Invitro and invivo antagonism of actinomycetes ... - Wiley Online Library

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De Vrije, T., Antoine, N., Buitelaar, R.M., Bruckner, S.,. Dissevelt, M., Durand, A., Gerlagh, M., ... Anton Leeuw 81, 557–564. Noling, J.W. and Becker, J.O. (1994) ...
Journal of Applied Microbiology ISSN 1364-5072

ORIGINAL ARTICLE

In vitro and in vivo antagonism of actinomycetes isolated from Moroccan rhizospherical soils against Sclerotium rolfsii: a causal agent of root rot on sugar beet (Beta vulgaris L.) R. Errakhi1,2, A. Lebrihi2 and M. Barakate1 1 Laboratory of Biology and Biotechnology of Microorganisms (LBBM), Department of Biology, Faculty of Sciences Semlalia, Marrakech, Morocco 2 Laboratoire de Ge´nie Chimique, UMR 5503 (CNRS ⁄ INPT ⁄ UPS), Ecole Nationale Supe´rieure Agronomique de Toulouse, INPT, Castanet-Tolosan Cedex, France

Keywords biological control, Morocco, root rot, S. rolfsii, Streptomyces. Correspondence Mustapha Barakate, Laboratory of Biology and Biotechnology of Microorganisms (LBBM), Department of Biology, Faculty of Sciences Semlalia, PO Box 2390 Marrakech, 40000 Morocco. E-mail: [email protected]

2008 ⁄ 0389: received 6 March 2008, revised 5 January 2009 and accepted 6 January 2009 doi:10.1111/j.1365-2672.2009.04232.x

Abstract Aims: To evaluate the ability of the isolated actinomycetes to inhibit in vitro plant pathogenic fungi and the efficacy of promising antagonistic isolates to reduce in vivo the incidence of root rot induced by Sclerotium rolfsii on sugar beet. Methods and Results: Actinomycetes isolated from rhizosphere soil of sugar beet were screened for antagonistic activity against a number of plant pathogens, including S. rolfsii. Ten actinomycetes out of 195 screened in vitro were strongly inhibitory to S. rolfsii. These isolates were subsequently tested for their ability to inhibit sclerotial germination and hyphal growth of S. roflsii. The most important inhibitions were obtained by the culture filtrate from the isolates J-2 and B-11, including 100% inhibition of sclerotial germination and 80% inhibition of hyphal growth. These two isolates (J-2 and B-11) were then screened for their ability to protect sugar beet against infection of S. rolfsii induced root rot in a pot trial. The treatment of S. rolfsii infested soil with a biomass and culture filtrate mixture of the selected antagonists reduced significantly (P £ 0Æ05) the incidence of root rot on sugar beet. Isolate J-2 was most effective and allowed a high fresh weight of sugar beet roots to be obtained. Both antagonists J-2 and B-11 were classified as belonging to the genus Streptomyces species through morphological and chemical characteristics as well as 16S rDNA analysis. Conclusion: Streptomyces isolates J-2 and B-11 showed a potential for controlling root rot on sugar beet and could be useful in integrated control against diverse soil borne plant pathogens. Significance and Impact of the Study: This investigation showed the role, which actinomycete bacteria can play to control root rot caused by S. rolfsii, in the objective to reduce treatments with chemical fungicides.

Introduction Sclerotium rolfsii is a soil borne fungus that survives long term in soil as sclerotia. It is a serious plant pathogen, with a host range of over 200 plant species (Punja 1988). This plant pathogen can cause several types of damage, 672

including seedling damping-off, crown and root rot as well as dry rot canker in older plants (Punja 1988; Abada 2003). In Morocco, S. rolfsii is a major constraint to sugar beet production in the irrigated region of Doukkala that represents 31% and 33% of sugar beet cultivation area

