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Soil Biology & Biochemistry 38 (2006) 15051520 www.elsevier.com/locate/soilbio

Review

Non-streptomycete actinomycetes as biocontrol agents of soil-borne fungal plant pathogens and as plant growth promoters
Khaled A. El-Tarabilya, Krishnapillai Sivasithamparamb,
b

Department of Biology, Faculty of Science, United Arab Emirates University, Al-Ain 17551, United Arab Emirates Soil Science and Plant Nutrition, School of Earth and Geographical Sciences, Faculty of Natural and Agricultural Sciences, University of Western Australia, 35 Stirling Highway, Crawley, W.A., 6009, Australia Received 7 October 2005; received in revised form 8 December 2005; accepted 9 December 2005 Available online 3 March 2006

Abstract Among soil microorganisms, bacteria and fungi and to a lesser extent actinomycetes, have received considerable attention as biocontrol agents of soil-borne fungal plant pathogens and as plant growth promoters. Within actinomycetes, Streptomyces spp. have been investigated predominantly, mainly because of their dominance on, and the ease of isolation from, dilution plates and because of the commercial interest shown on the antibiotics produced by certain Streptomyces spp. Many of non-streptomycete actinomycetes (NSA) taxa are therefore rarely reported in literature dealing with routine isolations of biocontrol agents and/or plant growth promoters from plant and soil. It is clear that special isolation methods need to be employed in routine isolations to selectively isolate NSA. Some interesting information exists, albeit in relatively few reports compared to that on other microorganisms, on the biological activities of NSA, especially in relation to their mechanisms of action in the biological control of soil-borne fungal plant pathogens and plant growth promotion. This review presents an overview of this information and seeks to encourage further investigations into what may be considered a relatively unexplored area of research. Certain soil environmental factors, especially in horticultural systems, could be manipulated to render the soil conducive for the biological activities of NSA. A variety of NSA isolated by selective methods have not only shown to be rhizosphere competent but also adapted for an endophytic life in root cortices. Some of the NSA, including endophytic strains that have shown potential to suppress soil-borne fungal plant pathogens, are able to employ one or more mechanisms of antagonism including antibiosis, hyperparasitism and the production of cell-wall degrading enzymes. Strains of NSA promote plant growth by producing plant growth regulators. Enhancement of plant growth by the antagonists are considered to help the host by producing compensatory roots that mask the impact of root diseases. r 2006 Elsevier Ltd. All rights reserved.
Keywords: Biological control; Disease suppression; Endophytic actinomycetes; Microbial interaction; Pathogen suppression; Plant growth regulators; Rhizosphere competence

1. Introduction The study of actinomycetes has been the subject of thorough and comprehensive reviews by Cross (1982), Williams and Wellington (1982a, b), Williams et al. (1984), Nolan and Cross (1988). All these reviews, however, deal with the different methods of isolation, enumeration and identication of streptomycete actinomycetes and non-

streptomycete actinomycetes (NSA). Ensign et al. (1993) reviewed the physiology of some NSA as a component of soil microora. Although Lechevalier (1988) and Doumbou et al. (2001) reviewed the literature on the biological control of soil-borne fungal plant pathogens and plant growth promotion by actinomycetes, they covered activities mainly of Streptomyces spp. This is at least partly due to relatively poor attention paid to biocontrol and plant

Corresponding author. Tel.: +61864882497; fax: +61864881050.

E-mail address: [email protected] (K. Sivasithamparam). 0038-0717/$ - see front matter r 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.soilbio.2005.12.017

