bergera Koenigii Mangifera indica Psidium guajava Vinca rosea Glycine max Azadirachta indica Cicer arietinum Bougainvillae "Buttiana" Ocimum sanctum Brassica juncea Tagetes patula Aloe vera Tagetes erecta rp rp rp rp rp rs rs rp rp rp rs rs rs rp rs rp rp rs rs rs rp rs rs Isolate no. Growth difference Y ­ X = Dfp [mm] Zone of inhibition, Z [mm] 4.38 3.50 3.38 3.00 2.88 2.63 2.63 2.38 2.25 2.13 2.00 2.00 1.75 1.75 1.63 1.63 1.63 1.25 1.25 1.13 1.13 1.00 0.55 P5 P4 P22 P17 P19 P21 P23 P18 P6 P1 P7 P16 P9 P11 P10 P13 P20 P12 P15 P2 P3 P8 P14 Isolates of Pseudomonas showing visibly considerable inhibition to Fusarium were selected and recoded X = Growth of Fusarium towards Pseudomonas Y = Growth of Fusarium away from Pseudomonas Data given is an average of quadruplicates Arranged in the order of zone of inhibition measurements Readings were taken after about 55 hours of beginning the experiment when the zone of inhibition was visible distinctly Positive correlation (0.96568) between Dfp and Z obtained through MS Excel Data Analysis ToolPak application Agriculture (Ponohospodárstvo), 58, 2012 (4): 138­145 difference to the zone of inhibition. Several isolates showed clearly distinct zone of inhibition. Zone of inhibition in relation to the growth of Fusarium oxysporum The difference in the growth of F. oxysporum towards and away from Pseudomonas (Dfp) quite well corresponded to the zone of inhibition (Z). An overall directly proportional relationship between Dfp and Z was observed with a correlation value of 0.96568. This confirmed the inhibitory effect of Pseudomonas isolates on F. oxysporum. Pseudomonas isolate P5 showed best activity against F. oxysporum followed by P4, P22 and P17 as evident (Table 1). The effect of Pseudomonas on F. oxysporum can be visualized in Figure 2 (a & b). Most of the Pseudomonas isolates were not able to grow on PDA medium used for dual culture, but some isolates (P24, P25 and P26) grew unrestrictedly and completely covered the Fusarium colony. Although, such a swathe of Pseudomonas interfered with the growth of F. oxysporum but it was not confirmed if it completely restricted the growth of F. oxysporum beneath the swathe. The antagonistic effect of Pseudomonas on F. oxysporum observed in this research may be attributed to the ability of antibiotic synthesis by Pseudomonas. Pseudomonas species are known to produce a number of antibiotics such as Amphisin, Oomycin A, Tensin, Tropolone and cyclic lipopeptides (Compant et al. 2005). Pyoluteorin, Pyrrolnitrin, hydrogen cyanide, phenazine-1-carboxylate are also produced by Pseudomonas. Lactone, 2,3-de-epoxy-2,3-didehydra-rhizoxin is also in the list (Shalini & Srivastava 2008). Zone of inhibition in relation to growth of Pseudomonas The effect of F. oxysporum on Pseudomonas has not been evaluated in this work. The interaction is assumed to show unidirectional antibiosis. Since Pseudomonas was cultured separately and then inoculated in the wells of the dual culture plate after about 48 hours, Pseudomonas is assumed to synthesize enough antibiotics to inhibit F. oxysporum by this time period. Therefore, evaluating the effect of F. oxysporum on Pseudomonas in this experiment might be futile. The broth culture of Pseudomonas aliquot in wells may still produce antibiotics in the initial stages of inoculation until the broth soaks and diffuses around the well with time and F. oxysporum begins to release toxins to interfere with antibiotic production by Pseudomonas. Although, in cases where Pseudomonas isolates were overwhelming the Fusarium colony, it is interpreted that F. oxysporum was unable to restrict the growth of Pseudomonas. Other researchers such as Duffy and Defago (1997) found in their experiment that biosynthesis of impor- Figure 2 (a & b). Zone of inhibition created by bioagent Pseudomonas sp. Isolate no.P5 and P22 against pathogen Fusarium oxysporum after 48 hours of experiment tant antibiotics, 2,4-diacetylphloroglucinol and pyoluteorin, by Pseudomonas is retarded even at low concentrations of fusaric acid. They stated that toxin fusaric acid produced by Fusarium spp. affected the biosynthesis