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Journal of Environmental Management 95 (2012) S280eS284

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Journal of Environmental Management

journal homepage: www.elsevier .com/locate/ jenvman

Isolation and identification of actinomycetes from a compost-amended soilwith potential as biocontrol agents

Gonzalo Cuesta a, Rosana García-de-la-Fuente b, Manuel Abad b, Fernando Fornes b,*

aDepartamento de Biotecnología, Universidad Politécnica de Valencia, P.O. Box 22012, E 46071 Valencia, Spainb Instituto Agroforestal Mediterráneo, Universidad Politécnica de Valencia, P.O. Box 22012, E 46071 Valencia, Spain

a r t i c l e i n f o

Article history:Received 17 August 2009Received in revised form28 April 2010Accepted 29 November 2010Available online 28 December 2010

Keywords:StreptomycesLechevalieriaPhytopathogenic fungiComposted two-phase olive mill waste

* Corresponding author: Tel.: þ34 96 3879 413; faxE-mail address: ffornes@bvg.upv.es (F. Fornes).

0301-4797/$ e see front matter � 2010 Elsevier Ltd.doi:10.1016/j.jenvman.2010.11.023

a b s t r a c t

The search for new biocontrol strategies to inhibit the growth of phytopathogenic microorganisms hasbecome widely widespread due to environmental concerns. Among actinomycetes, Streptomyces specieshave been extensively studied since they have been recognized as important sources of antibiotics.Actinomycete strains were isolated from a calcareous soil, 2 two-phase olive mill waste (‘alperujo’)composts, and the compost-amended soil by using selective media, and they were then co-cultured with5 phytopathogenic fungi and 1 bacterium to perform an in vitro antagonism assay. Forty-nine actino-mycete strains were isolated, 12 of them showing a great antagonistic activity towards the phytopath-ogenic microorganisms tested. Isolated strains were identified by 16S rDNA sequence analysis andphenotypic procedures. Eleven isolates concerned the genus Streptomyces and 1 actinomycete withchitinolytic activity belonged to the genus Lechevalieria.

� 2010 Elsevier Ltd. All rights reserved.

1. Introduction

Composted materials have traditionally been applied to agri-cultural and horticultural soils as means of improving soil fertilityand crop growth, mainly by enhancing soil physical and chemicalproperties. These organic amendments also contribute to improvesoil biological characteristics, often providing an effective control ofsoil-borne diseases (Vargas-García and Suárez-Estrella, 2008).

Compost capability to suppress soil-borne plant pathogens hasbecome an interesting subject as a strategy for reducing the adverseeffects of massive fungicides’ applications on the environment.Several mechanisms have been proposed to explain plant diseasecontrol by compost such as competition, hyperparasitism, activa-tion of disease-resistance genes, or antibiotic production bybeneficial microorganisms (Hoitink and Boehm, 1999; Maher et al.,2008).

In this context, actinomycetes have received considerableattention as biocontrol agents, particularly Streptomyces species.Streptomyces are Gram-positive aerobic members of the orderActinomycetales within the classis Actinobacteria which producean extensive branching substrate and aerial mycelium (Andersonand Wellington, 2001). Since the first antibiotic discovery in 1942,there have been continued efforts towards screening compounds

: þ34 96 3877 419.

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from the genus Streptomyces (Watve et al., 2001) which is known tobe the largest antibiotic-producing genus. In fact, about 60% of theantibiotics developed for agriculture and horticulture have beenisolated from Streptomyces spp. (Hwang et al., 2001). Lechevalieria(formerly classified as Saccharothrix), like Streptomyces, are Gram-positive aerobic members of the order Actinomycetales within theclassis Actinobacteria (Labeda et al., 2001). Until now, only oneLechevalieria species has been described as antibiotic producer(Onaka, 2009). In addition, no chitinolytic activity has been repor-ted for this genus.

The aim of this investigation was to isolate actinomycetes froma soil, 2 two-phase olive mill waste (‘alperujo’) composts, anda compost-amended soil to perform an in vitro antagonism assay,and to identify the actinomycete strains with the highest inhibitoryactivity.