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growth promotion by NSA. For this reason, the current review will only concentrate on the role of NSA as biocontrol agents of soil-borne fungal plant pathogens and as plant growth promoters. This review will not cover the symbiotic relationship of Frankia spp., although they promote plant growth essentially through nitrogen xation (Benson and Schultz, 1990; Dawson, 1990) and sometimes through the suppression of root diseases (Gopinathan, 1995), as this topic has been comprehensively reviewed (e.g. Dawson, 1990). 2. Soil actinomycetes 2.1. Habitat of actinomycetes in bulk, rhizosphere soils and as endophytes Actinomycetes are ubiquitous and are considered to prefer the soil constituents such as humus, litter, dung and even rock surfaces (Lechevalier, 1981). Viable counts of several millions per gram soil are common and over 20 genera have been isolated from soil (Williams and Wellington, 1982a). It is generally accepted that in relatively dry, humic, calcareous soils, actinomycetes form the dominant fraction of the microora with viable counts reaching 106 g1 dry weight soil (Goodfellow and Williams, 1983). In contrast, numbers in waterlogged, anaerobic soils and acidic soils are often found to be relatively low (102103 g1 dry weight soil) (Williams and Wellington, 1982a). Williams and Wellington (1982a) found that among actinomycetes, frequencies of isolation for Streptomyces were 95.3%, Actinoplanes 0.2%, Actinomadura 0.1%, Microbispora 0.18%, Micromonospora 1.4%, Nocardia 1.98%, Pseudonocardia 0.06%, Streptosporangium 0.10%, Thermoactinomyces 0.14%, and Thermomonospora 0.22%. Frequency of isolation must be regarded as tentative, because any isolation procedure is to some extent selective (Williams and Wellington, 1982a). Streptomyces spp. normally account for 7090% and rarely as low as 5% of the actinomycete colonies recovered on isolation media inoculated with dilute suspensions of soil (Alexander, 1977). This variation, in part, could be due to the nature of the isolation media employed. Lechevalier and Lechevalier (1967) examined 5000 isolates of actinomycetes from the soil, and found that genera of NSA which can be considered as rare, such as Thermomonospora, Actinoplanes, Microbispora, Thermoactinomyces, Streptosporangium, Micropolyspora, Pseudonocardia, and Microellobosporia, constitute less than 0.2% of the total isolations. In Japan, Nonomura and Ohara (1971) found that NSA such as Microbispora and Thermomonospora constitute less than 103 propagules g1 dry weight soil. All these reports further support the view that isolation techniques can bias the proportion of NSA isolated in such studies. Often a signicantly higher number of actinomycetes in comparison to bulk soil, are found in the rhizosphere

(Goodfellow and Williams, 1983) depending, however on the species and age of the plant. Rhizosphere effects on actinomycetes have been expressed as R:S (Rhizosphere:Soil) ratios. For example, soybean and maize harboured 1018 times as many actinomycetes in their rhizosphere compared to bulk soil (Abraham and Herr, 1964), with R:S ratios of sand dune plants ranging from 16 to 50 (Watson and Williams, 1974). Vruggink (1976), however, found, little qualitative differences between soil and rhizosphere populations, although differences between root surface populations and those in the rhizosphere were detectable. Basil et al. (2004) reported that there were no statistical differences between the relative numbers of culturable actinomycetes in the rhizosphere of sagebrush (Artemisia tridentata) versus bulk soils, but PCR amplication of the 16S rRNA gene sequences of the total soil DNA and denaturing gradient gel electrophoresis showed that the community structure was different between the rhizosphere and the bulk soil. A high percentage of actinomycetes produced antimicrobial antibiotics and the percentage of active producers was signicantly higher among the rhizosphere isolates, as compared with the bulk soil isolates (Basil et al., 2004). Endophytic streptomycetes (OBrien et al., 1984; Sardi et al., 1992; Tokala et al., 2002; Coombs and Franco, 2003; Taechowisan et al., 2003; Coombs et al., 2004; Tian et al., 2004; Cao et al., 2005) have been isolated from inside live tissues of various plant species. Since the early work of Smith (1957), only a few, recent studies have highlighted the endophytic nature of strains among NSA (Sardi et al., 1992; de Araujo et al., 2000; Stamford et al., 2001; Kunoh, 2002; Coombs and Franco, 2003; El-Tarabily, 2003; Coombs et al., 2004). 2.2. Environmental factors Actinomycetes are widespread in the environment and most actinomycete species are chemo-organotrophic, aerobic, mesophilic and grow optimally at a pH near neutrality (Williams and Wellington, 1982a; Goodfellow and Williams, 1983). The important factors controlling the abundance and activity of actinomycetes in the soil have been suggested to be the availability of nutrients, nature and abundance of organic matter, salinity, relative moisture content, temperature, pH and soil vegetation (Goodfellow and Williams, 1983; McCarthy and Williams, 1990). Actinomycetes in general, prefer neutral to alkaline soils as a natural habitat (Flaig and Kutzner, 1960; Goodfellow and Williams, 1983). Most soil actinomycetes grow in the pH range of 5.09.0, with an optimum close to neutrality (Goodfellow and Williams, 1983). The addition of lime to acidic soils led to a 100-fold increase of actinomycetes as compared with untreated soils (Tsao et al., 1960). In Western Australia, application of agricultural lime increased soil pH, populations of streptomycete and NSA and increased the recovery of species of NSA such as Actinoplanes, Micromonospora, Microbispora,