of antibiotics but not preformed antibiotics. Also, bacterial growth was not affected even at high concentrations of fusaric acid. Tentative identification of bacterial biocontrol agent The Gram negative rod-shaped bacterial isolates showed fluorescence on Pseudomonas selective agar medium (Figure 3) and also, yellow green pigmentation was noticed in Pseudomonas selective broth medium. The isolates grew profusely at about 30°C. On the basis of literature available, Pseudomonas aeruginosa build colonies surrounded by a yellow to greenish-yellow zone due to fluorescein production which fluoresces under UV light. If pyocyanin is also synthesized, a bright green colour is produced. Most pyocyanin-producing Pseudomonas strain synthesize fluorescein also and others produce just one of the pigments. Temperature can be a determining factor as most fluorescent strains will not grow at 35°C and higher. Rather, they grow between 25°C and 35°C (King et al. 1954; Blazevic et al. 1973). The findings in this research reconfirm the biocontrol potential of Pseudomonas. Research by several workers over a period of time has elicited interest and faith in the prospects of bacterial agents like Pseudomonas in plant disease control. Production of antibiotics, inhibition of several pathogens such as Fusarium oxysporum, solubilization of inorganic phosphate and plant growth promotion are the attributes investigated upon by several researchers (Landa et al. 2002; Sarathchandra et al. 1993; Compant et al. 2005; Hallman et al. 1997; van Loon et al.1998; Negi et al. 2005). Production of siderophores and competition for nutrients is another mechanism of pathogen control (Buysens et al. 1996). Though Pseudomonas and other biocontrol agents have been studied in detail over the years, often the results obtained in laboratory experiments may not be reproducible on agricultural lands due to various reasons or for the fact that the effect of biocontrol agent may be seen after repeated application of the agent on field over a period of time. Under such circumstances where using biocontrol agent alone may not be very effective, Integrated Pest Management (IPM) can be adopted. Integrated Pest Management is a strategy that focuses on long-term solution of the pests through a combination of techniques such as biological control (Pseudomonas and Trichoderma), habitat manipulation, modification of agronomic practices, and use of resistant varieties (Birthal & Sharma 2004). Preferably, pesticides should be used as a last remedy in IPM programmes because of their potential negative effect on the environment. Botanical pesticides can be used as raw crushed plant leaves, extracts of plant parts or chemicals purified from the plants (Birthal & Sharma 2004; Ehler 2006). CONCLUSION In the present research, several isolates of Pseudomonas showed inhibitory effect on the growth of pathogenic Fusarium oxysporum. Pseudomonas Isolates P5, P4, P22 and P17 gave best results followed by other listed isolates. In the present time, natural antagonists of agricultural pests are an important alternative to synthetic pesticides. Bacterial and fungal agents have been studied as biocontrol agents with promising results. Proper strategy of implementation and commercialization of these agents is necessary for their efficient outcome in agricultural fields. Acknowledgements: I thank all individuals who supported my research and offered necessary help throughout my study. 143 Figure 3. Glimpse of fluorescence noticed in Pseudomonas sp. Isolate no. 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Pseudomonas as a Prospective Candidate For Minimization in use of Chemical Pesticides or their Gradual Replacement with Biocontrol Agents on Agricultural Fields

Agriculture , Volume 58 (4) – Dec 1, 2012

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Abstract

