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Crop Protection 30 (2011) 770e775

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Crop Protection

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Combined use of Streptomyces sp. A6 and chemical fungicides against fusariumwilt of Cajanus cajan may reduce the dosage of fungicides required in the field

Anil Kumar Singh, H.S. Chhatpar*

Department of Microbiology and Biotechnology Centre, Faculty of Science, Maharaja Sayajirao University of Baroda, Vadodara 390 002, India

a r t i c l e i n f o

Article history:Received 17 May 2010Received in revised form7 March 2011Accepted 7 March 2011

Keywords:Biological control agents (BCAs)FungicidesFusarium wiltIntegrated pest management (IPM)Mycolytic enzymesStreptomycesSynergism

* Corresponding author. Tel.: þ91 265 2794396; faxE-mail address: chhatparhs@yahoo.co.in (H.S. Chh

0261-2194/$ e see front matter � 2011 Elsevier Ltd.doi:10.1016/j.cropro.2011.03.015

a b s t r a c t

Biological control agents offer one of the best alternatives to reduce the use of pesticides. This investi-gation studied the tolerance to fungicides and integrated use of the potential biocontrol agent Strepto-myces sp. A6 for control of Fusarium wilt of pigeon pea, Cajanus cajan. Streptomyces sp. A6 exhibitedstrong tolerance towards most of the fungicides used in the study at concentrations higher than thoserecommended for field applications. The isolate showed enhanced growth and mycolytic enzymeproduction in the presence of sulphur, mancozeb, carbendazim, fosetyl aluminium and triadimefon. Thefungicides mancozeb, sulphur and carbendazim were selected for further studies. Effective concentra-tions (EC50 values) of the test fungicides that reduced Fusarium spore germination and fungal biomass by50% were determined. Similarly, the EC50 for inhibiting fungal spore germination and reducing fungalbiomass to 50% by Streptomyces sp. A6 and culture filtrate (CF) were also determined. Combining the EC50dose of the culture and CF with test fungicides was found to be more effective for controlling Fusariuminfection in C. cajan compared to the sum of the effects of the individual treatments. Such combined useof biocontrol agent with fungicides can reduce the dosage of toxic fungicides in agricultural fields,thereby reducing environmental risks. Tolerance and synergistic interaction of Streptomyces sp. A6 withfrequently used fungicides suggested its potential in integrated pest management. To the best ourknowledge, this is the first extensive study on integrated use of Streptomyces species with fungicides.

� 2011 Elsevier Ltd. All rights reserved.

1. Introduction

Excessive use of pesticides in modern agriculture has led toseveral problems related to pollution, environmental degradationand emergence of resistant strains (Fox et al., 2007). Thus, in recentyears, search for alternative methods that can minimize the use oftoxic agrochemicals has been a focus. Integrated pest management(IPM) has emerged as an eco-friendly and economic alternativeto conventional use of chemical pesticides for controlling plantdiseases in agricultural fields. IPM’s main feature is minimumuse ofsynthetic pesticides and maximum reliance on natural regulatorymechanisms to keep pest populations below the level at which theycan cause economic damage (Gray et al., 2009). One of the strate-gies used in IPM is integrated use of biocontrol agents (BCAs) withpesticides. This approach reduces input of pesticides and, if app-ropriately chosen, provides better control of plant diseases than thechemical control. Several reports have indicated better control of

: þ91 265 2792508.atpar).

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plant diseases by integrated use of BCAs and pesticides (Kiewnicket al., 2001; Someya et al., 2007).

The first requirement for any BCA to be used in IPM is its toler-ance to the pesticideswithwhich it is to be integrated. Secondly, it isnecessary to determine the type of interaction between the BCAand pesticide in control of the test pathogen. A synergistic interac-tion between the two control agents can provide better protectionagainst the pathogen.

