The Antagonistic Activity of Bioactive Compound Producing ... · characteristics and purified onto ISP-2 agar [10]. To induce sporulation, purified colonies were subcultured on ISP-3

1680 Chiang Mai J. Sci. 2018; 45(4)

Chiang Mai J. Sci. 2018; 45(4) : 1680-1698http://epg.science.cmu.ac.th/ejournal/Contributed Paper

The Antagonistic Activity of Bioactive CompoundProducing Streptomyces of Fusarium Wilt Diseaseand Sheath Blight Disease in RiceMathurot Chaiharn* [a], Nikhom Sujada [b], Wasu Pathom-aree [b] and

Saisamorn Lumyong [b]

[a] Division of Biotechnology, Faculty of Science, Maejo University, Chiang Mai 50290, Thailand.

[b] Departments of Biology, Faculty of Science, Chiang Mai University, Chiang Mai 50200, Thailand.

* Author for correspondence; e-mail: [email protected]

Received: 20 December 2016

Accepted: 19 April 2017

ABSTRACT

Rhizospheric actinomycetes were screened for bioactive compound production suchas antibiotic, siderophore and cell wall degrading enzyme against Fusarium wilt disease andsheath blight disease in rice which are caused by Fusarium oxysporum and Rhizoctonia solaniAG-2, respectively. A total of 150 strains was classified based on their cultural and physiologicalcharacterization such as color of aerial mycelium, color of substrate mycelium and sporechain morphology combined with cell wall composition analysis. They were classified asStreptomyces (52.7 %), non-Streptomyces (35.3 %) and unidentified (12 %). Involvement of cellwall degrading enzymes such as chitinase, cellulase and β-1,3-glucanase was investigated.Results showed that isolate RHI-43 produced high levels of chitinase (3.50 U/ml), cellulase(4.90 U/ml) and β-1,3-glucanase (0.45 U/ml) while isolate RHI-114 produced high levels ofprotease (15 U/ml). On the basis of dual culture assays, isolate RHI-39 and isolate RHI-146were selected on the basis of broad spectrum activities and identified using cell wall compositionanalysis combined with 16S rDNA analysis and phylogenic classification. These isolateswere identified as Streptomyces lydicus (for isolate RHI-39) and Streptomyces corchorussi (for isolateRHI-146). Further, culture filtrates of isolate RHI-146 inhibited the growth of all fungi testedwhich showed hyphal swelling and abnormal shapes of tested fungi under microscope.These results suggest that Streptomyces isolate RHI-39 and Streptomyces isolate RHI-146 may beutilized as an environmental friendly biocontrol agent against some important rice-pathogenicfungi in vitro.

Keywords: biological control, fusarium wilt disease, sheath blight disease, bioactive compound,Streptomyces

1. INTRODUCTION

Rice (Oryza sativa L.) is a major foodcrop of Asia. Rice diseases are caused bydifferent groups of pathogen such as fungi,

viruses, bacteria and nematodes [1]. Fusariumwilt disease and sheath blight disease are causedby Fusarium oxysporum and Rhizoctonia solani,

Chiang Mai J. Sci. 2018; 45(4) 1681

respectively, which lead to high losses of ricein many countries of Asia. Fusarium oxysporumenters the plant from young roots andblocks water and nutrient transportation inthe vessels via macro and micro conidia.The fungus causes leaf chlorosis and slightvein clearing on outer leaflets, followed bywilting and dropping of leaves, then xylembrowning of stem and finally death of theabove ground parts [2]. There are no effective,economical and practical ways to controlFusarium wilt disease due to re-colonizationof air-borne spores of Fusarium oxysporumunder favorable conditions [2]. Rhizoctoniasolani attacks rice at maximum till ring stageby sclerotium over winter in soil and plantdebris and causes poor filling of the grainsand emergence of panicles [3]. Control ofboth pathogens is difficult because of theirecological behavior, their extreme broadhost range and the high survival rate offungal spore under various environmentalconditions and no rice variety capable ofresistant completely to these pathogen [4].The most common approach to control ricedisease is to use chemical fungicides but theycan destroy the balance of ecosystems andthe contamination by their toxic residuesmay be harmful to human and animals.Biological control may be effective inminimizing the incidence of fungal pathogens.

Streptomyces sp. have been explored overseveral decades for antibiotic production.In the last few years strong antimicrobialpotential of Streptomyces has received attentionfor plant-growth promotion. Streptomycesdegrade a broad type of biopolymers bysecreting hydrolytic enzymes and effectivecolonizer of plant root systems and endureunfavorable conditions by forming spores.Actinobacteria produce a variety of antibioticspossessing polyketides, β-lactams, peptideand other secondary metabolites such as

antifungal, antibacterial and anti-tumorcompounds [5]. Thus, Streptomyces arerecognized as antimicrobial agents whichinhibit plant pathogens such as soil-bornefungi [6]. The mode of action includesantifungal and antibiotics production,competition for iron by siderophoreproduction, cell wall degrading enzymeproduction such as chitinase and glucanase,phenylacetic and sodium acetate production[7]. Various groups of bioactive compoundswere produced by Streptomyces such asmacrolide, benzoquinones, aminoglycosides,polyenes and nucleoside antibiotics [8].Application of Streptomyces as biocontrolagents is prolonging survival duringcommercial process and formulation afterapplying in the field. This is becauseStreptomyces are able to produce spores tomaintain their viability at non-specificconditions [9]. Therefore, Streptomyces growin a wide range of temperature which is aidsthe survival in the soil after application [9].

In this study, Streptomyces sp. were isolatedfrom rice rhizosphere soils, and screenedfor antagonistic activities such as antibioticproduction, siderophore production andhydrolytic enzyme activity against Fusariumoxysporum, and Rhizoctonia solani AG-2, thecausal agents of Fusarium wilt disease andsheath blight disease in rice, respectively.

2. MATERIALS AND METHODOLOGY

2.1 Fungal Strains and Culture ConditionsFusarium oxysporum and Rhizoctonia solani

were isolated from root and leave of lowlandrice in the north of Thailand and tested forpathogenicity. The virulent strains weregrown on potato dextrose agar (PDA) andincubated at 28°C for 3-5 days. Stock cultureswere maintained on PDA slants and storedat 4°C.

1682 Chiang Mai J. Sci. 2018; 45(4)

2.2 Isolation of RhizosphericStreptomyces

Streptomyces strains were isolated from ricerhizospheric soil by serial dilution and spreadplate technique. Briefly, soil sample (10 g)was suspended in 9 ml of basal salt solution(5.0 g/l KH

2PO

4 and 5.0 g/l NaCl), and the

suspension was mixed using rotator shaker(150 rpm) at 28°C for 24 hr. Then, the dilutionsamples were spread on starch-casein-agarand incubated at 28°C for 2 weeks. Afterincubation, colonies of Streptomyces wereselected on the basis of their morphologicalcharacteristics and purified onto ISP-2 agar[10]. To induce sporulation, purified colonieswere subcultured on ISP-3 agar [10].