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and production, respectively (Fidah 1995). It can cause severe and significant economic damage that occurs just prior to harvest (Fidah 1995). The yield reduction can be more than 50% in some fields (Khettabi et al. 2004). Our observation of susceptible sugar beet plants indicates that S. rolfsii grows and attacks plants at or near the soil line. Before the pathogen penetrates host tissue, it produces a considerable mass of mycelium on the plant surface. The first indications of infection, usually undetectable, are dark-brown lesions on the root at or just beneath the soil level and the first visible symptoms are progressive yellowing and wilting of leaves. Following this, the fungus produces abundant white fluffy mycelia on infected tissues and soil (Fidah 1995). Sclerotia of relatively uniform size are produced on the mycelium. Sclerotia start out white to tan and become dark-brown to black at maturity. The disease may lead to plant death. Punja et al. (1985) reported that it is difficult to control S. rolfsii, and they speculated that this is due in part to the pathogen’s extensive host range, prolific growth and ability to produce large numbers of sclerotia that may persist in the soil for many years. Several methods have been used to control S. rolfsii. Chemical treatment is currently the major control method to reduce the incidence of S. rolfsii root rot. Methyl bromide is the major fumigant used globally for soil disinfestations preplanting. Because of its high vapour pressure and specific gravity, methyl bromide rapidly reaches pests and pathogens and effectively penetrates soil and plant tissue to considerable soil depths (Chakrabarti and Bell 1993; Noling and Becker 1994). However, it is believed that agricultural use of methyl bromide is a potential threat to the stratospheric ozone layer due to brome atom catalysis, as methyl bromide escapes from fumigated soils into the atmosphere (Chakrabarti and Bell 1993; Singh and Kanakidou 1993; Noling and Becker 1994). Moreover, Punja et al. (1985) indicated that chemical treatment has reduced the effectiveness of fungicides due to the tolerance developed by S. rolfsii. Cultural approaches, including enhanced soil drainage, reduced mechanical compaction of soil, crop rotation and soil solarization all contribute to successful disease management (Pulli and Tesar 1975; Swaminathan et al. 1999). However, S. rolfsii produces sclerotia, which can survive in the soil for long periods in the absence of their hosts. Consequently, these treatments may have only a minor effect on S. rolfsii. In general, effective control of fungal plant pathogens requires integrated strategies (Xiao et al. 2002). One component of an integrated strategy is biological control using antagonistic micro-organisms. A number of antagonistic fungi and bacteria have been shown to provide control against S. rolfsii in controlled experiments, though

Biocontrol of sugar beet rot root

field results have varied. Some of the commonly used organisms are Trichoderma harzianum (Khettabi et al. 2004), T. koningii (Tsahouridou and Thanassoulopoulos 2002) and Pseudomonas fluorescens (Singh et al. 2003). However, to our knowledge, no studies have been published on the control of S. rolfsii root rot on sugar beet using actinomycete bacteria. Actinomycetes are Gram-positive filamentous bacteria. The majority of this group is saprophytic and found widely distributed in the soil (Katsifas et al. 1999). Actinomycetes are recognized for their ability to produce a wide variety of extracellular enzymes and antibiotics, and to colonize plants (Wohl and McArthur 1998; Barakate et al. 2002). The efficacy of antagonistic actinomycetes in biological control has been shown against many plant pathogenic fungi such as Verticillium dahliae (Entry et al. 2000), and Fusarium oxysporum (Getha and Vikineswary 2002), and oomycetes such as Pythium ultimum (El-Tarabily et al. 1997), and Phytophthora sp. (Xiao et al. 2002). Antagonistic actinomycetes may control plant pathogenic fungi by many mechanisms, such as hyperparasitism (Sabaou and Bounaga 1987; El-Tarabily and Sivasithamparam 2005), antibiosis (Gyenis et al. 1999; Xiao et al. 2002; El-Tarabily and Sivasithamparam 2005), Cell-wall degrading enzymes (El-Tarabily and Sivasithamparam 2005) and induction of plant resistance (Hassanin et al. 2007). Many strategies have been used to find potential antagonistic agents to control plant pathogens. Chan et al. (2003) reported that an in vitro agar diffusion assay used as an initial selection method for putative microbial antagonism is a preferred and practical means of screening a large number of isolates for antibiosis, rather than using plant bioassays. Plant tests, however, remain essential for verifying the effectiveness of potential candidates, as in vitro antibiotic activities often do not correlate with in vivo biocontrol activities (Fravel 1988). Our aim in this investigation was to (i) isolate actinomycetes from the rhizosphere of sugar beet, (ii) to study their activity against S. rolfsii and other plant pathogens, (iii) determine the ability of culture filtrates from selected isolates to inhibit S. rolfsii sclerotial germination and hyphal growth and (iv) evaluate in vivo the efficacy of potential antagonistic isolates as biological control agents against S. rolfsii root rot on sugar beet. Materials and methods Sampling Samples were collected from two rhizosphere soils of sugar beet; one from Doukkala (Mid-West-Morocco), where the diseases caused by S. rolfsii are very important

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(Fidah 1995) and the other from Beni-Mellal (Mid-EastMorocco), which a region free of S. rolfsii diseases. Three samples from each rhizosphere soil were taken with an auger (up to 10 cm depth) after removing 3 cm of the soil surface. Each sample was taken independently from one field and from rhizosphere of five healthy sugar beet roots along the diagonal of the field. Samples were first mixed and 500 g of soil were placed in sterile polyethylene bags, closed tightly and stored at 4C for 1 day before use. Microbial and pedological analysis of soil samples Samples were first mixed, suspended in sterile distilled water (1 g in 100 ml) and shaken on rotatory shaker (200 rev min)1, 30 min), serially diluted up to 10)6, and spread (0Æ1 ml) over the surface of nutrient agar (Difco) and potato dextrose agar (PDA, Difco), respectively, for total bacterial and fungal counts. Plates were incubated at 28C and the number of colonies was determined after 7 days. The pH, moisture content and organic-matter content of the samples were determined as described by Aubert (1978).