Streptomyces sp. are themost abundant soil microorganisms andarewell known for production of extracellular enzymes and a rangeof antimicrobials (Crawford et al., 1993). Moreover, Streptomyces sp.can promote plant growth by atmospheric nitrogen fixation,mineral solubilisation (Chang and Yang, 2009) and production ofsiderophores or phytohormones (Dimkpa et al., 2008). Strainsbelonging to genus Streptomyces can, therefore, act as biocontrolagent with plant growth promoting ability. Futhermore, theirpotential metabolic diversity, mycelia growth habit, rapid growthrate, colonization of semi-selective substrates and ability to begenetically manipulated make them well-suited for soil inocula-tion. Additionally, ability to form desiccation resistant spores whichassists their spread, persistence and formulation makes thempreferred biocontrol agents (Benimeli et al., 2007).

A.K. Singh, H.S. Chhatpar / Crop Protection 30 (2011) 770e775 771

Although Streptomyces sp. have been studied extensively forbiocontrol, studies on their fungicide tolerance and the fungicidetolerance of their mycolytic enzymes are limited.

This study was focused on the pesticide tolerance of Strepto-myces sp. A6 towards commonly used fungicides and to investigateits potential in IPM to control Fusarium wilt of Cajanus cajan.Fusariumwilt of pigeon pea caused by Fusarium udumwas taken asmodel disease as it is the most important pulse crop of Gujarat,India.

2. Materials and methods

2.1. Organism and culture conditions

The bacterium used in this studywas isolated from the intertidalzone 2 km away from the sea coast of Diu (Gujarat, India) and wasidentified as Streptomyces sp. A6 bymorphological, biochemical and16S rDNA sequence analysis. The GenBank Accession No. for thenucleotide sequence is DQ908927.1 (Singh and Chhatpar, 2010a).

The medium used for mycolytic enzyme production by Strep-tomyces sp. A6, spore germination inhibition assay and fungalbiomass reduction assays consisted of (g/l): Shrimpwaste,14; FeCl3,0.035; ZnSO4$7H2O, 0.065 and pH, 8.0 (Singh and Chhatpar, 2010a).

Spores from F. udum and Streptomyces sp. A6 were harvested in0.001% aqueous triton X-100 by cultivating the fungus in mediumcontaining (g/l): Potato infusion, 200.0; Dextrose, 20.0; Rose Ben-gal, 0.0084; Agar, 15.0; pH 5.6 � 0.2 for 7 days at 25 �C andbacterium inmedium containing (g/l): chitin, 5.0; yeast extract, 0.5;(NH4)2SO4, 1.0; MgSO4$7H2O, 0.3; KH2PO4, 1.36; Agar, 15.0; pH, 7.2for 7 days at 30 �C (Monreal and Reese, 1969).

2.2. Pesticide tolerance of Streptomyces sp. A6

The list of fungicides used in this study, their active ingredients,classification based on Fungicide Resistance Action Committee(FRAC), mode of action and recommended field concentration (RFC)according to the manufacturer is given in Table 1. The effect offungicides on growth and mycolytic enzyme production by Strep-tomyces sp. A6 was investigated by growing the culture in pesticideamended media. The concentrations of pesticides used were RFCand 100 mg/ml of active ingredient.

2.3. Enzyme assays

The culture was centrifuged at 10,000g for 10 min (4 �C) and theculture supernatant was used as a source of enzymes. Protease,chitinase and glucanase activities were determined as described byOceguera-Cervantes et al. (2007), Vyas and Deshpande (1989) andGohel et al. (2007), respectively. One unit of protease, chitinase andglucanase activity was defined as the amount of enzyme required to

Table 1Fungicides used in the present study.

Trade name Active ingredient Group (www.frac.info) F

Dithane Mancozeb M3 MTaqat Captan M4Sultaf Sulphur M2Benfil Carbendazim MBC 1Aliette Fosetyl Aluminium Phospho-nates 3Follicur Tebuconazole DMI 3Dhan PropioconazoleScore DifenoconazoleKrizoleþþ5 HexaconazoleBalyton Triadimefon

RFC, Recommended field concentration; FRAC, Fungicide Resistance Action Committee.

liberate 1 mmole of tyrosine, N-acetyl-D-glucosamine and glucose,respectively, per hour at 55 �C (protease) or 50 �C (chitinase andglucanase activity).