2.3 Streptomyces Screening forAntagonistic Activity Against Fusariumwilt Disease and Sheath Blight Disease

All strains were screened for their in vitroantagonism against Fusarium oxysporumand Rhizoctonia solani according to the methodof Crawford et al., (1993). Briefly, a 20 μlspore suspension of Streptomyces sp.(106 spore/ml) was spotted on one side ofPDA plate and incubated at 28°C for 5 days.After incubation, mycelium’s plugs of fungi(6.0 mm) were transfer to PDA plate andincubated at 28°C for 5 days. For controlgroup, fungal plug was inoculated on PDAplate without Streptomyces sp. The radialfungal control and dual culture plates weremeasured at 5 days after incubation andlevels of inhibition were calculated usingthe equation as previously mentioned by Yangand Crawford (1995) [11]. Briefly, R1

-R2/R

1

× 100, where R1 is the radial distance (mm)

grown by plant pathogenic fungi in directionof antagonist (a control value), and R

2 is

the radial distance (mm) grown by fungalin dual culture. All strains were tested intriplicates.

2.4 Evaluation of the ParasiticMechanism

The mycoparasitism of Fusariumoxysporum and Rhizoctonia solani cell wall byStreptomyces strain RHI-39 and Streptomycesstrain RHI-146 was studied using lightmicroscopy (CHS, Olympus optical Co. Ltd,Japan) and scanning electron microscopy(SEM) (JEOL JSM-6610CV, Japan). Forscanning electron micrographs, the cultureson the dual culture plates were pre-fixedovernight in 2% CH

2(CH

2CHO)

2 in 0.1 M

sodium cacodylate buffer pH 7.2-7.4 at 4°C;washed three times with the same bufferfor 15 min; post-fixed at 4°C for 1h in 1%OsO4 in 0.1 M sodium cacodylate buffer;washed three times with 0.1 M sodiumcacodylate buffer, pH 7.2-7.4 at 4°C for15 min; dehydrated in a graded series ofethanol-amylacetate. Samples in the amylacetate were critical point dried withHitachi HCP-2 critical point dryer (HCP-2;Hitachi Co.Ltd., Japan); coated with Pt-Pdmixture for 4 min in Hitachi E-102 ionsputter (E-102; Hitachi Co. Ltd., Japan) andexamined in a Hitachi S-510 SEM (JSM-6610CV; Japan Electron Optics Laboratory Co.Ltd.).

2.5 Quantitative Determination of CellWall Degrading Enzymes2.5.1 Assay for chitinase

Selected strains which show inhibitoryeffect on test fungi were grown in ISP-2 brothwith continuous shaking at 150 rpm at28°C for 10 days. After incubation, cell-freesupernatant was collected by centrifugationat 8,000 rpm for 20 min at 4°C. Chitinaseactivity was determined by the reducingend group of N-acetylglucosamine (NAG)using DNS method [12].

Chiang Mai J. Sci. 2018; 45(4) 1683

2.5.2 Assay for cellulaseCellulase activity was measured by

spectrophotometric assay. A 500 ml aliquotof crude enzyme was incubated with 500 mlCMC (1% w/v-Carboxy Methyl Cellulose)in 0.1 M sodium phosphate buffer (pH 5.5).After incubation at 37°C for 60 min,the reaction was stopped by adding 2 ml of3,5-dinitrosalicyclic acid and absorbancewas measured at 530 nm in UV/Visspectrophotometer. One Unit (U) is definedas the amount of enzyme that releases1 mmol of reducing end group of glucoseper minute [12].

2.5.3 Assay for β-1,3-glucanaseβ-1,3-glucanase activity was measured

according to the method of Singh et al.,(1996) [13] using laminarin from Laminariadigitata (Sigma USA) as substrate. Briefly,500 l of 0.1% laminarin (in 50 mM citratebuffer pH 5.5) was mixed with 500 ml ofculture supernatant. The mixture wasincubated at 37°C for 60 min andthe reaction was stopped with 2 ml of3,5-dinitrosalicyclic acid [12]. The amountof reducing sugar released was measured at530 nm in UV/Vis spectrophotometer.One unit of β-1,3-glucanase activity wasdefined as the amount of enzyme thatreleases 1 mmol of glucose per min in theseconditions.

2.5.4 Assay for proteaseFive hundred ml of 0.5% casein in

Tris-HCl buffer with 100 ml of enzymesolution were incubated at 37°C for 60 min.Reaction was stopped by adding 500 ml of15% tricholoroacetic acid with shaking for15 min. After incubation, the mixture wascentrifuged at 3,000 rpm for 5 min at 40°Cand one ml of supernatant was addedwith one ml of 1M NaOH and the amountof reducing sugar released was measured at

440 nm. Enzyme activity was calculatedfrom standard curve of L-tyrosine and oneunit of enzyme activity is equivalent to 1 mgof L-tyrosine min/ml under the assayconditions.

2.5.5 Siderophore productionThe strains were assayed for the

siderophore production on the ChromeAzurol S (CAS) agar according to themethod of Alexander and Zuberrer (1991).CAS agar plates were prepared as followed.A dye made of CAS, Fe, and hexadecryl-trimethyl-ammonium bromide (HDTMA)was mixed with M9-based growth media.For 1 L of CAS-agar, 100 ml of CAS-Fe-HDTMA dye was mixed with 900 ml ofprepared media. The CAS-Fe-HDTMA dyewas prepared as followed; for 1 L, 10 ml ofa 10 mM ferric chloride (FeCl3) in 100 mMhydrochloric acid (HCl) solution was mixedwith 590 ml of a 1 mM aqueous solutionof CAS. The Fe-CAS solution was added to400 ml of a 2 mM aqueous solution ofHDTMA. The resulting CAS-Fe-HDTMAsolution was autoclaved for 25 min in apolycarbonate bottle that had previouslybeen soaked overnight in 10 % (v/v) HClthen rinsed five times with Milli Q water.The CAS-Fe-HDTMA dye was stored atroom temperature and covered from lightuntil use.