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and Streptosporangium (El-Tarabily et al., 1996a). A number of these genera were shown to be antagonistic to Pythium coloratum a causal agent of cavity spot of carrots in Western Australia (El-Tarabily et al., 1996a). Actinomycetes are affected directly by the presence of available carbon sources and their number is especially high in land rich in organic matter. In general, sites high in carbonaceous materials and humus harbour a larger number than habitats poor in organic matter (Alexander, 1977). Amendment with organic nutrients such as crop residues and animal manure increases the abundance of actinomycetes (Alexander, 1977). Henis (1986) also pointed out that the number of actinomycetes in soil is positively correlated with the level of organic matter. Clay and humic colloids can differentially affect the distribution and activity of soil actinomycetes. Ruddick and Williams (1972) found that streptomycete spores adsorbed readily to kaolin but not to montmorillonite clays. Growth and respiration of Streptomyces, Micromonospora and Nocardia were stimulated by the incorporation of calcium montmorillonite into the culture medium (Mara and Oragui, 1981). Moisture content can also be a critical environmental factor that inuences the population structure of actinomycetes in soils. In waterlogged or in very wet soils, for example at 85100% of the water-holding capacity, actinomycetes may appear only rarely (Alexander, 1977). In contrast, actinomycetes are not as equally inuenced by semi-dry conditions as are bacteria, and actinomycetes are favored by relatively low moisture levels. The colonyforming units of actinomycetes, in general, remain high as soils dry out while the relative incidence of bacteria are adversely affected as they lack tolerance to arid conditions (Alexander, 1977). The high counts in drying soils may also be due to the enhanced sporulation of actinomycetes under these conditions which could compensate for the death of mycelial propagules, resulting in the maintenance of high numbers of colony-forming units on enumeration media. Williams et al. (1972) also showed that well-drained soils such as sandy loam support larger populations of actinomycetes than heavy clay soils. Large populations of actinomycetes were shown to coincide with relatively high levels of organic matter, whatever the degree of salinity (Zahran et al., 1992). 2.3. Taxonomy of actinomycetes The classical criteria used for the identication of actinomycetes into genera and species have been thoroughly reviewed and discussed, including ecological, morphological, cultural, and physiological aspects (Goodfellow and Cross, 1984; Goodfellow, 1989). Wall chemotype, whole cell sugar pattern, peptidoglycan type, fatty acid pattern, major menaquinone, phospholipid type and molecular % of G+C of DNA are also useful tools in the identication of actinomycetes (Goodfellow and Cross, 1984; Goodfellow et al., 1988; Goodfellow, 1989). Recent

molecular biology techniques including 16S RNA analysis and DNA/DNA-hybridization techniques are now commonly employed (Rainey et al., 1996; Maidak et al., 1999).

3. Isolation of actinomycetes from soil, rhizosphere and live plant tissues Cross (1982), Williams and Wellington (1982a, b) and Goodfellow and Williams (1983) outlined the stages in the isolation of actinomycetes from bulk or rhizosphere soils. A wide range of antibiotic amendments and substrate pretreatments has been used for the selective isolation of actinomycetes from a range of substrates (Cross, 1982; Williams and Wellington, 1982a). The types of culture media used to selectively isolate actinomycetes vary considerably. Incubation period of isolation plates can be critical with NSA usually taking a longer period to emerge as colonies, in comparison to Streptomyces spp. (Cross, 1982). Methods commonly used for the isolation and enumeration of soil and rhizosphere actinomycete strains used in biocontrol and plant growth promotion activities deal almost exclusively with those suitable for Streptomyces species. Streptomyces spp. grow relatively rapidly and compete successfully on soil-dilution plates and present an unbalanced picture of the spectrum of actinomycetes which inhabit the soil and rhizosphere (Goodfellow and Williams, 1983). Other methods, including the application of the dry heat technique (Nonomura and Ohara, 1969) and Streptomyces phage (Kurtboke et al., 1992; Long and Amphlett, 1996), have been successfully used to reduce the dominance by the fast-growing streptomycete actinomycete colonies on isolation plates and to facilitate the recovery of slow growing and relatively less competitive NSA. Bacteriophages were also included by Kurtboke et al. (1993) and McKenna et al. (2002) to reduce the dominance of fast growing bacteria on isolation plates. Recent development of techniques such as the use of coalvitamin medium with soil pre-treatment with peptone and lauryl sulfate at 50 1C for 10 min (You and Park, 1996), microwave radiation (Bulina et al., 1997), electric pulses (Bulina et al., 1998), differential centrifugation (Hayakawa et al., 2000), high frequency radiation (Li et al., 2002), and succession analysis (Li et al., 2003) are all aimed at increasing the recovery of actinomycete genera from isolation plates. However these methods are yet to be used in the selective isolation of biocontrol agents or plant growth promoters among NSA. Certain endophytic bacteria (Hallmann et al., 1997; Sturz et al., 2000; Bacon and Hinton, 2002) and fungi (Sivasithamparam, 1998; Mucciarelli et al., 2003; Narisawa et al., 2004) have been shown to be highly suited both as biocontrol agents and plant growth promoters. Although endophytic streptomycete and NSA actinomycetes have been shown to have potential as biocontrol agents (e.g. El-Tarabily, 2003; Coombs et al., 2004) there have been no