DOI: 10.2478/v10207-012-0015-6 POORNIMA SHARMA Government Model Science College, Pachpedi, Jabalpur, M.P. Mata Gujri Women's College, Marhatal, Jabalpur, M.P. SHARMA, P.: Pseudomonas as a prospective candidate for minimization in use of chemical pesticides or their gradual replacement with biocontrol agents on agricultural fields. Agriculture (Ponohospodárstvo), vol. 58, 2012, no. 4, pp. 138­145. Isolates of Pseudomonas species obtained from rhizosphere, rhizoplane and endophytic habitats of various plants were tested for their antagonistic activity against plant pathogenic Fusarium oxysporum isolated from the roots of a diseased pea plant. Pseudomonas isolate P5 showed best activity against F. oxysporum followed by P4, P22 and P17. Some isolates of Pseudomonas, P24, P25 and P26, grew unrestrictedly and completely covered the Fusarium colony. The antagonistic effect of Pseudomonas on F. oxysporum observed in this research can be attributed to the ability of antibiotic synthesis by Pseudomonas and also the ability to counter the toxic metabolites of Fusarium oxysporum. Key words: plant disease, Fusarium oxysporum, biological control, Pseudomonas Human has developed an agricultural ecosystem which is an artificial ecosystem for the cultivation of plants and animals appropriate for food. This artificial ecosystem has disturbed a steady balance between the host plant and pathogen allowing development of organisms pathogenic to the host crop plant in excess; therefore, the cultivated plants are more vulnerable to disease (Oku 1994). To overcome this problem, new varieties of crops are developed. But, after a period of time these new varieties also become susceptible to the pathogen, as new races of the pathogen develop with time (Oku 1994; Trigiano et al. 2004). Fusarium wilts and root rot diseases have been a problem throughout the world for a long period of time. Padwick (1938), Buxton and Storey (1954), Haware (1971), Clarkson (1978), Manzies and Koch (1990), Keinath (1994) and many other researchers have discussed Fusarium wilt as a major problem in several crops. The problem still exists in the present times. Walsh et al. (2010), Polizzi et al. (2011) and several researchers have reported the spread of Fusarium wilt diseases to new host varieties of crops and in new regions of the world. A number of Fusarium species have been implicated in this problem, majorly F. oxysporum. The control of this organism has been an important confront as the pathogenic forms of Fusarium oxysporum show great genetic variability (Leslie & Summerell 2006). There is an increasing consideration in using physical methods and cultural practices in disease management as alternatives to pesticides for the control of soil-borne pathogens (Katan 2000). But, the physical methods being unfeasible for the general masses of farmers and unsuitable for large areas may not be acceptable in the long run. Crop rotation is a cultural method of disease control which may be effective in some cases. Poornima Sharma, Institutional Address 1: Government Model Science College, Pachpedi, Jabalpur-482001, Madhya Pradesh, India. Institutional Address 2: Mata Gujri Women's College, Civic Center, Marhatal, Jabalpur-482002, Madhya Pradesh, India. E-mail: poornima.sharma.india@gmail.com Chemical treatment has been the most effective and popular method of disease control till date. Fungicides such as methyl bromide, captan and carbendazim have been used effectively against Fusarium oxysporum (Melero-Vara et al. 2005; Mukhtar 2007). But with time, the uncontrolled use of chemicals has posed many environmental problems and risks to the health of humans and other organisms. Therefore, there is a need to minimize the use of hazardous chemicals and gradually increase the use of natural and safe methods of controlling pathogen. Widespread investigation on the biological interactions that occur in the rhizosphere of plants has revealed a number of agents which are able to control plant diseases. The mechanisms involved by these biocontrol agents have been classified as antibiosis, competition, parasitism and induced resistance to plant diseases. The concept of rhizosphere competence of these agents is a major consideration. These micro-organisms called biological control agents are also implicated in plant growth promotion (Whipps 2001). A number of bacteria and fungi have been studied which are known to control