The growth media was prepared; for 1 lof CAS-agar, 30.24 g of 1, 4-piperazine-diethanesulfonic acid (Pipes), together with1 g of ammonium chloride (NH

4Cl), 3 g

potassium phosphate (KH2PO

4), and 20 g

sodium chloride (NaCl) was dissolved intoMilli Q water by adjusting the pH with 10MNaOH to 6.8. As a solidifying agent, 9 g ofagar noble (Difco), agar or agarose was addedto the solution. The volume was adjustedto 860 ml and the solution was autoclaved.After cooling, 30 ml of a sterile 10 % (w/v)

1684 Chiang Mai J. Sci. 2018; 45(4)

casamino acid (Difco) aqueous solution and10 ml of a sterile 20 % (w/v) glucose aqueoussolution were added. Finally, the 100 ml ofCAS-Fe-HDTMA were added to the growthmedia. The final concentrations of the CASagar components were as followed: 100 mMPipes, 18 mM NH

4Cl, 22 mM KH

2PO

4, 2 %

(w/v) NaCl, 0.3 % casamino acids, 0.2 %(w/v) glucose, 10 M FeCl

3, 58 mM CAS,

and 80 mM HDTMA. All chemicals wereobtained from Sigma-Aldrich.

CAS agar was spot inoculated withactinobacterial strain and incubated at28°C for 7 days. When the actinomycetesconsumed iron, they were present in theblue-colored CAS media. The coloniesproducing yellow to orange halos wereconsidered positive for siderophoreproduction.

2.6 Streptomyces Identification2.6.1 Primary identification2.6.1.1 Cultural and physiologicalcharacteristics

The genetic diversities of actinomycetesisolated from rhizospheric soil wereclassified using Bergey’s Manual of SystemicBacteriology [14]. The arrangement of sporeswas examined microscopically using a coverslip-implanting technique. The cover slipwas implanted on ISP-2 agar and incubatedat 28°C for 14 days, which allowed aerialmycelia to grow on the cover slip. The coverslip was removed from the culture plateand the mycelia were stained with crystal-violetdye for 5 min. The fixed mycelia wereobserved under a light microscope.

Taxonomic properties of isolate RHI-39and isolate RHI-146 were evaluated by themethods of the international Streptomycesproject (ISP) [14]. Morphology of the matureculture and color was observed on yeastextract-malt extract agar (ISP-2), inorganicsalts-starch agar (ISP-4) and glycerol-

asparagine agar (ISP-5). Melanin productionwas determined on peptone-yeast extract-ironagar (ISP-6), tyrosine agar (ISP-7) andtryptone-yeast extract broth (ISP-1), whilethe carbon utilization was carried out incarbon utilizing medium (ISP-9) withthe addition of one of the following sugars;D-glucose (positive control), L-arabinose,sucrose, D-xylose, Inositol, D-mannitol,D-fructose, D-sorbitol, cellubiose and inthe absence of a carbon source (negativecontrol). The colony morphology of theactinomycetes grown was observed under astereomicroscope (Olympus CH30, Japan).

2.6.1.2 Color determinationAerial mass color on ISP-3 and ISP-4,

substrate mycelia color and diffusible solublepigments on ISP-5, melanin production onISP-6 and ISP-7 were observed at 28°C for14 days using a reference color key [14].

2.6.1.3 Cell wall composition analysisAnalysis of 2,6-diaminopimelic acid

(DAP) was carried out to determine thecell wall chemotype. The isomeric type ofDAP with the colony morphology wasused to divide non-Streptomyces andunidentified genera from Streptomyces group.The unidentified genera were determinedaccording to the limit formation of aerialmycelia and spores on the agar mediaused. In order to determine the genus ofantagonistic actinomycetes isolate RHI-39and isolate RHI-146, the 2,6- diaminopimelicacid, one of the cell wall components ofactinomycetes mycelia was analyzed usingthe method of the ISP as described byShirling and Gottlieb (1966) [15] and Bergey’sManual of Systemic Bacteriology [14].Potential actinomycetes were cultured inISP-2 medium (4.0 g yeast extract, 10.0 g maltextract, 4.0 g dextrose, 20.0 g agar and 1 Lsterile water, pH 7.3) at 150 rpm, 28°C for

Chiang Mai J. Sci. 2018; 45(4) 1685

14 days. After cultivation, the culture brothwas centrifuged to collect cell debris andcell debris were hydrolyzed with 6 N HCland heated at 70°C for 18 h in water bath.The hydrolysate was filtrated with WhatmanNo.1 filter paper and evaporated to drynessin order to remove the residue HCl.Residue was dissolved in 1 ml of distilledwater and applied onto TLC plate (15 × 20cm, Merck Co., USA). Twenty microliters of0.01 M DL-DAP (Sigma Chemical Co., USA)containing meso- and LL-DAP isomers andamino acids (alanine, glycine and glutamate)were loaded on the plates as a standard [15].

2.6.2 Secondary identification2.6.2.1 DNA preparation

Secondary identification was determinedbased on phylogenetic position. The genomicDNA was extracted from biomass.The extraction was performed followingthe manufacturer’s instructions forthe FavorPrep™ Tissue Genomic DNAExtraction Mini Sample Kit (FAVORGEN®,Taiwan). The selected actinomycetes weregrown for 5 days at 28°C with agitation in100 ml of ISP-2 medium. Biomass washarvested by centrifugation (8000 g, 10 min)and washed twice with double-distilledwater. Two hundred milligrams of myceliawere used for DNA extraction as followed;the sample was dispersed in 800 μl of theaqueous lysis solution (100 mmol / l Tris-HCl,pH 7; 20 mmol/l EDTA; 250 mmol/l NaCl;2 % m/v SDS; 1 mg/ml lysozyme). Fivemicroliters of 950 mg/ml RNase solution wasadded, and the suspension was incubated at37°C for 60 min. Ten microliters of aproteinase K solution (20 mg/ml) was addedand the lysis solution was re-incubated at 65°Cfor 30 min. The lysate was extracted with anequal volume of phenol and centrifuged (8000g, 10 min). The aqueous layer was re-extractedwith phenol (50-50 % v/v), then by

chloroform (50-50 % v/v). DNA wasrecovered from the aqueous phase by theaddition of NaCl (150 mmol/l finalconcentration) and two volumes of cool95% (v/v) ethanol period to centrifugation.The precipitated DNA was cleaned with50 μl of TE buffer (10 mmol/l Tris-HCl,pH 7.4; 1 mmol / l EDTA, pH 8), and storedat 20°C. The quantities of DNA solutionswere measured at 260 nm.