2. Materials and methods

2.1. Sample collection

Actinomycetes were isolated from one calcareous soil located atLliria (Valencia, Spain), �39�4500400 in latitude and 0�4101000 inlongitude, 2 composts prepared from two-phase olive mill waste(85% by dry weight) mixed with fresh horse manure (15% dry wt) eone irrigated with water and the other with an animal fattyproteinaceous waste sludge during the first 38 days of composting,

G. Cuesta et al. / Journal of Environmental Management 95 (2012) S280eS284 S281

and the soil amended with each of the composts at two rates(12 Mg ha�1 and 24 Mg ha�1).

2.2. Isolation of actinomycete strains

The samples were firstly homogenized in buffered peptonewater at 100 rpm for 30 min, serially diluted, and cultured on starchcasein agar (SCA), arginine glycerol salts agar (AGSA), and glycerolasparagine agar (International Streptomyces Medium No. 5 [ISP-5])(Shirling and Gottlieb, 1966), all supplemented with cyclohexamide(50 mg L�1) to reduce fungal contamination (Labeda and Shearer,1990). Sample dilution plates were incubated at 28 �C for 14e21days until sporulated or non-sporulated actinomycete colonieswere observed. Selected colonies were then inoculated onto yeastextractemalt extract agar (ISP-2) for purification and stored at 4 �Cin slant agar and in 20% glycerol at �80 �C.

2.3. Phytopathogenic strains

To assess the actinomycete potential suppressive effects, 5phytopathogenic fungi e Fusarium oxysporum f. sp. melonis (CECT20474), Phytophthora cinnamomi (CECT 20186), Pythium debar-yanum (CECT 2362), Sclerotinia sclerotiorum (CECT 2823), andThanatephorus cucumeris (CECT 2813) e and 1 bacterium e Agro-bacterium tumefaciens (CECT 4119) e obtained from the “ColecciónEspañola de Cultivos Tipo” (CECT) were included in the in vitroantagonism experiment. Previous experiments using six differentculture media showed that the maximum antifungal activities ofactinomycetes were obtained with the potato dextrose agar (PDA)and the yeast-malt extract agar (YMA) (data not shown). Then,fungi strains were incubated at 28 �C on PDA and YMA media for5e7 days. The A. tumefaciens strain was cultured on nutrient agar(NA) at the same temperature for 24 h.

2.4. Fungal antagonism assay

With the aim of obtaining active growing microorganisms,actinomycete strains were incubated on PDA (Castillo et al., 2002;Getha et al., 2005) and YMA (Gomes et al., 2000) at 28 �C for 7days or until sporulation was detected. From these media, 4 agarplugs of 6 mm diameter (corresponding to 4 different isolatedstrains) were transferred at equidistant positions to Petri dishescontaining YMA and PDA, and incubated for 7 days at 28 �C. Afterthis period, a plug with fungal mycelia was placed in the centre ofthe plate and co-cultured at 28 �C for 7e14 days. Antagonism wasdetermined by measuring the distance between the growing edges

Table 1Antagonistic activity of the actinomycete strains on YMA and PDA culture media.

Isolate Fusarium oxysporumf. sp. melonis

Phytophthoracinnamomi

Sclescle

YMA PDA YMA PDA YMA

CO2-9 ea e þþ þ e

CO2-16 þþ þþ þþþ þþþ þS-1 þþþ þþþ þþþ þþþ þþS-2 þþ þþþ þþþ þþþ þþþS-3 þþþ þþþ þþþ þþþ þþS-5 þþ þþ þþ þþ þS-6 þþþ þþþ þþþ þþþ þþþS-7 e e e e e

T2-10 e e þþþ þþþ e

T2-19 e e þþ þþ þT6-32 e e þþ e þþT8-2 e þ e þ e

a e, þ, þþ, þþþ: no inhibition, minimum, intermediate and maximum antagonism, r

of actinomycetes and fungi, hence establishing 4 levels of inhibi-tion: maximum (þþþ), scored as an inhibition distance >2 cm,intermediate (þþ) as a distance of 2e1 cm, minimum (þ) asa measurement <1 cm, and no antagonism (e) when contactbetween actinomycetes and fungi occurred. Two replicates werecarried out for each of the presumptive antagonist strains and thephytopathogenic fungi.