2.4. Determination of 50% effective concentration (EC50) dose ofStreptomyces sp. A6, culture filtrate (CF) and fungicides forinhibition of Fusarium spore germination and fungal biomassreduction

The dose of test fungicides (mancozeb, sulphur and carbenda-zim), Streptomyces sp. A6 and CF for inhibiting the Fusarium sporegermination by 50% was determined as described earlier by Loritoet al. (1994). F. udum conidial population of 105 was incubatedfor 96 h at 30 �C with varying concentrations of test fungici-des (10e80 mg/ml), Streptomyces sp. A6 (104e106 spores) and CF(10e50%) in a 10.0 ml system. Samples from each mixture wereanalyzed for spore germination. Germination of the first 100 sporesobserved was evaluated. EC50 dose for inhibition of fungal sporegermination was determined from the dosage response curve witheach of the test agents.

To verify the results of the fungal spore inhibition assay, the EC50dose of test fungicides, Streptomyces sp. A6 and CF were also deter-mined in terms of reduction in fungal biomass. To determine theEC50 dose of Streptomyces sp. A6, different percentages (0.25e4%) ofStreptomyces sp. A6 spore suspension (108 spores/ml) were incu-bated with 106 spores of F. udum in 100 ml medium at 30 �C undershaking (180 rpm) conditions. After 96 h, fungal biomass wasobtained by filtering the media throughWhatman filter paper No.1.Biomass was dried at 60 �C till constant weight. Similarly, the EC50dose of CF and test fungicides were determined by supplementingdifferent volumes (10e50%, v/v) of filter sterilized CF and varyingconcentrations (10e80 mg/ml) of the active ingredients of fungicidesin medium.

2.5. Seed protection assay

Seeds of a wilt susceptible variety of C. cajan (T15-15) weresurface sterilized by treatment with 0.01% HgCl2 and 4% NaOCl for90 s with intermittent washes using sterile distilled water. The EC50doses of Streptomyces sp. A6 and test fungicides for seed protectionstudies were determined by incubating seeds with spore susp-ension of Streptomyces sp. A6 having varying spore densities(106e1010 spores/ml) in the presence of 1% carboxymethyl cellulose(CMC) and different concentrations of test fungicides (mancozeband carbendazim) (20e100 mg/ml) for 4 h. After incubation, theseeds were air dried under sterile conditions and placed on wateragar plates (5 seeds per plate) containing 105 spores/ml of F. udum.Plates were incubated in the dark for 96 h and observed for inf-ection. The experiment was conducted with six replicates pertreatment.

RAC group Mode of action RFC (mg/ml)

Multisite contact action 657585

Inhibition of mitosis and cell division 453 Unknown 75

Inhibition of sterol biosynthesis 2015102060

A.K. Singh, H.S. Chhatpar / Crop Protection 30 (2011) 770e775772

2.6. Field trials

The field trials were conducted (2006e2008) in a rain irrigatedand well maintained Fusarium wilt sick plot at the pulse researchcentre model farm, Vadodara, India, during the monsoon season ofeach year. A complete randomized block designwas used with ninedifferent treatments and four replicates and three 6m rows per plotper treatment. Seeds were surface sterile and treated as describedin the previous section. Treated seeds (100) were sown manually ata depth of 2e3 cm in 36 (9 � 4) rows in wilt sick plots with a 60 cmdistance between each plant and 40 cm distance between each row.Untreated seeds were also sown for the control. Each replicate plotwas separated from others by 2 m distance. Wilting was observedat intervals of 1 month till harvesting.

2.7. Determination of type of interaction between Streptomyces sp.A6 or CF and fungicides for control of F. udum

The percentage reduction in fungal biomass and wilt incide-nce during seed protection studies and field trials, in presence ofcombined EC50 dose of Streptomyces sp. A6 or CF and test fungicideswas analyzed for synergism using Limpel’s formula (Eq. (1)) (Richer,1987).

Ee ¼ ðX þ YÞeðXYÞ=100 (1)

where, Ee is the expected effect from additive responses of twoinhibitory agents (say, Streptomyces sp. A6/CF and fungicide); X andY represent the percentage inhibition caused by Streptomyces sp.A6/CF and fungicide, respectively.