2.6.2.2 16S rDNA sequence analysisThe concentration and purification

of DNA samples were evaluated byelectrophoresis on a 1% (w/v) agarose geland visualized by staining with ethidiumbromide. Taq DNA polymerase and a pairof universal primers (forward primer 27(5’-AGT TTG ATC CTG GCT CAG GACGAA CG-3’) and reverse primer 1,525(5’-AGC CGG TCC CCC TGC AAG-3’)were used. PCR was performed as followed;PCR amplication was carried out in 20 μlreaction mixture containing 4 μl of lysedbacterial suspension, 1X PCR bovineserum albumin, 5 % dimethyl sulfoxide(DMSO), 100 μM dNTP and 1.4 Uof Taq DNA polymerase. Amplificationwas performed with a cycler and initialdenaturation (2 min at 94°C) was followedby 30 PCR cycles (94°C for 30s, 60°C for30s and 72°C for 60s) and final extensionat 72°C for 10 min. Amplified DNA waspurified using the NucleoSpin® Gel andPCR Clean-up kit (MACHEREY-NAGEL,Germany). Purified PCR products weresequenced using the facilities of First BaseLaboratories, Malaysia. Nearly complete 16SrRNA gene sequences (approximately 1,500bp) were determined and compared withthe corresponding sequences available inthe GenBank database (http://www.ncbi-nlm-nih.gov/) using the BLAST program.The 16S rDNA sequences of isolate RHI-39

1686 Chiang Mai J. Sci. 2018; 45(4)

and isolate RHI-146 were aligned usingCLUSTAL W and compared to publishedsequences of species with standing innomenclature available from GenBankdatabases using the BLASTN algorithm(http://blast.ncbi.nlm.nib.gov/Blast.cgi ).The DNA sequences of the isolates weredeposited in GenBank at the National Centerfor Biotechnology Information (NCBI).The phylogenetic tree of the 16S rDNAsequence was constructed by the neighbor-joining method using MEGA 4.0 softwarepackage [16]. In pairwise comparisons,sequence similarities were computed usingPHYDIT program Version 1.0 (http://plaza.snu.ac.kr/jchun/phydit ).

2.7 Production of Antifungal MetabolitesSecondary metabolites produced by

Streptomyces isolates were extracted accordingto the method of Ellaiah et al., (2005) [17].Pure cultures of these isolate were transferredinto ISP-2 medium and incubated at 30°Cfor 1 week under agitation at 150 rpm.After incubation, the cultures were harvestedusing centrifugation at 8,000 × g, 4°Cand biomass was separated from broth.Culture filtrates were extracted with ethylacetate to dryness under vacuum to yield acrude residue and dissolved in 10 % DMSO.Crude extracts were used for antifungalactivity against Fusarium oxysporum andRhizoctonia solani by agar well diffusion method.DMSO extracted at a concentration of100 μg/ml was added to each well as negativecontrol. After incubation at 30°C for48-72 hr, diameters of inhibition zonesof the tested fungi were determined.

2.8 Effect of Streptomyces Crude Extracton Mycelium Morphology of TestedFungi

The tested fungi were separately grownon PDA plate at 28°C for 3 days and mycelia

were cultured on surface of microscope slidescovering the layers of PDA containing 20 %(v/v) culture extract. Inoculated slides wereincubated at 28°C in the dark for 3 days andfungal hyphae present on the slides werestained with lactophenol cotton blue (FlukaSwitzerland). Subsequent changes in themorphology of young emerging hyphae wereobserved under light microscope.

2.9 Statistical AnalysesThe data were analyzed by variance

analysis (ANOVA), and the mean separationwas achieved by the DUNCAN’s multiplerange tests. All numeric differences in the datawere considered significantly different at theprobability level of p ≤ 0.01.

3. RESULTS

3.1 Isolation and Screening ofAntagonistic Streptomyces

A total of 150 Streptomyces isolates wasobtained from rice rhizospheric soil samplescollected from northern parts of Thailand.Colonies of Streptomyces were picked on thebasis of morphological characteristics andidentified by their form of opaque, rough,non-spreading morphology embedded inagar medium. Color of substrate and aerialmycelia were observed.

3.2 Antifungal Activity of StreptomycesIn this report, Streptomyces were selected

for three traits that were often associated withbiocontrol agents, ability to hydrolyze cellwall and cell membrane of pathogens, andproduction of siderophore and activemetabolites against Fusarium oxysporum andRhizoctonia solani AG-2. Among 150 isolates,101 isolates (63.7 %) showed antagonisticability by inhibiting the growth of tested fungiand the actinomycete isolates. Of them, 22isolates (14.6 %) showed siderophore andantifungal activity as presented in Table 1.

Chiang Mai J. Sci. 2018; 45(4) 1687

Percentages of antagonistic ability againstFusarium oxysporum and Rhizoctonia solaniAG-2 were 32 % and 40 % respectively.Streptomyces colony color series showedantagonistic activities against Fusarium wiltdisease and sheath blight disease. Resultsshowed that grey colony gave the highestinhibition (35 and 40 isolates), followed bybrown (27 and 30 isolates), white (8 and 9isolates), yellow (6 and 6 strains) and greencolony (not detected and 1 isolate), respectively(Table 2). The screening results for biocontroltraits are depicted in Table 1. Only 14.6 % of150 antagonistic actinomycetes were positivefor siderophore production and antifungalactivity. The siderophore production wasdetected in all potential actinomycete isolates,showing yellow or orange halo zone

on CAS-blue agar with 10.7-30.3 mm,respectively. Isolate RHI-39 and isolateRHI-146 gave the highest value of antifungalactivities. All isolates demonstrated at leastone trait of biocontrol potentials (Table 1).Thus, isolate RHI-146 exhibited maximuminhibition against pathogenic fungi Rhizoctoniasolani AG-2, followed by isolate RHI-39(Figure 2). Isolate RHI-39 showed maximuminhibition against to Fusarium oxysporum,followed by isolate RHI-146 (Figure 1).Further, 40 isolates (26.7%) had strongantagonistic activity against Fusarium oxysporumand Rhizoctonia solani AG-2. Results indicatedthat Streptomyces were released extracellulardiffusible metabolites which inhibited hyphalgrowth of pathogenic fungi.

Table 1. CAS assay for analysis of siderophore produced by broad spectrum Streptomycesisolate and antifungal activity.