2.5. Bacterial antagonism assay

Prior to the test, A. tumefaciens was grown on NA (see Section2.3) and suspended in sterile nutrient broth (NB) with a concen-tration adjusted to approximately 1.5�108 CFUmL�1. This bacterialinoculum was homogenized and serially diluted to 1.5�107,1.5�106 and 1.5�105 CFUmL�1, respectively. One millilitre of theresulting suspensions was added to a test tube containing 19 mLliquid sterile NA at ca. 50 �C, vortexed, and transferred to Petridishes. Once solidified, 6 plugs (6 mm in diameter) of actinomy-cetes grown on YMA and PDA were equidistantly positioned oneach inoculated medium and incubated at 28 �C. After 24 and 48 hof incubation, inhibition zones were measured as the radialdistance between bacterial and actinomycete growth.

2.6. Identification of antagonistic actinomycete strains

Actinomycete strains which showed the highest inhibitoryeffects towards the phytopathogenic microorganisms tested wereselected to perform their identification by using molecular andphenotypic procedures.

Total genomic DNA was extracted according to the CTAB(cetyltrimethylammonium bromide, Sigma) procedure (Wilson,1987) and subsequently subjected to PCR (polymerase chain reac-tion) amplification using primers 27f and 1525r as described byLane (1991). Each 50 mL PCR contained 1 mL DNA extract, 1.5 mMMgCl2, 0.2 mM of each dNTPs (Ecogen), 0.4 mM of each primer, and1.5 U Taq DNA polymerase (Ecogen) with 1� PCR buffer. Amplifi-cationwas performed in a PTC-100 Peltier Thermal Cycler using theprogram: initial denaturation at 95 �C for 5 min and 30 cycles at95 �C for 1 min; annealing at 54 �C for 1 min; and, primer extensionat 72 �C for 1 min followed by a final extension at 72 �C for 10 min.Controls e where template DNA was replaced by sterile waterewere also included in each PCR experiment. The PCR product waspurified with the GenElute PCR Clean-up Kit (Sigma) with the samesets of primers. The 16S rDNA gene sequences were obtained usingthe ABI PRISM� BigDye� Terminator Cycle Sequencing Kit (version3.1) and the automatic sequencer Applied Biosystems 3730xl DNA

rotiniarotiorum

Pythium debaryanum Thanatephoruscucumeris

PDA YMA PDA YMA PDA

e e e þþ þþþþþþ þþþ þþþ þþþ þþþþþþ þþþ þþþ þþþ þþþþþþ þþþ þþþ þþþ þþþþþþ þþþ þþþ þþþ þþþþþ þ þþ þþþ þþþþþþ þ þþþ þþþ þþþþþ þþþ þþþ þþþ þþþe e e þþ þþe þ þ þ þe þþ e þ e

þ e þ þþ þþespectively.

Table 2Width (mm) of the inhibition zone between Agrobacterium tumefaciens (at threeconcentrations) and actinomycete isolates grown on YMA and PDA media for 48 h.

Isolate Bacterial inoculum concentration (CFUmL�1)

1.5� 107 1.5� 106 1.5� 105

YMA PDA YMA PDA YMA PDA

S-6 3 0 4 0 6 0T2-10 2 2 3.5 3.5 2 3.5T8-2 1 3 5 5 1 2

G. Cuesta et al. / Journal of Environmental Management 95 (2012) S280eS284S282

Analyzer. The 16S rDNA gene sequences were manually assembledfrom the combination of separate fragments generated withforward and reverse sequencing primers using the PHYDITprogram (Chun, 1995). The sequences were presumptively identi-fied using the BLAST (Basic Local Alignment Tool) program (NCBI;http://www.ncbi.nlm.nih.gov/). The almost complete sequenceswere aligned against sequences of reference strains. Phylogenetictrees were inferred using the neighbour-joining algorithm (Saitouand Nei, 1987) from the PHYLIP suite programs (Felsenstein,1993), and evolutionary distance matrices prepared after Jukesand Cantor (1969). The topologies of the resultant unrooted treeswere evaluated in a bootstrap analysis (Felsenstein, 1985) based on1000 resamplings of the neighbour-joining dataset using thePHYLIP package.