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reports of endophytic streptomycete or NSA actinomycetes as plant growth promoters. Isolation of endophytic actinomycetes has been successful (Coombs and Franco, 2003; El-Tarabily, 2003) using the method developed by Hallmann et al. (1997) for the isolation of endophytic bacteria. This method involved surface-disinfesting plant organs by exposing them to ethyl alcohol and sodium hypochlorite. The surface-disinfested organs are then rinsed and triturated in buffer with a sterile mortar and pestle under aseptic conditions. The slurry is then ltered through sterile cotton cloth, and serially diluted in buffer and spread onto a selective agar medium (Hallmann et al., 1997). The major key to success in isolating and studying endophytes is to ensure sterility of the plant surface (Hallmann et al., 1997), therefore sterility checks must be carried out for each sample to monitor the effectiveness of the disinfestation procedures. For these checks, surface-disinfested plant organs are dried and pressed on to tryptic soy agar plates. In addition, aliquots of the nal buffer from the nal rinse solutions are transferred to tryptic soy broth and incubated to check for presence of surface contaminants (Coombs and Franco, 2003; El-Tarabily, 2003). 4. Mechanisms of action Certain microbial antagonists of soil-borne fungal plant pathogens have been shown to have totally unrelated mechanisms of action (Cook and Baker, 1983). Although much work has been done on the mechanisms of activity by microbial antagonists, most of it has been focused on fungal and bacterial antagonists (Whipps, 1997, 2001) with relatively less attention paid to the complexes of mechanisms employed by actinomycete antagonists, especially NSA (Doumbou et al., 2001; Whipps, 2001). 4.1. Antibiosis Studies on the mechanisms of action of actinomycetes have focused mainly on in vitro screens especially in relation to antibiosis (Cooper and Chilton, 1950; Knauss, 1976; Rose et al., 1980; Turhan and Grossmann, 1986; Crawford et al., 1993; Yuan and Crawford, 1995; El-Tarabily et al., 1997; Berg et al., 2001; Getha et al., 2005). Such assays, routinely used in screening of microbial antagonists have been most valuable as screens for antibiotic activity (Broadbent et al., 1971; Turhan, 1981; Yuan and Crawford, 1995; Trejo-Estrada et al., 1998a; Barakate et al., 2002; Getha et al., 2005). In routine screening tests, antibiosis is determined by pairing colonies on agar plates (Cooper and Chilton, 1950; Johnson and Curl, 1972). There have been several modications of this method including the Herrs tripleagar-layer plate technique (Herr, 1959), agar-ring method (Williams and Willis, 1962), reversed layer method (Hasegawa et al., 1990), and mycelial spray technique (Alvarez et al., 1995), all of which give an indication of the