Isolate

RHI-19RHI-27RHI-29RHI-33RHI-35RHI-39RHI-40RHI-42RHI-43RHI-55RHI-62RHI-67RHI-72RHI-80RHI-81RHI-91RHI-99RHI-104RHI-107RHI-116RHI-145RHI-146

Color change onCAS agar

orangeorangeorangeorangeorangeyellowyellowyelloworangeorangeyelloworangeyellowyelloworangeyelloworangeorangeorangeorangeorangeyellow

Zone width(mm)

15.7±1.2 b16.0±1.0 b12.3±1.5 c11.3±1.2 c10.0±1.0 d29.0±1.0 a28.3±3.2 a

23.0±1.0 ab17.7±1.5 b26.3±1.2 ab15.3±2.1 b26.3±1.2 ab27.3±2.5 ab28.7±2.1 a13.7±0.6 c11.3±0.6 c11.0±1.0 c11.3±0.6 c15.3±0.6 b12.7±1.5 c10.7±1.5 c30.3±0.6 a

% mycelia inhibition in PDA in dual culture assayFusarium oxysporum

42.2±0.7 e60.1±8.8 c21.8±0.2 g53.3±2.3 d54.7±2.3 d80.1±4.4 a48.5±1.3 e54.0±5.3 d33.8±4.6 f59.2±3.2 c

270.0±9.1 b70.0±9.1 b37.3±0.3 f50.7±1.2 d62.3±0.3 c45.8±3.6 e67.9±7.5 c31.5±0.5 f37.7±0.3 f33.2±3.7 f24.1±1.6 g75.0±6.3 b

Rhizoctonia solani AG-280.0±2.2 c81.8±6.1 c42.7±3.3 f

70.9±5.2 de78.8±3.5 d83.4±0.3 c82.0±4.8 c

71.4±7.6 de70.8±1.4 de86.9±3.6 b67.5±1.2 e85.7±1.2 b66.0±5.2 e

74.5±3.8 de85.6±3.4 b80.3±1.1 c87.7±6.0 b78.6±2.2 d83.9±4.8 b81.6±2.6 c65.1±2.4 e97.0±1.7 a

* Means with the same letter are not significantly different (LSD) according to ANOVA test (p ≤ 0.01).

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Table 2. Effect of Streptomyces colony color series on antagonistic activities against Fusariumwilt disease and sheath blight disease of rice.

Parameter

Number of isolatePercentage ofcolony colorNumber ofactive isolateNumber of

active isolate againstFusarium oxysporumNumber of active

isolate againstRhizoctonia solani AG-2

Color seriesGrey

5134

49

35

40

Brown

4530

35

27

30

White

1510

14

8

9

Yellow

1310

8

6

6

Green

58.6

2

ND

1

Black

33.3

2

2

2

Orange

162

11

7

5

Noproduced

32

1

1

1

Total

150100

113

86

94

Figure 1. Scanning electron micrographs of the deformation of fungal mycelium of Fusariumoxysporum and Rhizoctonia solani AG-2 by the antagonist at 5 days after co-culturing1. Streptomyces isolate RHI-39

(A) mycelium of fungal pathogen (bar = 1 mM)(B) small and thinner mycelium and abnormal branching of fungal pathogen caused by

Streptomyces isolate RHI-392. Streptomyces isolate RHI-146

(A) mycelium of fungal pathogen (bar = 1 mM)(B) small and thinner mycelium and abnormal branching of fungal pathogen caused by

Streptomyces isolate RHI-146.

Chiang Mai J. Sci. 2018; 45(4) 1689

When growing Streptomyces isolateRHI-39 and Streptomyces isolate RHI-146with Fusarium oxysporum and Rhizoctoniasolani in PDA, the penetration of myceliumof Streptomyces isolate RHI-39 and Streptomycesisolate RHI-146 into the colony ofFusarium oxysporum were revealed underSEM (Figure 1). The area where bothStreptomyces isolates grew had a clearinhibition zone. Thus, the clear inhibitionzone was observed on the mycelia massof Fusarium oxysporum indicating possibleproduction of antibiological compoundsand extracellular enzyme. The degradationof fungal mycelium by the antagonistwas observed. The fungal mycelia weresmaller and thinner which causedabnormal branching of hyphae anddeformation of the fungi than controlplate (Figure 1).

3.3 Production of Cell Wall DegradingEnzymes

Out of 150 Streptomyces isolates, 37isolates produced chitinases (24.7 %), 79isolates produced cellulase (52.7 %), 109isolates produced protease (72.7 %), 56 isolatesproduced β-1,3-glucanase (62.7 %) and allisolates produced catalase (100 %). Screeningof cellulase and β-1,3-glucanase producerswere conducted by Congo red test as apreliminary study. After 5 days of incubation,79 isolates showed clear zone on CMC agarwhich indicated cellulose degradation. IsolateRHI-41, RHI-42, RHI-43 showed maximumcellulase activity (4.25, 5.00, 4.84 U/ml).Further, 58 isolates produced β-1,3-glucanase(38.7 %) and glucanolytic activity were varied(0.03 to 2.53 U/ml). Moreover, 109 isolatesexhibited protease activity (72.7 %) and isolatesRHI-6, RHI-120, RHI-131 showed maximumprotease activity (130, 112, 95 U/ml)(Table 3).

Figure 2. Morphology observation of Streptomyces lydicus isolate RHI-39 and Streptomycescorchorussi isolate RHI-146 on ISP-2 medium incubated at 30 °C for 7 days

(A) Color of substrate mycelium(B) Color of aerial mycelium(C) Aerial mycelium under light microscopy at 400× magnification(D) Spores surface and spore chain under scanning electron microscopy at 10,000×

magnification.

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Table 3. Bioactive compounds and hydrolytic enzyme activity of potential Streptomyces.

Isolates

RHI-6RHI-16RHI-23RHI-40RHI-41RHI-42RHI-43RHI-44RHI-45RHI-57RHI-58RHI-60RHI-72RHI-80RHI-110RHI-112RHI-114RHI-120RHI-128RHI-149

Hydrolytic enzyme activity (U/ml)Cellulase

2.25±0.60 eND

0.90±0.65 h3.58±1.25 c4.25±1.50 b5.00±1.25 a

4.84±1.35 ab2.98±0.65 d

ND2.87±1.25 d0.95±0.65 h1.32±0.45 g1.45±0.6 f0.87±1.25 h

ND1.42±1.25 f1.00±0.65 h

NDNDND

ChitinaseND

3.58±1.25 aND

3.15±1.25 bND

3.00±1.25 b2.85±1.00 c

NDNDNDNDND

2.53±0.65 d0.87±0.35 fg0.95±0.15 f

ND0.58±1.25 ef

ND1.79±1.00 e0.35±0.15 g

Protease130±1.75 a

NDND

122±1.50 bNDNDNDND

115±1.25 cNDNDND

95±0.65 d119±1.50 b60±0.35 f

ND85±0.45 e112±1.00 c

NDND

β-1,3-glucanase0.10±0.15 g

NDND

0.45±0.35 d1.00±0.65 c2.50±1.25 a

ND1.83±1.25 b

ND0.10±0.35 g0.06±0.25 h0.36±0.25 e0.30±0.15 f0.29±0.15 f

ND0.37±0.25 e0.04±0.15 h

NDNDND

*Means with the same letter are not significantly different (LSD) according to ANOVA test(p ≤ 0.01)**ND = not detected, + = produced fluorescent pigment, - = not produced fluorescentpigment.