Diaminopimelic acid isomers from whole-cell extracts weredetermined on ISP-2 cultured actinomycete strains (5 days at 28 �C)according to standard procedures (Staneck and Roberts, 1974).Aerial spore-mass colour, substrate mycelial pigmentation, diffus-ible pigments, and melanin production were recorded on theInternational Streptomyces Project culture media (Shirling andGottlieb, 1966) after 14 days of incubation at 28 �C. Spore-chainsmorphology from cultures incubated for 10e14 days on ISP-5 wereobserved by light microscopy.

Chitinase activity was determined by streaking actinomycetestrains on a culture medium as described by Kawase et al. (2004).Chitinase productionwas assessed by visual examination of clearedzones developed around colonies incubated for 7 and 14 days.

3. Results and discussion

3.1. Isolation of actinomycetes

A total of 49 actinomycete strains were isolated from thecomposts, soil and amended soil samples using the selective mediaSCA, AGSA and ISP-5, and included in the in vitro screen against the6 phytopathogenic strains.

Table 3Identification of actinomycete strains by using 16S rDNA analysis and phenotypic metho

Isolate Actinomycete species DAPisomer

Aerial spore-masscolour

CO2-9 S. lincolnensis L Light greenCO2-16 S. aureoverticillatus L Dark orangeS-1 S. variegatus L Dark orangeS-2 S. variegatus L Orange-redS-3 S. variegatus L Light orangeS-5 S. griseoruber L Blue-greenS-6 S. lusitanus L Dark yellowS-7 S. olivochromogenes L Light greyT2-10 S. coeruleorubidus L White greenT2-19 Lechevalieria sp. meso naT6-32 S. griseoruber L Blue-greenT8-2 S. albogriseolus L Blue-green

a RF, S: Rectus-Flexibilis or Spira, respectively. na: not applicable.

3.2. Fungal antagonism assay

From the in vitro assays, 12 actinomycete strains were found tobe highly effective in the suppression of most of the 5 referencephytopathogenic fungi tested, thus providing 24.5% isolates withstrong inhibitory effects (Table 1). This percentage is similar tothose reported by other authors (Larkin and Fravel, 1998). It isremarkable that 7 strains (CO2-16, S-1, S-2, S-3, S-5, S-6, and T8-2)showed high antifungal activity against the 5 pathogenic strains,whereas antagonistic activity towards 2e4 fungal strains wasobserved in the 5 remaining actinomycete isolates (CO2-9, S-7, T2-10, T2-19, and T6-32).

3.3. Bacterial antagonism assay

The assay with A. tumefaciens disclosed that 3 actinomycetestrains e S-6, T2-10, and T8-2 e had antimicrobial activity againstthis phytopathogenic species. Inhibition radial zones controlledafter 48 h of incubation were wider than those recorded afterincubation during 24 h, thus suggesting that actinomycetes werestill producing antibiotic compounds. The distance between acti-nomycetes and bacterial growth after 48 h is presented in Table 2.

3.4. Identification of antagonistic actinomycete strains

After studying the morphology and pigmentation properties ofcolonies, all isolates e except the strain T2-19 e were presump-tively assigned to the genus Streptomyces (Table 3). As shown inFig. 1, the identification achieved from 16S rDNA gene sequencesrevealed that 11 actinomycete isolates belonged to the genusStreptomyces with 8 different species being represented: 3 strainswere identified as Streptomyces variegatus (S-1, S-2, and S-3;Fig. 1d), 2 as Streptomyces griseoruber (S-5 and T6-32; Fig. 1f), 1 asStreptomyces lincolnensis (CO2-9; Fig. 1f), 1 as Streptomyces lusitanus(S-6; Fig. 1c), 1 as Streptomyces aureoverticillatus (CO2-16; Fig. 1e), 1as Streptomyces olivochromogenes (S-7; Fig. 1b), 1 as Streptomycescoeruleorubidus (T2-10; Fig. 1a), and 1 as Streptomyces albogriseolus(T8-2; Fig. 1f). Nucleotide similarities between the Streptomycesisolated and the corresponding reference strains (Fig. 1) rangedfrom 99.43% for S-1 to 100% for T2-10 and T8-2. These resultssupport that streptomycetes have been investigated predominantlyas biocontrol agents, since they are frequently and easily isolated,and their antibiotics’ production arouses significant commercialinterest (Anderson and Wellington, 2001).