level of effectiveness of the antagonist to inhibit the vegetative growth of a pathogen. These are usually followed by tests involving culture ltrates or puried fractions of the ltrates (Smith, 1957; Turhan, 1981; Rothrock and Gottlieb, 1984; Upadhyay and Rai, 1987; El-Abyad et al., 1993; Yuan and Crawford, 1995; TrejoEstrada et al., 1998b). Possible involvement of competition for nutrients in such plates can be effectively negated by the use of the dialysis membrane overlay technique (Gibbs, 1967; Dennis and Webster, 1971a). In such studies it is also possible to eliminate the confusion over fungistatic versus fungicidal effects by testing the viability of the pathogen plug on a fresh agar plates, following exposure to the antifungal metabolites (Dennis and Webster, 1971a; El-Tarabily et al., 1997). Positive results from such screening on agar however, do not rule out other mechanisms that may operate in concert. This could happen even in simple in vivo screens such as the carrot bioassay performed by El-Tarabily et al. (1997) with NSA antagonistic to P. coloratum, where the possible involvement of other mechanisms, such as induced resistance, can not be ruled out. The modications of the paired plate technique are also commonly used for assays of volatile antifungal metabolites (Dennis and Webster, 1971b) produced by NSA antagonists which are inhibitory to the vegetative growth of the pathogens (e.g. Upadhyay and Rai, 1987; ElTarabily et al., 1997). In comparison to the reports of volatiles produced by antagonistic Streptomyces spp., there is little or no evidence of volatiles produced by NSA that are inhibitory to the vegetative growth of soil-borne fungal plant pathogens (Rai et al., 1981; Upadhyay and Rai, 1987; El-Tarabily et al., 1997, 2000). The standard tests used for the in vitro assays for antibiotic production involve relatively nutrient-rich media although it has been shown by a number of researchers (e.g. Sivasithamparam and Parker, 1980; Whipps, 1987) that the availability of nutrients has a signicant effect on the antagonistic activities of the microorganisms screened. This is also true for NSA (El-Tarabily et al., 1997). There is also a relative paucity of information on the biological activity of puried antifungal metabolites of NSA on soil-borne fungal plant pathogens although several antibiotics of NSA have been puried and characterized (Table 1). Although antifungal antibiotics produced by Streptomyces spp. in soil have been detected (Rothrock and Gottlieb, 1984; Trejo-Estrada et al., 1998a), no attempt to date has been made to elucidate the production of antifungal antibiotics by NSA in soil. Detailed studies on metabolites of NSA in relation to the growth and pathogenic activities of soil-borne fungal plant pathogens is clearly warranted. 4.2. Hyperparasitism Although parasitism of hyphae by Streptomyces spp. have been reported (e.g. Tu, 1988; Tapio and

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Pohto-Lahdenpera, 1991; Yuan and Crawford, 1995), very few reports exist on hyphal parasitism by NSA. Sabaou et al. (1983) reported that a strain of Nocardiopsis dassonvillei showed antibiotic, mycolytic and parasitic activites against the vegetative hyphae of Fusarium oxysporum f.sp. albedinis. They observed that the parasitized fungus produced resistant structures in the form of polymorphous thallospores or chlamydospores. Upadhyay and Rai (1987) found that a strain of Micromonospora globosa parasitized the hyphae of Fusarium udum in vitro. The hyphal interaction included coiling, penetration, branching of the growing hyphae of the hyperparasite inside the fungal host leading to granulation, coagulation of the cytoplasm and hyphal lysis. Antagonistic NSA are capable of multiplying in the presence of the resting spores of plant pathogens (Fig. 1a) and can also parasitize the vegetative mycelium (ElTarabily et al., 1997) or the resting oospores (Sneh et al., 1977; Sutherland and Lockwood, 1984; Khan et al., 1993; El-Tarabily et al., 1997) of oomycetes. The mycelia of a pathogen can be parasitized by the antagonists in its active phase in soil or in plant, while the oospores can be parasitized by the antagonists in the survival phase of the pathogen. Colonization of hyphae of plant pathogens (Fig. 1b) and hyphal parasitism has been established in relation to NSA such as Actinoplanes philippinensis which produces motile zoospores (El-Tarabily et al., 1997). It has been reported that zoospores of Actinoplanes spp. have a chemotactic response to the presence of the fungal conidia, chlamydospores and sclerotia (Arora, 1986). In the presence of hyphae of P. coloratum, the zoospores of A. philippinensis not only swim towards the hyphae but also lodge themselves on the surface of the hyphae and proceed to germinate and penetrate the mycelium (El-Tarabily et al., 1997). Although, Sneh et al. (1977) used oospores to successfully bait NSA-parasitizing oospores of oomycetes, no attempt to date have been made to isolate NSA from living or dead fungal hyphae using the mycelial bait technique described by Kobayashi et al. (1995). In addition, it is also not known whether parasites of oospores are equally effective as parasites of hyphae or vice versa. Oospore parasitism can be studied either in a noncompetitive environment in plates where only the parasite and the pathogen oospores interact (Sutherland and Lockwood, 1984; Khan et al., 1993; El-Tarabily et al., 1997; El-Tarabily, 2006) or in a competitive environment (non-sterile soil) (Sutherland and Lockwood, 1984; Khan et al., 1993). On plates, the parasitism has been observed to be as either external colonization of oospore wall (Khan et al., 1993; El-Tarabily et al., 1997), or as the penetration and the internal colonization of oospores (Sutherland et al., 1984). Transmission electron microscopy studies of hyperparasitism of Phytophthora megasperma var. glycinea by Actinoplanes missouriensis have shown that invasion occurred without appressorial or haustorial formation. The hyphae of the