3.4 Characterization and Identification ofPotential Streptomyces3.4.1 Phenotypic characteristics

The growth of isolate RHI-39 and isolateRHI-146 was checked on various testedmedia, including yeast extract-malt extract(ISP-2), oatmeal (ISP-3), inorganic salt-starch(ISP-4), glycerol-asparagin (ISP-5), tyrosine(ISP-7) and Bennett’s modified agar. Ondifferent agar, both isolates produceddifferent colony colors and aerial spore massesdepending on the substrate. The characteristicsof isolate RHI-39 and isolate RHI-146 wereclosely related to the genus Streptomyces.

3.4.2 Cultural and physiologicalcharacterization, cell wall composition(DAP) and microscopic determination

Streptomyces isolates were checked forchemical analysis of DAP isomer in wholecell hydrolysates. It revealed that 79 isolates(52.7 %) were assigned to the group ofStreptomyces as their whole cell hydrolysateswere rich in LL-DAP and formed coloniescharacteristics of Streptomyces. Fifty-threeisolates (35.3 %) contained meso-DAP whichwere classified as non-Streptomyces. Eighteenisolates (12 %) were classified as unidentifiedgroup. Cell wall peptidoglycans of isolate

Chiang Mai J. Sci. 2018; 45(4) 1691

RHI-39 and isolate RHI-146 containedLL-diaminopimelic acid, typical of cell wallchemotype I, as well as glutamic acid, alanineand glycine. Cell contained no diagnosticsugar components.

After grouping by colony color, eightmain classes were observed and main colorswere grey (34 %), brown (30 %), white(10 %), orange (10 %), yellow (8.6 %), green(3.3 %), black (2 %) and no-produced (2 %)(Table 2). On the basis of in vitro antagonisticactivities against Fusarium wilt diseaseand sheath blight disease, we selected isolateRHI-39 and isolate RHI-146 for identification.Under light microscope and SEM, bothsubstrate and aerial mycelia structure of isolateRHI-39 and isolate RHI-146 were observed.The substrate mycelium of isolate RHI-39

was brown and aerial mycelium was white.SEM observation, spores showed smoothsurface and spore chains were straight spiralwhich contained generally 8-10 spores perchain. RHI-39 colonies appeared powderyin consistency with brown mycelium,produced brown pigment on ISP-2 mediumwhich was classified as Streptomyces (Table 4).The substrate mycelium of isolate RHI-146was yellow and aerial mycelium was white.Microscopic determination by SEM revealedthat spores showed smooth surface andspore chains were straight spiral whichcontained generally 6-11 spores per chain(Figure 2). In addition, RHI-146 coloniesappeared powdery in consistency withyellow mycelium, produced yellow pigmenton ISP-2 medium and other media (Table 5).

Table 4. Morphological and biochemical characteristics of Streptomyces isolate RHI-39 ondifferent culture media.

Culture media

ISP-1ISP-2ISP-3ISP-4ISP-5ISP-6ISP-7ISP-8ISP-9

Hickey-TresnerNutrient agar

PDAModified Bennett agar

Streptomyces isolate RHI-39Growth rate

MediumHigh

MediumMedium

HighMedium

HighMedium

RareMediumMediumMediumMedium

Aerial myceliumBrownWhiteBrownBrownWhiteYellowWhiteYellowBrownYellowYellow

Light orangeYellow

Substrate myceliumLight orange

OrangeBrownYellowOrange

Light orangeOrange

Light orangeOrangeBrown

Light orangeOrangeBrown

Pigment-

Brown-----------

* Color compared with Standard ISCC-NBS color chart [14]. * * ISP-1, Tryptone-yeastextract agar; ISP-2, Yeast extract-malt extract agar; ISP-3, Oatmeal agar; ISP-4, Inorganicsalts-starch agar; ISP-5, Glycerol-asparagine agar; ISP- 6, Peptone-yeast extract-iron agar; ISP-7, Tyrosine agar; ISP-8, Nitrate medium; ISP-9, Carbon utilization agar

1692 Chiang Mai J. Sci. 2018; 45(4)

Figure 3. Neighbor-joining tree based on 16S rRNA gene sequences showing relationshipamong Streptomyces isolate RHI-146 and related Streptomyces. The numbers on branches indicatedpercentage bootstrap values of 1,000 replicates (only values > 50% are indicated). Bar represents0.005 substitutions per nucleotide position.

Table 5. Morphological and biochemical characteristics of Streptomyces isolate RHI-146 ondifferent culture media.

* Color compared with Standard ISCC-NBS color chart [14] * * ISP-1, Tryptone-yeast extractagar; ISP-2, Yeast extract-malt extract agar; ISP-3, Oatmeal agar; ISP-4, Inorganic salts-starchagar; ISP-5, Glycerol-asparagine agar; ISP-6, Peptone-yeast extract-iron agar; ISP-7, Tyrosineagar; ISP-8, Nitrate medium; ISP-9, Carbon utilization agar

Culture media

ISP-1ISP-2ISP-3ISP-4ISP-5ISP-6ISP-7ISP-8ISP-9

Hickey-TresnerNutrient agar

PDAModified Bennett agar

Streptomyces isolate RHI-146Growth rate

MediumHigh

MediumMedium

HighMedium

HighMedium

RareMediumMediumMedium

high

Aerial myceliumBrownWhiteBrownOrangeBrownYellowBrownYellowBrownBrownWhite

Light orangeBrown

Substrate myceliumLight orange

YellowLight brown

BrownOrange

Light orangeLight orange

YellowBrownOrange

Light yellowOrange

Light orange

Pigment-

Yellow-----------

Chiang Mai J. Sci. 2018; 45(4) 1693

3.4.3 Molecular identification andphylogenic tree analysis

The 16S rDNA amplicons weresequenced and identified using the BLASTalgorithm. The identified cultures belongedto the genus Streptomyces. BLAST search resultsindicated 16S rDNA sequence of RHI-39isolate was closely related to Streptomyces lydicuswith 90 % (1,267 bp) identity and RHI-146isolate had close relationship to Streptomycescorchorusii with 99 % (1,349 bp) identity.Comparison of isolate RHI-39 and isolateRHI-146 nucleotide sequence with membersof actinomycetes clearly showed that theseisolates belonged to the genus Streptomyces.A neighbor-joining tree based on 16S rDNAsequences showed that the isolates occupieda distinct phylogenetic position with theradiation including representatives of theStreptomyces family. The phylogenetic treebased on Maximum-parsimony method also

showed that the isolates formed a separateclade. Physicochemical parameters and 16SrDNA sequencing showed that RHI-146isolates were identified as Streptomyces corchorusii(Figure 3).