Strain T2-19 (accession number FN808347) was the only non-streptomycete isolate belonging to genus Lechevalieria. Phyloge-netic position of strain T2-19 (Fig. 2) was between Lechevalieria

ds.

Melaninproduction

Diffusible pigmentproduction

Spore chainsmorphology

Chitinaseproduction

þ þ RFa e

þ e RF e

þ e RF e

þ e RF e

þ e RF e

þ þ S e

e e RF e

e e S e

þ þ S e

e e na þe þ S e

þ e S e

0.02

S. albus subsp. albus (AJ621602)

S. fimbriatus (AB184659)

S. flavoviridis (AB184842)

S. speibonae (AF452714)

S. albogriseolus (AJ494865)

Isolate T2-10

S. coeruleorubidus (AB184849)

S. cyaneus (DQ028984)

S. carpinensis (AB184574)

100

73

81

77

83

100

79

a 0.02

S. albus subsp. albus (AJ621602)

S. diastatochromogenes (D63867)

S. mirabilis (AF112180)

S. neyagawaensis (D63869)

S. griseorubiginosus (AJ399488)

S. bottropensis (D63868)

S. scabiei (D63862)

Isolate S-7

S. olivochromogenes (AY094370)

S. seoulensis (Z71365)

S. chartreusis (AJ399468)

100

97

77

58

100

50

b

0.02

S. albus subsp. albus (AJ621602)

S. thermoviolaceus (Z68096)

S. pactum (AB184398)

S. griseoluteus (AY999751)

S. aurantiacus (AJ781383)

S. badius (AY999783)

S. galbus (X79852)

S. capoamus (AB045877)

Isolate S-6

S. lusitanus (AB184424)

100

91

83

72

8175

100

100

c0.02

S. albus subsp. albus (AJ621602)

S. coeruleofuscus (AJ399473)

S. odorifer (Z76682)

S. variegatus (AJ781371)

Isolate S-2

S. intermedius (Z76686)

Isolate S-3

Isolate S-1

S. gougerotii (Z76687)

S. rutgersensis (Z76688)

S. eurythermus (D63870)

S. nogalater (AB045886)

100

72

100

100

98

75

100

d

0.02

S. albus subsp. albus (AJ621602)

S. coelicolor (AB184196)

S. griseoaurantiacus (AB184676)

S. spiralis (EF178683)

S. ruber (AB184604)

S. roseiscleroticus (AB184251)

S. poonensis (DQ445792)

S. fumigatiscleroticus (DQ442499)

S. intermedius (AB184277)

S. diastaticus (AB184785)

Isolate CO2-16

S. aureoverticillatus (AY999774)

100

56

51

80

98

66

99

100

e 0.02

S. albus subsp. albus (AJ621602) S. sparsogenes (AJ391817)

S. acidiscabies (D63865)

S. cinnabarinus (AJ399487)S. galbus (X79852)

S. griseoruber (AY094585)

Isolate T6-32Isolate S-5

S. capoamus (AB045877) S. hygroscopicus (X79853)

S. indigocolor (AJ399464) S. bicolor (AJ276569)

Isolate CO2-9S. lincolnensis (X79854)

Isolate T8-2S. albogriseolus (FJ486338)

100

58

97

51

99

80

100

75

86

100

100

f

Fig. 1. Neighbour-joining tree based on nearly complete 16S rDNA gene sequences showing relationships between Streptomyces isolates and related Streptomyces type strain species.Numbers at nodes indicate levels of bootstrap support based on a neighbour-joining analysis of 1000 resampled datasets; only values >50% are given. The scale bar indicates 0.02substitution position.