et al. 2000); Botrytis cinerea Tu¨157 the causal agent of grey mold of grapevine (Paul et al. 1998); Pythium ultimum causal agent of damping-off of pea and sugar beet (Bardin et al. 2004) (strains collection of Laboratory of Biology and Biotechnology of Microorganisms, Marrakech), and Streptomyces scabies EF35 causal agent of common scab (Paradis et al. 1994) (kindly provided by Pr. C. Beaulieu, University of Sherbrooke, Canada). Pure actinomycete isolates were grown on Bennett’s agar medium (beef extract 1 g l)1; glucose 10 g l)1; peptone 2 g l)1; yeast extract 1 g l)1 and agar 15 g l)1). After incubation for 14 days at 30C and two mycelia plugs (6 mm in diameter) were cut and placed on PDA (for fungal and oomycetes) and Bennett’s medium (for S. scabies EF35), which were seeded with the appropriate test organism. Plates were first kept for 4 h in a refrigerator (at 4C) for at least 4 h to allow the diffusion of any antibiotics produced, then incubated at 30C in the dark. Inhibition diameters were determined after 48 h of incubation. Only the isolates that showed an inhibition zone larger than 8 mm were considered as active isolates (two replicates were used for each treatment). Production of sclerotia

Isolation and characterization of actinomycetes Actinomycetes were isolated from both soils by the dilution agar plating method, as described above, using a soil extract medium (Barakate et al. 2002) supplemented with actidione (40 lg ml)1) found to inhibit the growth of fungi (Goodfellow and Williams 1988) and nalidixic acid (10 lg ml)1), which inhibits the bacteria capable of swarming without affecting the growth of actinomycetes (Nonomura and Hayakawa 1988). Actinomycetes colonies were recognized on the basis of morphological and physiological characteristics following directions given by the International Streptomyces Project (Shirling and Gottlieb 1966). Morphological characteristics were studied under a light microscope after 15 days of growth on oatmeal agar (ISP3) (Shirling and Gottlieb 1966). Colour of aerial mycelium was determined according to the scale adopted by Prauser (1964). Screening for actinomycete isolates inhibiting Sclerotium rolfsii and other plant pathogens The antimicrobial activity of isolated actinomycetes was determined by the agar diffusion method (Bauer et al. 1966) against S. rolfsii Sr1, and other plant pathogens: Fusarium oxysporum f. sp. albedinis f.o.a.133 a causal agent of vascular wilt of date-palm tree (Sabaou and Bounaga 1987); V. dahliae V72 a causal agent of verticillium wilt of tomato, potato, olive-tree and other plants (Entry 674

Mycelial disks (8 mm in diameter) obtained from a 5-day-old PDA culture of S. rolfsii were transferred onto agar plates containing PDA and incubated at 25C in the dark for 3 weeks. The sclerotia formed were dislodged from the surface of the agar plates and used immediately. Inhibition effect of culture filtrates on sclerotia germination and hyphal growth of Sclerotium rolfsii The ten actinomycete isolates having the largest average in vitro inhibition against S. rolfsii (data not shown) were selected for the following experiments. Each isolate was grown separately in 500 ml Bennett’s liquid medium on a rotary shaker (160 rev min)1) at 30C and after 7 days the cultures were passed through a sterile 0Æ45 lm Millipore filter. The cell-free culture filtrate of the selected isolates was mixed separately with PDA, just before the agar started to set, in proportions of 0, 1, 2 and 3 v ⁄ v (v ⁄ v: actinomycetes culture filtrate ⁄ PDA medium). For the sclerotial germination test, sclerotia were disinfected with sodium hypochlorite (2%) for 3 min and washed three times with sterile distilled water. The sclerotia were disinfected to ensure no living hyphae were present for the subsequent sclerotia germination test. Ten sclerotia, which were obtained from S. rolfsii culture of 10 days, were placed on the surface of the prepared agar plate (PDA mixed with actinomycete culture filtrate) and the number of germinated sclerotia was recorded after

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72 h of incubation at 25C. Five replicate plates were used for each concentration of culture filtrate. For the hyphal growth test, an 8 mm diameter mycelial disk was removed from the margin of 5-day-old colonies of S. rolfsii and transferred into prepared plates. Mycelial growth was assessed by measuring the diameter of each colony 72 h after incubation at 25C. Five replicate plates were used for each concentration. Preparation of actinomycete inocula for in vivo antagonism assays Based on the results of the in vitro assays, two actinomycete isolates (J-2 and B-11) were selected to test, under greenhouse conditions, for their ability to control rootrot diseases of sugar beet caused by S. rolfsii. The biomass and culture filtrate mixture of each antagonistic actinomycete isolate was obtained after growing each isolate in a 5 l fermentors containing 3 l of Bennett’s liquid medium for 7 days at 30C. The soil used for this study was collected from the sugar beet rhizosphere from the Doukkala (mid-west-Morocco), which is recognized as having a high level of S. rolfsii infestation. The soil was first dried for 2 days at 28C to remove excess moisture, and sieved (