The synergy factor (SF) is calculated by Abott’s formula (Eq. (2))(Abbott, 1925).

SF ¼ Observed Inhibition=Expected Inhibition (2)

where, SF > 1 for Synergistic reaction; SF < 1 for antagonisticreaction; SF ¼ 1 for additive reaction.

2.8. Statistical analysis

The data were analyzed statistically by Fisher Least SignificantDifference (LSD) test with a significance level of p < 0.05.

3. Results

3.1. Effect of pesticides on growth and mycolytic enzyme productionby Streptomyces sp. A6

Streptomyces sp. A6 was found to be tolerant to all tested fungi-cides. Growth and production of all mycolytic enzymes (chitinase,protease and glucanase) were enhanced in presence of the fungi-cides sulphur, mancozeb, carbendazim, fosetyl aluminium andtriadimefon at both concentrations used, RFC (Fig.1A) and 100 mg/ml(Fig. 1B). Amongst these fungicides, sulphur, mancozeb and car-bendazim showed the maximum positive effects on growth as wellas enzyme production and hence were selected for further studies.

3.2. Determination of EC50 dose of Streptomyces sp. A6, CF and testfungicides

Fifty percent effective concentration (EC50) of Streptomyces sp.A6, CF and test fungicides for inhibition of spore germinationand reduction of fungal biomass were determined. The EC50 dosesof mancozeb, carbendazim and sulphur were 27.8, 16.78 and41.9 mg/ml for inhibition of fungal spore germination and 29.3, 18.3and 45.5 mg/ml for fungal biomass reduction, respectively. The EC50

dose of Streptomyces sp. A6 and CF was found to be 2 � 106 spores/ml and 14.8% (v/v) for fungal spore germination inhibition and1.44 � 106 spores/ml and 20% (v/v) for fungal biomass reduction,respectively. The spore count of 2 � 106 spores/ml served as EC50dose of Streptomyces sp. A6 for prevention of Fusarium infection ofC. cajan seeds. EC50 dose of test fungicides, used for seed dressing,mancozeb and carbendazim were determined as 40 and 30 mg/ml,respectively.

3.3. Interaction between Streptomyces sp. A6/CF and fungicides

The interaction between Streptomyces sp. A6 and fungicides wasfound to be synergistic (SF > 1) as their combined EC50 dosesreduced the fungal spore germination and fungal biomass betterthan expected. Streptomyces sp. A6 exhibited maximum synergismwithmancozeb for inhibition of fungal spore germination as well asfor reduction of fungal biomass (Table 2).

Table 3 shows the observed and expected values for fungalbiomass reduction obtained by combined EC50 dose of CF and testfungicides. The observed inhibition for all the combinations wasfound to be greater than the expected inhibition indicating syner-gism between CF and test fungicides. Maximum synergism of CFwas observed with carbendazim for both fungal spore germinationinhibition and biomass reduction.

Seed protection assays and field trials against Fusarium infectionalso revealed a synergistic interaction between Streptomyces sp. A6and the fungicides used for seed dressing (carbendazim and man-cozeb) with carbendazim showing maximum synergism. Fungalcontrol was better in combination of organism and fungicidescompared to their individual treatments (p value < 0.05) (Table 4).