Table 1 Reports of some antibiotics produced by non-streptomycete actinomycetes Producing species Actinoplanes sp. Actinoplanes sp. A. brasiliensis A. caeruleus A. deccanensis A. ferrugineus A. A. A. A. ianthinogenes missouriensis philippinensis teichomyceticus Antibiotic Xanthone Sch 54445 A/672 Heptaene Lipiarmycin L-azetidine-2carboxylic acid Naphthoquinone 5-azacytidine Macrocyclic lactone Lipoglycopeptide complex Echinocandin Simaomicin Pradimicin FA-1 SCH 31828 Glucosylquestiomycin Spartanamicins Rustmicin Micromonosporin Everninomicin Glutarimide Gentamicin Hazimicins Halomicin XK-41 Fortimicin SCH 42282 Formamicin HM17 H107 Aculeximycin AH7 Quinaldopeptin HA-94 Reference Cooper et al. (1992) Min et al. (1997) Palleroni (1989) Palleroni (1989) Palleroni (1989) Palleroni (1989) Palleroni (1989) Palleroni (1989) Palleroni (1989) Carelli et al. (1995) Boeck et al. (1989) Maiese et al. (1990) Sawada et al. (1990) Patel et al. (1988) Igarashi et al. (1998) Nair et al. (1992) Sigmund and Hirsch (1998) Thawai et al. (2004) Kawamoto (1989) BeomSeok et al. (1999) Kawamoto (1989) Marquez et al. (1983) Kawamoto (1989) Kawamoto (1989) Kawamoto (1989) Hegde et al. (1998) Igarashi et al. (1997) Hacene et al. (1994) Hacene et al. (2000) Ikemoto et al. (1983) Hacene et al. (1998) Toda et al. (1990) Paradkar et al. (1998)

A. utahensis Actinomadura madurae Actinomadura hibisca Microbispora sp. Microbispora sp. Micromonospora sp. Micromonospora sp. Micromonospora sp. M. carbonacea Micromonospora coerulea M. echinospora M. echinospora M. halophytica M. inositola M. olivasterospora Microtetraspora Saccharothrix sp. Spirillospora sp. Spirillospora sp. Streptosporangium albidum Streptosporangium roseum Streptoverticillium album Streptoverticillium cinnamoneum

hyperparasite penetrate the oogonial and oospore cell walls without appreciable differentiation, and many hyphae were seen in the periplasmic space (Sutherland et al., 1984). Soil-borne Actinoplanes spp. have been reported to parasitize oospores of P. megasperma f.sp. glycinea (Sutherland et al., 1984) and Pythium spp. (Khan et al., 1993; El-Tarabily et al., 1997; El-Tarabily, 2006) in in vitro studies. A. missouriensis was found to parasitize the oospores of Phytophthora megasperma var. sojae, Phytophthora cactorum, Pythium sp., and Aphanomyces euteiches in a natural soil (Sneh et al., 1977). Parasitism was favored in ooded soils compared to soil with moisture levels below water-holding capacity (Sneh et al., 1977).

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Fig. 1. (a) Stimulated production of sporangia of Actinoplanes sp. around oospore of Pythium coloratum ( 400). (b) Zoospore germination and subsequent growth of hyphae of Actinoplanes sp. on and around hyphae of Pythium coloratum ( 400). (c) Zoospore germination and subsequent growth of hyphae of Actinoplanes sp. on and around oospore of Pythium coloratum ( 400).

This indicates that the activity of antagonists that produce zoospores are clearly favored in a ooded soil. In addition, A. missouriensis has also been reported to parasitize oospores of P. megasperma var. glycinea, Aphanomyces cochlioides, A. euteiches, Phytophthora citrophthora, Pythium aphanidermatum and Pythium ultimum in vitro (Sutherland and Lockwood, 1984). They also found that oospores of P. megasperma var. glycinea were also parasitized in vitro by Actinoplanes utahensis, A. philippinensis, Amorphosporangium auranticolor, Ampullariella regularis, Spirillospora albida and Micromonospora sp. However, the age of the host or the parasite has no signicant effect on the hyperparasitic activity of the species (Sutherland and Lockwood, 1984). The activity of A. missouriensis is clearly affected by the environment (temperature and moisture) in the soil which is important for both the antagonist and the pathogen (Sutherland and Lockwood, 1984). While A. missouriensis can parasitize P. megasperma var. glycinea over a wide temperature and moisture range, 0 bars matric potential and temperatures of 1530 1C were most conducive for oospore parasitism (Sutherland and Lockwood, 1984). Sutherland and Lockwood (1984) reported that the A. missouriensis was an effective hyperparasite both in vitro and in greenhouse studies and reduced root rot of soybean caused by P. megasperma var. glycinea. Sutherland and Papavizas (1991), however, reported that although A. missouriensis, A. philippinensis, A. utahensis, A. auranticolor, A. regularis, and S. albida were effective hyperparasites of oospores of Phytophthora capsici in vitro, they were ineffective in the control of crown rot of pepper under greenhouse conditions. This clearly indicates that the interaction between the pathogen, host and the NSA can be different depending on the pathogen and the environment in which they are tested.