pathogenic micro-organisms in the rhizosphere. Among fungi, Trichoderma has been considered as a promising biocontrol agent (Harman et al. 2004; Zeilinger & Omann 2007; Sharma 2011). Bacteria such as Bacillus subtilis, B. cepacia Serratia plymuthica, Pseudomonas putida and P. fluorescens, P. aureofaciens (Duijff et al. 1999; Whipps 2001) have also been evaluated with promising results. These bacterial agents especially Pseudomonas spp. are known to show strong antibiosis to pathogens. They produce antibiotics such as butyrolactones, HCN, kanosamine, viscosinamide and zwittermycin A and many other antibiotics some of which are yet to be characterized (Kim et al. 1999; Whipps 2001). Another mechanism of biological control is the competition for nutrients. In case of Pseudomonas, it is recognized that bacterial iron chelators can impound iron when it is present in limited quantity and make it unavailable to pathogenic fungi (Loper & Henkels 1999). The pathogenic forms of Fusarium oxysporum require available Fe for the germination and penetration of root tips of susceptible host. Both, fluorescent Pseudomonas and F. oxysporum produce specific Fe chelating compounds (siderophores). But, siderophores of F. oxysporum have less affinity for Fe than the siderophores of Pseudomonas. Thus, Pseudomonas can easily deprive F. oxysporum of Fe under conditions of iron scarcity and result in decreased likelihood of root infection (Baker 1986). Parasitism is yet another mechanism involved in biocontrol. Certain bacteria such as Arthrobacter are known to parasitize the fungus Pythium debaryanum and cause complete lysis of fungal host (Mitchell & Hurwitz 1965). Rhizosphere bacteria, specifically, Bacillus and Pseudomonas are believed to induce systemic resistance in plants by enhanced expression of stress related genes. Some of these bacteria are also able to colonize root tissue internally (van Loon 1998; Whipps 2001). The role of rhizospheric bacteria in plant health promotion may be indirect by suppressing pathogens in the rhizosphere of plants or by the supporting growth of mycorrhizal fungi. Plant growth enhancement may also be achieved directly by associative nitrogen fixation and solubilization of nutrients by these bacteria. Thereafter, these nutrients are made available to plants (Whipps 2001). With such an intensive investigation on the attributes of bacterial agents and their promising prospective in agriculture attempts to popularize biological methods of pest control is necessary. This research aims at reinforcing faith in biocontrol agents and a gradual reduction in use of synthetic chemicals in agriculture. MATERIAL AND METHODS The pathogen taken under study was Fusarium oxysporum (isolate FP-02/G) isolated from the diseased pea plant root. The pathogen has been deposited at ARI, Pune (India) under accession no. NFCCI2195. Isolation and screening of microbes to obtain potential antagonists of the pathogen Fungi and bacteria both were screened to isolate antagonists of the pathogen F. oxysporum. A similar line of investigation was followed for both. Amongst fungi, several strains of Trichoderma showed considerable activity against F. oxysporum. Activity of Trichoderma has been discussed in another paper (Sharma 2011). The present paper focuses on bacterial isolates showing biocontrol potential. The screening for bacterial agents was performed in two phases. In the first as well as the second phase screening, healthy pea plants and other plants from var- ious regions of Jabalpur district (India) were arbitrarily selected. The roots of these plants were employed for isolation of microbial agents from rhizosphere, rhizoplane and endophytic habitats with expected potential to control the growth of pathogen under study. All the isolated microbes were screened for their antagonism on the pathogen. Dual culture technique was followed for the first and second phase screening. All isolations in the first phase were done on nutrient agar medium (3.0 g Beef Extract, 5.0 g Peptone, 2.0 g Sodium chloride, 15.0 g Agar) and the inoculated medium plates were incubated at 30°C for 24 hours. Modified