3.5 Determination of Antifungal Activityof Potential Streptomyces

To determine antifungal activityproduced by Streptomyces, crude extractswere prepared by solvent extraction andtest for antifungal activities using agar welldiffusion assay. Inhibition of mycelia growthwas observed in the presence of crudeextracts after incubation for 5 days. Crudeextract of Streptomyces (14 isolates) inhibitedmycelia growth of Fusarium oxysporum andRhizoctonia solani AG-2. Crude extract ofisolate RHI-146 exerted a high inhibitoryactivity over other crude extract samples(Table 6).

Table 6. Antifungal activity of culture filtrate and crude extract of Streptomyces against Fusariumoxysporum and Rhizoctonia solani AG-2 using agar well diffusion method.

Isolates

RHI-11RHI-12RHI-27RHI-35RHI-39RHI-55RHI-67RHI-79RHI-81RHI-99RHI-110RHI-111RHI-146RHI-147

Fusarium oxysporumCulture broth13.3±1.5 def11.7±1.5 f12.7±1.2 ef

13.0±1.0 def22.7±0.6 ab15.3±0.6 d

13.7±1.5 def19.7±1.2 c

13.7±1.2 def20.0±1.0 c

21.0±2.0 bc14.0±1.0 def24.3±1.5 a

15.0±2.0 de

Crude extract13.7±2.1 de12.3±1.2 e15.0±1.0 d14.3±1.2 de24.7±0.6 a15.7±1.5 d

14.0±1.0 de20.3±1.2 b12.3±0.6 e

19.3±1.5 bc19.0±1.0 bc15.3±0.6 d26.3±0.6 a17.7±1.5 c

Rhizoctonia solani AG-2Culture broth

22.3±1.2 c20.0±1.0 de21.7±1.2 cd19.0±1.0 e22.3±0.6 c

23.0±1.0 bc21.0±1.7 cd24.7±0.6 ab21.3±0.6 cd22.7±0.6 c18.3±1.2 e22.3±0.6 c26.0±1.0 a20.0±1.7 de

Crude extract23.3±1.5 bc21.0±1.7 cd23.3±0.6 bc20.0±1.7 d24.0±1.0 b24.0±1.0 b23.0±1.7 bc26.7±0.6 a

22.3±2.1 bc24.3±1.2 b18.7±1.2 d23.7±1.2 b26.7±1.2 a18.7±0.6 d

*Means with the same letter are not significantly different (LSD) according to ANOVA test(p ≤ 0.01)

1694 Chiang Mai J. Sci. 2018; 45(4)

This research showed that Streptomyces sp.exhibited antifungal activities to Fusariumoxysporum and Rhizoctonia solani AG-2 in vitro,which proved that rhizospheric soil of ricecan serve as an effective source of biocontrolmicroorganisms.

4. DISCUSSION

Actinomycetes are abundantly distributedin the rhizosphere and colonized plantroots which play a role in plant growthpromotion. In the present study, we isolatedand characterized actinomycetes from ricerhizosphere samples. Rhizosperic soil wasselected in this study because it containedtwice as much Streptomyces isolates asnon-rhizospheric soils [10]. Results indicatedthat Streptomyces sp. constituted the majorityof total actinobacteria in the rice rhizosphere(106 cfu/g). The search for novel metabolitesfrom Streptomyces sp. was done fromrhizosphere of rice because Streptomyces havebeen found to have higher occurrences inrhizosphere soil than in bulk soil [18].Plant exudates may play a role in theoccurrence of microbial diversity as reportedby Coombs and Franco (2003) [18] thatactinobacteria were the abundant rhizospheremicroflora and effective colonizers of plantroot.

The use of actinobacteria to preventplant pathogens and insect pests issupplementary for biological control ofplant diseases. Phytopathogenic fungi areone of the major problems to agriculturalproduction. Streptomyces sp. produced variousbioactive compounds including antibioticswhich used as agrochemicals [18]. Resultsindicate that 98 % of isolates were activeagainst all tested fungi and percentage ofactive isolates varied by each color series.The most active compound producingisolates belong to grey and brown color seriesand 98 % were active against one of tested

organisms. Most active Streptomyces werefound in white and grey colony color series[18], however, some Streptomyces isolates (2 %)did not show any antibiotic activity (Table 2).It could be due to the possibility that theseisolates produced other compounds whichwere not screened for activities in this study.Comparison of antifungal activity amongall color classes against tested organismsshowed that isolates in grey and brown seriesdisplayed highest antibiosis against fungaltested. Different percentage of antifungalactivity could belong to different speciesof Streptomyces or different bioactivecompounds. Current use of Streptomyces tocontrol pathogenic fungi is little. Streptomycesisolate RHI-39 and Streptomyces isolateRHI-146 inhibited mycelium growth andspore germination of Fusarium oxysporumand Rhizoctonia solani AG-2. Our researchagreed with Streptomyces violaceusniger G10which showed antagonistic effects againstFusarium oxysporum f. sp. cubense and madehyphal swelling and inhibited sporegermination of fungi [19]. Chitinaseproducing strain of Streptomyces griseusMG 3 possessed antifungal activity againstAspergillus, Botrytis cinerea, Fusarium culmorum,Guignardia bidwelli and Sclerotia sclerotiorum [20].Also, Streptomyces strain CDRIL-312 andS. purpueofuscus showed good antifungal activityagainst filamentous fungi [21]. However, thereare limited reports of Streptomyces sp. whichshowed antagonistic effect to rice pathogens,i.e. Fusarium oxysporum and Rhizoctonia solaniAG-2 which caused yield losses up to 50 %[22]. The effects of Streptomyces sp. on fungalmycelia in dual culture assay were checkedunder SEM. They showed mycelia deformitiesnear the zone of inhibition that were causedby antibiological compounds and extracellularenzymes (Figure 1). There are many reportsrelated to antifungal substances whichinduce malfunctions such as stunting,

Chiang Mai J. Sci. 2018; 45(4) 1695

distortion, and swollen hypha protuberancesof highly branched appearance of fungalgerm tubes [22].

Biocontrol is one of the most desirabletraits for Streptomyces. It has been reportedthat antifungal mechanism has been attributedto the action of hydrolytic enzymes such aschitinase, β-1,3-glucanase and protease [23].Production of chitinase and β-1,3-glucanaseby Streptomyces sp. was related to fungalgrowth inhibition and biocontrol of fungalpathogens due to the cell wall degradingenzyme activity [23]. Phytopathogenic fungicell wall contains β-1,3-glucan, chitin andprotein which are essential for diseasetransmission and pathogenesis. Cell lysis byβ-1,3-glucanase/chitinase/protease-producingmicrobe leads to leakage of cell contentsand collapse of fungal cell wall [24].Streptomyces isolate RHI-16 produced themaximum chitinase (3.85 U/ml), followedby Streptomyces isolate RHI-40 (3.15 U/ml)and Streptomyces isolate RHI-42 (3.00 U/ml)(Table 3). Chitinase degraded fungal cellwall and limited growth of fungal pathogens.Streptomyces sp. produced various levels ofchitinase. Rhizospheric Streptomyces withchitinolytic activity have been reported tosignificantly reduce the risk of pathogenicinfection [24]. Bressan (2003) [25] reportedthe effectiveness of Streptomyces sp. isolatedfrom maize rhizosphere that they inhibitedseed pathogenic fungi, i.e. Aspergillus sp. andFusarium subglutinans but did not suppressPenicillium sp. The culture filtrate of Streptomycessp. inhibited mycelium growth of Pythiumutimum, Fusarium oxysporum f. sp. albedinis,Scherotium rolfsii, Verticillium dahlia and Botryliscinerea [26]. The non-significant chitinaseproducer, Streptomyces isolate RHI-39 andStreptomyces isolate RHI-146, inhibited alltested fungal pathogens indicating achitinase-independent antifungal activity.This was consistent with results by Jog et al.