Khan et al. (1993) found that many species of the genus Actinoplanes parasitized the oospores of Pythium spp. including P. aphanidermatum, P. ultimum, P. arrhenomanes, P. irregulare and P. myriotylum, both in vitro and in sterile and non-sterile soils. Sporulation of Actinoplanes spp. from infected oospores incubated in soil was more abundant than that observed with oospores in pure culture on agar (Khan et al., 1993). This indicates the presence in soil of factor(s) favoring sporulation of Actinoplanes spp. A. philippinensis and Micromonospora carbonacea which are capable of colonizing P. coloratum oospores in vitro (Fig. 1c) and were effective in suppressing the cavity spot incidence in carrots (El-Tarabily et al., 1997). Hyperparasitism could be an important mechanism of biocontrol of P. coloratum by NSA. As in many other instances of hyperparasitism, it is a component of antagonism rather than the sole mechanism of biocontrol. With the mycoparasite Gliocladium spp. it has been proven that antibiosis and hyperparasitism function together, the antibiotic destroying the integrity of the host cell wall enabling the hyperparasite (Gliocladium sp.) to penetrate otherwise resistant fungal cells (Di Pietro et al., 1993). In the study of El-Tarabily et al. (1997), A. philippinensis and M. carbonacea showed considerable promise as hyperparasites, and both species were also shown to produce antibiotics. Antibiotics produced by Trichoderma spp. have been proposed to predispose the hyphae of pathogens to mycoparasitic activity of the antagonists (Ghisalberti and Sivasithamparam, 1991). 4.3. Cell-wall degrading enzymes NSA have been recorded to produce cell wall degrading enzymes including X-1,3, X-1,4, X-1,6-glucanases (Waldron et al., 1986; Palleroni, 1989; Valois et al., 1996; Gacto et al., 2000; El-Tarabily, 2006) and chitinases (El-Tarabily et al., 2000; Nawani et al., 2002; El-Tarabily, 2003). The importance of X-1,3, X-1,4, X-1,6-glucanases (El-Tarabily et al., 1996b; Valois et al., 1996; El-Tarabily, 2006) and chitinases (Lorito et al., 1993; Mahadevan and Crawford, 1997; Gomes et al., 2000; El-Tarabily, 2003) in the activity of cell wall degradation and resulting hyphal lysis in fungal pathogens can not be over emphasized. M. carbonacea has been reported to suppress Sclerotinia minor, the causal agent of basal drop disease of lettuce in the United Arab Emirates (El-Tarabily et al., 2000). This antagonist produced high levels of chitinase and X-1,3-glucanase, and when the live pathogen was presented as the sole carbon source, it caused extensive hyphal plasmolysis, cell wall lysis and signicantly reduced the level of disease incidence under controlled glasshouse conditions (El-Tarabily et al., 2000). In addition to the chitinase-producing M. carbonacea, there is one other study of an endophytic isolate of A. missouriensis on Plectosporium tabacinum the causal agent of lupin root rot in Egypt by El-Tarabily (2003). Here, the chitinase produced by A. missouriensis not only caused hyphal lysis