methods were adopted for isolation from rhizosphere and rhizoplane (Pandey & Palni 1998; Hasan 2002). a) Isolation of microbes from rhizosphere: Roots of freshly obtained plants were taken and the soil clumps adhered to roots were shed off. The roots were washed in a minimum amount of sterilized distilled water for 10 to 15 seconds. This washing was taken as sample suspension containing rhizosphere microbes; 0.1 ml of suspension was spread on the medium. b) Isolation of microbes from rhizoplane: Roots were washed under tap water for 2­3 minutes and cut into segments of 3­4 centimetre. The segments were washed in sterilized distilled water several times and left in sterilized distilled water for 5 minutes. Thereafter, the segments were placed on the medium. c) Isolation of endophytes: Modification of method by Paul et al. (2007) was adopted. Root segments were washed as mentioned in b) and then treated with 0.05% of HgCl2 for 30 seconds to one minute depending on the kind of root. The segments were rinsed several times in sterilized distilled water. About a half centimetre portion was cut off from either side of each root segment and then the segment was placed on the medium. Test of antagonism by dual culture technique on 90 mm petri-plate (modified methods adopted from Dhingra and Sinclair 1995; Nair and Anith 2009) The isolates obtained by the above method were subjected to dual culture technique to test their activity against the pathogen. Isolates showing zone of inhibition against pathogen were tentatively identified to be Pseudomonas when subjected to the gelatine liquefaction test. Also, they showed growth below 10°C (King et al. 1954; Blazevic et al. 1973). Based on the results obtained in the first screening phase, second screening phase was planned. In the second screening phase, Pseudomonas spp. were specifically isolated on Pseudomonas selective medium (for fluorescein) [HiMedium, M120, Lot-24035] and subjected to the dual culture technique. The test was performed on potato dextrose agar medium (Central Drug House (P) Ltd., JO 0013, Batch 51037). The fungal pathogen was point inoculated and 200 µL broth culture of the expected control agent was aliquoted in a well (7 mm diameter) at a distance of 12 mm opposite to the pathogen (Figure 1). C ­ control agent, Z ­ zone of inhibition X ­ growth towards control agent Y ­ growth in the opposite direction Z is directly proportional to difference (Dfp) between X and Y % Inhibition = (Dfp / Y) × 100 Figure 1. Diagrammatic representation of biocontrol activity Analysis of observations recorded in Pseudomonas-Fusarium dual culture Observations were taken as the pathogen approached the well. The difference (Dfp) in growth of the pathogen towards the well (control agent) from the point of inoculation and in the opposite direction from point of inoculation was associated with the zone of inhibition (Z) formed by the control agent against pathogen. This relation was analysed statistically by MS Excel Data Analysis ToolPak application for Correlation. Identification of Pseudomonas Pseudomonas was cultured in Pseudomonas selective (for fluorescein) broth medium for 48 hours till visibly sufficient density was obtained; thereafter, glycerol (20% v/v) was added to the culture and was stored at about 10°C. Pseudomonas was tentatively identified on the basis of its growth on Pseudomonas selective (for fluorescein) agar medium, gram staining, and gelatine liquefaction test (King et al.1954; Blazevic et al. 1973). from rhizoplane and eleven isolates obtained from rhizosphere of plants showed considerable results. In this research, endophytic isolates did not show comparably good results. Table 1 shows the potential of these twenty-three isolates. Unidentified antibiotics released from Pseudomonas culture aliquot in the well, diffused into the surrounding agar medium and inhibited the growth of F. oxysporum. This was evident by measuring the growth of F. oxysporum towards and away from Pseudomonas culture aliquot and relating the growth RESULTS AND DISCUSSION Forty five isolates of Pseudomonas were tested in both the phases of