4. Discussion

The isolate Streptomyces sp. A6 had strong antifungal activityagainst a wide spectrum of fungal plant pathogens, mainly attrib-uted to production of mycolytic enzymes and an unknown anti-fungal metabolite which makes it a potential biocontrol agent(Singh and Chhatpar, 2010b). Although BCAs have proved effectiveunder controlled conditions, results are highly variable whenapplied in fields due to environmental fluctuations and residualpesticides in fields. One way to overcome this ineffectiveness is toapply them in combination with chemical pesticides to which theyare tolerant as in integrated pest management (IPM) (Guetsky et al.,2001). Enhanced growth and mycolytic enzyme production byStreptomyces sp. A6 in presence of commonly used fungicidessuggested its compatibility with these chemicals for possibleapplication in integrated pest management to control phytopath-ogens. High tolerance towards mancozeb and carbendazim wasadvantageous as these fungicides are being used during seeddressing mainly for pigeon pea (C. cajan) seeds. Fungicides act bya number of different mechanisms inhibiting various metabolicfunctions specific to eukaryotic cells as depicted in Table 1. Suchtargets are absent in prokaryotes which explains the tolerance ofStreptomyces sp. A6 towards fungicides. Reports on tolerance ofbacteria towards fungicides are scarce. Di�grak and Kazanzi (2001)have reported an enhanced total viable count of soil bacteria onapplication of organophosphorous insecticides isofenphos, fonofosand phorate. We have recently reported tolerance of Paenibacillussp. D1 against a different group of pesticides (Singh et al., 2009).

Low EC50 doses of fungicides and CF for inhibition of fungalspore germination compared to EC50 dose required for fungalbiomass reduction suggested better interaction between fungicidesand antifungal agents present in the CF against fungus during staticincubation than under shaking (for fungal spore germination assaythe fungal spores were incubated with test fungicides/Streptomyces

Fig. 1. (A) Effect of recommended field concentration (RFC) and (B) 100 mg/ml of test fungicides on growth and mycolytic enzymes (chitinase, protease and glucanase) production byStreptomyces sp. A6. Values in graphs are results of triplicate determinations. Culture without pesticide treatment was considered as control (100% growth; chitinase, protease andglucanase production).

A.K. Singh, H.S. Chhatpar / Crop Protection 30 (2011) 770e775 773

sp. A6/CF under static conditions while the biomass reduction assaywas done under shaking conditions). However, the EC50 dose ofthe Streptomyces sp. A6 was found to be less for fungal biomassreduction. This may be due to better growth and mycolytic enzymeproduction by the bacterium under shaking conditions.

Table 2Interaction between Streptomyces sp. A6 and fungicides for fungal biomass reduction.

Treatment Fungal spore germination inhibition

Observedinhibition

Expected inhibitio

% S.D %

EC50 Streptomyces sp. A6 þ EC50 Mancozeb 79.8 0.88 74.18EC50 Streptomyces sp. A6 þ EC50 Sulphur 80.4 2.04 75.6EC50 Streptomyces sp. A6 þ EC50 Carbendazim 80.1 1.63 74.02

SD, Standard Deviation.

The interaction of Streptomyces sp. A6 and its culture filtratewithtest fungicideswas found to be synergisticwith respect to inhibitionof fungal spore germination and fungal biomass reduction. Highervalues of synergy factors (SF) for fungal biomass reduction duringinteraction of Streptomyces sp. A6 and test fungicides, suggested

assay Fungal biomass reduction assay

n Synergy factor Observedinhibition

Expected inhibition Synergy factor

% S.D %

1.075 83.3 1.54 76.41 1.091.064 81.3 2.20 76.19 1.0671.08 83.6 0.77 77.24 1.083

Table 3Interaction between the culture filtrate (CF) and fungicides for reduction of Fusarium biomass.

Treatments Fungal spore germination inhibition assay Fungal biomass reduction assay

Observedinhibition

Expected inhibition Synergy factor Observedinhibition

Expected inhibition Synergy factor

% S.D % % S.D %

EC50 CS þ EC50 Mancozeb 82.3 1.82 77.88 1.057 78.1 2.79 77.93 1.002EC50 CS þ EC50 Sulphur 83.0 1.6 79.1 1.049 79.6 2.32 78.72 1.011EC50 CS þ EC50 Carbendazim 83.3 2.02 77.73 1.072 83.6 0.77 79.66 1.05

SD, Standard deviation.

Table 4Interaction of Streptomyces sp. A6 and fungicides for control of Fusarium infection during seed protection assay and field trials.