(2014) [27].Protease activity in culture filtrates of

tested isolates was determined and resultswere presented in terms of enzyme units(Table 3). Isolate RHI-146 culture filtrateshowed maximum protease activity (130 U/ml), followed by Streptomyces exfoliates CFS1068(115.2 U/ml), and Streptomyces somaliensis GS1242 (102.0 U/ml) [28]. Siderophoreproduction of tested isolates were detectedby color change on CAS agar and productionof halo zone around colonies. Differentcolor changes in CAS agar suggest theproduction of siderophores by a variety ofmicroorganism isolated. Two major groupsof bacterial siderophores, hydroxamate andcatechol, at neutral pH are reddish-orangeto yellow color respectively [29]. Siderophoreis one of the compounds that stimulates plantgrowth by forming complex with iron (Fe3+)in the rhizosphere, making iron unavailablefor phytopathogens and developing growthof the plant. Our research, showed that14.6 % of isolate were positive forsiderophore production. Siderophore isusually produced by actinomycetes. It bindsFe3+ from the environment and makesFe3+ available for microbial growth. Plantsutilized siderophore-bound Fe3+ as an ironsource. Str eptomyces sp. isolated fromrhizosphere soil produce siderophoreand inhibit growth of phytopathogens.Rhizospheric Streptomyces sp. need to competewith other rhizosphere plant pathogensfor iron, hence competition for iron is apossible mechanism to control thephytopathogens.

Streptomyces isolate RHI-39 andStreptomyces isolate RHI-146 inhibitedmycelium growth and spore germination ofFusarium oxysporum and Rhizoctonia solaniAG-2 by various modes of action. Manystudies refer Streptomyces isolates for plantprotection from most phytopathogenic fungi.

1696 Chiang Mai J. Sci. 2018; 45(4)

They can survive in soils of various types,have long survival due to their spore formingability, are capable of producing variousbioactive compounds such as antibiotics,siderophores, chitinase, phytohormonesand of phosphate solubilization [30].Moreover, there are limited number of reportof Streptomyces sp. that showed antagonisticability to rice pathogens, i.e. Fusarium oxysporumand Rhizoctonia solani AG-2 which caused yieldlosses up to 50 %. Our study confirmed thatStreptomyces are able to produce a wide varietyof antifungal activity. The cultural andmorphological characterization indicatedthat the isolate belongs to Streptomyces sp. Atree constructed by the Maximum-parsimonymethod showed that the isolate forms adistinct phyletic line from other relatedisolates available in the database (Figure 3).The isolate RHI-146 showed 99 % similaritywith Streptomyces corchorusii NBRC 12850 onthe culture data bank.

Data from the research proved that thehighest antifungal activities not onlydisplayed antagonistic activity characteristicbut also displayed hydrolytic enzymeproduction. The potential for developmentof actinomycetes as a biocontrol agentwas primarily indicated by the antagonisticactivity mechanism including a). directmechanism via an antibiosis or lysis mechanism,or b). indirect mechanism by inducingplant deference’s and growth promotersubstances. The antibiosis mechanism isfacilitated by antibiotic compounds, andthe lysis mechanisms are facilitated byamylase, protease, chitinase, cellulase andlipase produced by the actinomycetes.Additionally, indirect mechanism such assiderophore production and hydrogencyanide characteristics combated diseasedevelopment. Results showed that acombination of hydrolytic enzymes or otherantagonistic mechanisms result in a higher

level of antagonism than single mechanismalone (Table 1). Preliminary assessmentsof these characteristics are important forthe best selection of biocontrol agentcandidates. Streptomyces sp. showed higherantagonistic to rice fungal pathogen. The ideathat ideal biological control agent suitesnative conditions is of the prime importance.Streptomyces sp. are especially significant forthe following reasons: they can survivein soils of various types, have longsurvival due to their spore forming ability,are capable of producing various bioactivecompounds, such as antibiotics, siderophores,chitinases, phytohormones and of phosphatesolubilization [29]. Nowadays, biopesticidesare applied using spray technologies, dippingor coating seed but the success is highlylimited because of improper equipment,poor formation efficacy or combinationof both [30]. The biocontrol agent whichuses for leaf surface is exposed to rapidlyfluctuating temperature and relative humidity.Leaf surface provides limited nutrientresources to bacterial colonies and most ofphyllosphere bacterial colonies are easilykilled by non-penetrating agents such asperoxide or UV light [31].

In conclusion, the research described thein vitro activity of actinomycetes whichsuppressed the growth of Fusarium oxysporumand Rhizoctonia solani AG-2, the causingagent of Fusarium wilt disease and sheathblight disease of rice in vitro, respectively.Preliminary analysis based on antifungalactivity, hydrolytic enzyme and siderophoreproduction facilitated the best selection ofactinomycete candidates. Further researchesare required to evaluate their potential asbiocontrol under greenhouse and fieldconditions and to develop appropriateformulation to maintain the survival ofbiocontrol agent on the leaves and roots.Streptomyces sp. are likely to be the potential

Chiang Mai J. Sci. 2018; 45(4) 1697

candidates for discovery of novel secondarymetabolites for various biocontrol application.Determination of the exact mechanisms ofaction of the biocontrol agents can assist theuse of eco-friendly bio-fungicides in the future.

ACKNOWLEDGEMENTS

A grant from The Thailand ResearchFund (TRF; Research no. MRG 5580102) isgreatly appreciated. We thank Dr. NakarinSuwannarash, Chiang Mai University,THAILAND for providing pathogenic strainof Fusarium oxysporum and Rhizoctonia solaniAG-2. The authors also acknowledge supportof the Center of Excellence in Bioresourcesfor Agriculture, Industry and Medicine,Chiang Mai University is greatly appreciated.We are thankful to Dr. Olivier Rasp ,National Botanic Garden of Belgium,BELGIUM for improving the text.

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