screening. Twelve isolates obtained T a b l e 1 Analysis of antagonistic activity of Pseudomonas spp. on Fusarium oxysporum % inhibition of growth posed by Pseudomonas sp. on F. oxysporum (Dfp / Y) ×100 44 36 24 24 19 24 18 16 15 13 15 10 9 13 8 9 9 7 7 8 7 3 3 Source plant / habitat rhizoplane (rp) rhizosphere (rs) Abelmoschus esculentus Datura spp. Annona squamosa Brassica oleracea Solanum molongera Solanum lycopersicum Carica papaya Pisum sativum Musa paradisiaca Bougainvillae "Barbara K" Bergera Koenigii Mangifera indica Psidium guajava Vinca rosea Glycine max Azadirachta indica Cicer arietinum Bougainvillae "Buttiana" Ocimum sanctum Brassica juncea Tagetes patula Aloe vera Tagetes erecta rp rp rp rp rp rs rs rp rp rp rs rs rs rp rs rp rp rs rs rs rp rs rs Isolate no. Growth difference Y ­ X = Dfp [mm] Zone of inhibition, Z [mm] 4.38 3.50 3.38 3.00 2.88 2.63 2.63 2.38 2.25 2.13 2.00 2.00 1.75 1.75 1.63 1.63 1.63 1.25 1.25 1.13 1.13 1.00 0.55 P5 P4 P22 P17 P19 P21 P23 P18 P6 P1 P7 P16 P9 P11 P10 P13 P20 P12 P15 P2 P3 P8 P14 Isolates of Pseudomonas showing visibly considerable inhibition to Fusarium were selected and recoded X = Growth of Fusarium towards Pseudomonas Y = Growth of Fusarium away from Pseudomonas Data given is an average of quadruplicates Arranged in the order of zone of inhibition measurements Readings were taken after about 55 hours of beginning the experiment when the zone of inhibition was visible distinctly Positive correlation (0.96568) between Dfp and Z obtained through MS Excel Data Analysis ToolPak application Agriculture (Ponohospodárstvo), 58, 2012 (4): 138­145 difference to the zone of inhibition. Several isolates showed clearly distinct zone of inhibition. Zone of inhibition in relation to the growth of Fusarium oxysporum The difference in the growth of F. oxysporum towards and away from Pseudomonas (Dfp) quite well corresponded to the zone of inhibition (Z). An overall directly proportional relationship between Dfp and Z was observed with a correlation value of 0.96568. This confirmed the inhibitory effect of Pseudomonas isolates on F. oxysporum. Pseudomonas isolate P5 showed best activity against F. oxysporum followed by P4, P22 and P17 as evident (Table 1). The effect of Pseudomonas on F. oxysporum can be visualized in Figure 2 (a & b). Most of the Pseudomonas isolates were not able to grow on PDA medium used for dual culture, but some isolates (P24, P25 and P26) grew unrestrictedly and completely covered the Fusarium colony. Although, such a swathe of Pseudomonas interfered with the growth of F. oxysporum but it was not confirmed if it completely restricted the growth of F. oxysporum beneath the swathe. The antagonistic effect of Pseudomonas on F. oxysporum observed in this research may be attributed to the ability of antibiotic synthesis by Pseudomonas. Pseudomonas species are known to produce a number of antibiotics such as Amphisin, Oomycin A, Tensin, Tropolone and cyclic lipopeptides (Compant et al. 2005). Pyoluteorin, Pyrrolnitrin, hydrogen cyanide, phenazine-1-carboxylate are also produced by Pseudomonas. Lactone, 2,3-de-epoxy-2,3-didehydra-rhizoxin is also in the list (Shalini & Srivastava 2008). Zone of inhibition in relation to growth of Pseudomonas The effect of F. oxysporum on Pseudomonas has not been evaluated in this work. The interaction is assumed to show unidirectional antibiosis. Since Pseudomonas was cultured separately and then inoculated in the wells of the dual culture plate after about 48 hours, Pseudomonas is assumed to synthesize enough antibiotics to inhibit F. oxysporum by this time period. Therefore, evaluating the effect of F. oxysporum on Pseudomonas in this experiment might be futile. The broth culture of Pseudomonas aliquot in wells may still produce antibiotics in the initial stages of inoculation until the broth soaks and diffuses around the well with time and F. oxysporum begins to release toxins to interfere with antibiotic production by Pseudomonas. Although, in cases where Pseudomonas isolates were overwhelming the Fusarium colony, it is interpreted that F. oxysporum was unable to restrict the growth of