Treatment Seed protection experiment Field trials

Observedprotection

Expected protection Synergy factor Observedprotection

Expected protection Synergy factor

% S.D % % S.D %

Control 0.0 0.00 0.0 0.00Streptomyces sp. A6 68.5 3.21 69.1 5.04Carbendazim 74.1 8.49 73.2 6.4Mancozeb 72.2 5.56 71.0 5.65EC50 Streptomyces sp. A6 51.9 6.41 42.6 6.49EC50 Carbendazim 55.6 5.55 46.3 10.1EC50 Mancozeb 53.7 6.41 45.4 7.63EC50 Streptomyces sp. A6 þ EC50 Carbendazim 81.5 3.61 78.6 1.037 77.8 5.61 69.17 1.124EC50 Streptomyces sp. A6 þ EC50 Mancozeb 77.8 6.25 77.71 1.00 74.7 6.41 68.63 1.088

SD, Standard deviation.

A.K. Singh, H.S. Chhatpar / Crop Protection 30 (2011) 770e775774

better growth and mycolytic enzyme production by the cultureunder shaking conditions. Whereas, high values of SF achievedduring interaction between CF and fungicides for fungal sporeinhibition indicted strong effect of antifungal metabolites producedunder static conditions. The bacterium also exhibited synergismwith test fungicides in controlling infection of seeds and wilt inci-dence in C. cajan caused by F. udum. Bacteria are known to inhibitgrowth of fungi by degrading their cell wall components byproducing mycolytic enzymes such as chitinases, proteases andglucanases as observedwith Streptomyces sp. A6.Moreover, a 20 kDaserine protease from Streptomyces sp. A6 was found to possessantifungal activity (Singh and Chhatpar, 2010b). Digestion of fungalcell walls by such enzymes may enhance the uptake of chemicalfungicides and serve as the basis of synergism. The level of syner-gism may also be affected by the mode of action of fungicides.Mancozeb has broad-spectrum activity and acts by contact whilesulphur, once taken up by the fungus, disrupts the transfer of elec-trons reducing sulphur to hydrogen sulfide (H2S), which is toxicto most cellular proteins (McCallen, 1949). Carbendazim acts byinterfering with tubulin function, which is crucial for fungal growth(McMahan et al., 2001). Streptomyces sp. are known to produceseveral antimicrobials. Streptomyces sp. A6 produced a strong anti-fungal metabolite, inhibiting a range of fungal pathogens (data notshown). Culture supernatant containing antifungal principles ofStreptomyces sp. A6 exhibited higher synergism with fungicidesunder static conditions.

The control of Fusarium infection by combined EC50 dose ofStreptomyces sp. A6 and fungicides, observed during laboratorystudies and field trials, was much higher than both these antifungalagents used alone. These results suggested that the dosage offungicides can be reduced by more than 50% without compro-mising the efficiency of disease control. Several other reports havealso indicated the synergistic phenomenon involved in control ofpathogens using the integrated application of fungicides andbiocontrol agents to be more efficient and long-lasting than thatachieved through biocontrol agents or fungicides alone. Lorito et al.

(1994) had reported enhanced inhibition of spore germination toBotrytis cinerea, due to synergism between fungal cell wall deg-rading enzymes and fungicides. Control of rhizoctonia crown androot rot of sugar beet with integrated use of fungicides andantagonistic bacteria was reported by Kiewnick et al. (2001). Effi-cient control of cabbage yellows caused by Fusarium oxysporum hasbeen achieved by combined application of Pseudomonas fluorescensand low dosage of benomyl (Someya et al., 2007). Chien-Jui andChen (2008) reported synergistic interaction between chitinaseChiCW and fungicides against plant fungal pathogens.

In conclusion, the present investigation revealed strong toler-ance of Streptomyces sp. A6 towards number of commonly usedfungicides. Furthermore, Streptomyces sp. A6 and its antifungalprinciples exhibited synergistic interaction with test fungicides forcontrolling Fusarium wilt of C. cajan caused by F. udum. Suchsynergistic interactions will be advantageous for developing newfungicide formulations and application strategies which can reducethe dosage of toxic agrichemicals in fields.

Acknowledgement

The work was supported by Gujarat State BiotechnologyMissiongrant(GSBTM/MD/PROJECTS/1450/2004e2005),Gandhinagar,Gujarat, India.

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