G. Cuesta et al. / Journal of Environmental Management 95 (2012) S280eS284 S283

xinjiangensis (Wang et al., 2007) and the cluster made up ofLechevalieria atacamensis, Lechevalieria roselyniae, and Lechevalieriadeserti (Okoro et al., 2010). Sequence similarities between strainT2-19 and L. xinjiangensis, L. atacamensis, L. roselyniae, and L. desertiwere 99.19%, 99.41%, 99.19%, and 99.04%, respectively. Although themost related reference strain is L. atacamensis (99.41%), DNA:DNAhybridization and accurate polyphasic taxonomy as well asphenotypic descriptions will be carried out in the near future inorder to clarify the taxonomic position of T2-19 strain.

Concerning the phenotypic characteristics studied (Table 3),actinomycete isolates were Gramþ, aerobic, and with no myceliumfragmentation. Streptomyces strains produced moderate to

abundant aerial hyphae, whereas isolate T2-19 showed a slightaerial mycelium development. In addition, the peptidoglycan layerin streptomycetes contained mainly L-diaminopimelic acid (cellwall type I), whilemeso-diaminopimelic acid (cell wall type III) wasdetected in Lechevalieria T2-19. Diffusible pigments were producedby strains CO2-9, S-5, T2-10, and T8-2; melanin production wasdetected by the brown pigmentation of ISP-6 culture medium in 8strains (CO2-9, CO2-16, S-1, S-2, S-3, S-5, T2-10, and T8-2).According to the morphology of the spore chains observed underlight microscopy, CO2-9, CO2-16, S-1, S-2, S-3, and S-6 were grou-ped as Rectus-Flexibilis (RF), and S-5, S-7, T2-10, T6-32, and T8-2 asSpira (S), whereas strain T2-19 did not produce aerial mycelium.

0.02

Actinosynnema mirium (X84447)

Lechevalieria fradiae (AY114175)

Lechevalieria aerocolonigenes (AF114804)

Lechevalieria flava (AF114808)

Lechevalieria xinjiangensis (DQ898283)

IsolateT2-19 (FN808347)

Lechevalieria atacamensis ((EU551684)

Lechevalieria deserti (EU551682)

Lechevalieria roselyniae (EU551683)

100

89

89

76

88

99

97

Fig. 2. Neighbour-joining tree based on nearly complete 16S rDNA gene sequencesshowing relationships between isolate T2-19 and the Lechevalieria type strain species.Other details are given in the legend of Fig. 1.

G. Cuesta et al. / Journal of Environmental Management 95 (2012) S280eS284S284

Regarding chitinolitic activity, strain T2-19 was the only onecapable to degrade colloidal chitin. Nevertheless, chitinasesynthesis seems not to be the main fungi-inhibiting mechanismof Lechevalieria T2-19 since it inhibited mainly the oomyceteP. cinnamomi and P. debaryanum (Table 1) which do not have chitinin their cell walls. The antifungal activities showed by the Strepto-myces and the Lechevalieria isolated in this studywere probably dueto the synthesis of antibiotic compounds.

4. Conclusions

The in vitro assay carried out showed that nearly 25% of theactinomycete strains exhibited high activity to suppress thephytopathogenic microorganisms tested, a rate which is similar tothe percentages reported by several studies. This rate of effectiveantagonists indicates that both the isolation media and the culturemedia used in the in vitro experiment were appropriately selected.In addition, the analysis of 16S rDNA sequences provided a rapidand reliable identification of the actinomycete strains, and it alsoproduced results whichwere in linewith the phenotypic propertiesobserved for the isolates. Further research should focus on in vivoassays with the potential biocontrol isolates obtained, especiallyLechevalieria sp. (strain T2-19), to develop an effective biologicalcontrol strategy in commercial crop production systems.

Acknowledgements

This research was carried out within the framework of theproject UPV-PPI-05-05-5542 subsidized by the PolytechnicUniversity of Valencia (UPV) and a PhD fellowship supported by theValencian Council of Education.

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