Pseudomonas. Other researchers such as Duffy and Defago (1997) found in their experiment that biosynthesis of impor- Figure 2 (a & b). Zone of inhibition created by bioagent Pseudomonas sp. Isolate no.P5 and P22 against pathogen Fusarium oxysporum after 48 hours of experiment tant antibiotics, 2,4-diacetylphloroglucinol and pyoluteorin, by Pseudomonas is retarded even at low concentrations of fusaric acid. They stated that toxin fusaric acid produced by Fusarium spp. affected the biosynthesis of antibiotics but not preformed antibiotics. Also, bacterial growth was not affected even at high concentrations of fusaric acid. Tentative identification of bacterial biocontrol agent The Gram negative rod-shaped bacterial isolates showed fluorescence on Pseudomonas selective agar medium (Figure 3) and also, yellow green pigmentation was noticed in Pseudomonas selective broth medium. The isolates grew profusely at about 30°C. On the basis of literature available, Pseudomonas aeruginosa build colonies surrounded by a yellow to greenish-yellow zone due to fluorescein production which fluoresces under UV light. If pyocyanin is also synthesized, a bright green colour is produced. Most pyocyanin-producing Pseudomonas strain synthesize fluorescein also and others produce just one of the pigments. Temperature can be a determining factor as most fluorescent strains will not grow at 35°C and higher. Rather, they grow between 25°C and 35°C (King et al. 1954; Blazevic et al. 1973). The findings in this research reconfirm the biocontrol potential of Pseudomonas. Research by several workers over a period of time has elicited interest and faith in the prospects of bacterial agents like Pseudomonas in plant disease control. Production of antibiotics, inhibition of several pathogens such as Fusarium oxysporum, solubilization of inorganic phosphate and plant growth promotion are the attributes investigated upon by several researchers (Landa et al. 2002; Sarathchandra et al. 1993; Compant et al. 2005; Hallman et al. 1997; van Loon et al.1998; Negi et al. 2005). Production of siderophores and competition for nutrients is another mechanism of pathogen control (Buysens et al. 1996). Though Pseudomonas and other biocontrol agents have been studied in detail over the years, often the results obtained in laboratory experiments may not be reproducible on agricultural lands due to various reasons or for the fact that the effect of biocontrol agent may be seen after repeated application of the agent on field over a period of time. Under such circumstances where using biocontrol agent alone may not be very effective, Integrated Pest Management (IPM) can be adopted. Integrated Pest Management is a strategy that focuses on long-term solution of the pests through a combination of techniques such as biological control (Pseudomonas and Trichoderma), habitat manipulation, modification of agronomic practices, and use of resistant varieties (Birthal & Sharma 2004). Preferably, pesticides should be used as a last remedy in IPM programmes because of their potential negative effect on the environment. Botanical pesticides can be used as raw crushed plant leaves, extracts of plant parts or chemicals purified from the plants (Birthal & Sharma 2004; Ehler 2006). CONCLUSION In the present research, several isolates of Pseudomonas showed inhibitory effect on the growth of pathogenic Fusarium oxysporum. Pseudomonas Isolates P5, P4, P22 and P17 gave best results followed by other listed isolates. In the present time, natural antagonists of agricultural pests are an important alternative to synthetic pesticides. Bacterial and fungal agents have been studied as biocontrol agents with promising results. Proper strategy of implementation and commercialization of these agents is necessary for their efficient outcome in agricultural fields. Acknowledgements: I thank all individuals who supported my research and offered necessary help throughout my study. 143 Figure 3. Glimpse of fluorescence noticed in Pseudomonas sp. Isolate no. P5

Journal

Agriculturede Gruyter

Published: Dec 1, 2012

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