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but also reduced conidial germination and, where they did germinate, reduced the length of the germ tube of the pathogen. This antagonist produced only chitinase and no other cell wall degrading enzymes. El-Tarabily (2003) also established that an isolate of Actinoplanes italicus incapable of producing chitinase and a mutant strain of A. missouriensis that did not produce detectable levels of chitinase, failed to lyse hyphae of P. tabacinum or to reduce root rot in glasshouse experiments, indicating the importance of chitinases in the suppression of the disease. The hyphae of Pythium (Cooper and Aronson, 1967; Sietsma et al., 1975) and Phytophthora (Bartnicki-Garcia and Wang, 1983) have been reported to contain mainly the non-cellulosic X-1,3, and X-1,6 linked glucans as the predominant glucans in addition to the cellulosic X-1,4 linked glucan which is present as a relatively small proportion of the total glucans. The composition of cell walls of oomycetes selectively encourages the colonization of vegetative mycelia by NSA capable of producing these enzymes. Valois et al. (1996) reported that a strain of Nocardioides capable of producing X-1,3, X-1,4 and X-1,6 glucanases hydrolyzed glucans from Phytophthora fragariae cell walls, caused hyphal lysis and reduced the severity of the root-rot disease of raspberry caused by this pathogen. In addition, El-Tarabily et al. (1996b) found that attack by Phytophthora cinnamomi on Banksia grandis was controlled by the application of a cellulase and X-glucosidase-producing isolate of M. carbonacea which caused hyphal lysis and reduced the severity of root-rot under controlled glasshouse conditions. In that study, the activity of the M. carbonacea was enhanced by the presence of an antagonistic antibiotic-producing strain of Streptomyces violascens and the performance of the combined inoculum was superior to the treatment where these antagonists were applied singly (El-Tarabily et al., 1996b). These authors also concluded that once cell wall damage has occurred by the action of M. carbonacea, the pathogen is more likely to be susceptible to attack by the antifungal metabolites produced by S. violascens, and this may explain the improved disease control when both antagonists were combined. The large-scale screening of NSA for X-1,3, X-1,4, and X-1,6-glucanases involved in hyphal lysis of oomycete pathogens has been facilitated by the use of selective agar for the production of these enzymes in which mycelial fragments of the pathogen are provided as the sole carbon substrate for these antagonists (Valois et al., 1996). The mycelial fragments not only provide the substrate for the production for these enzymes but may also provide the signal necessary for the induction of the enzymes. The study of Valois et al. (1996) on P. fragariae and of ElTarabily (2006) on P. aphanidermatum using such media containing mycelial fragments has been most valuable in the screening and the selection of the isolates of NSA for the production of these enzymes. El-Tarabily (2006) reported that isolates of Microbispora rosea, Micromonospora chalcea and A. philippinensis produced X-1,3, X-1,4 and

X-1,6-glucanases in vitro, caused lysis of P. aphanidermatum hyphae in vitro and reduced damping-off disease of cucumber under controlled glasshouse conditions. These isolates were able to suppress damping-off in soil amended with or without cellulose. The treatment which combined all isolates in soil amended with cellulose was superior to all other treatments, in suppressing damping-off (El-Tarabily, 2006). It is noteworthy that production of siderophores, competition for nutrients and induction of systemic resistance are yet to be reported as mechanisms of biocontrol of soil-borne fungal plant pathogens by NSA. These mechanisms, however, have been reported to be involved in the biological control of soil-borne fungal plant pathogens by other antagonists, especially by bacteria (Elad and Chet, 1987; Buysens et al., 1996; Benhamou et al., 2002) and streptomycete actinomycetes (Tokala et al., 2002). While siderphores have been reported to be produced by NSA (Fiss and Brooks, 1991; Arahou et al., 1998), such isolates or their mutants were not tested against soil-borne fungal plant pathogens. 5. Pathogen suppression versus disease suppression In a review on the biological suppression of the take all fungus Gaeumannomyces graminis var. tritici, Simon and Sivasithamparam (1989) proposed that pathogen suppression occurs either in soil without the involvement of plants or occurs outside the inuence of plant roots, and that disease suppression by the antagonist suppressing the disease only occurs in the presence of or through the mediation of the plant host. Experimentally, they demonstrated that pathogen suppression is effective when the pathogen and the antagonist are introduced to the soil prior to sowing or planting and that a period of incubation preceding seeding or planting is necessary for effective bicontrol. This can be contrasted to disease suppression that occurs where all three components, the antagonist, pathogen and the plants, are introduced into the system concurrently. Greater suppression of the disease in the former treatment indicates the occurrence of pathogen suppression. Failure of pre-incubation to enhance suppression is considered to indicate disease suppression (Simon and Sivasithamparam, 1989). No such tests have been conducted to date with NSA. Rai and Upadhyay (1983) reported that competitive saprophytic colonization of pigeon-pea substrate by F. udum was strongly suppressed by M. globosa when used in inoculum mixtures or on substrates pre-colonized by M. globosa. Filonow and Lockwood (1985) reported that the application of A. missouriensis, A. utahensis, A. auranticolor or Micromonospora sp. reduced the inoculum density of P. megasperma f. sp. glycinea in a eld soil. Sabaou and Bounaga (1987) found that an isolate of N. dassonvillei reduced propagule numbers of F. oxysporum f.sp. albedinis, a pathogen of date palm, in autoclaved and non-autoclaved soil. The action was found

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to be less pronounced in the non-autoclaved soil where the antagonism was extenuated by the presence of other soil microorganisms. It is quite likely that as with antagonistic bacteria and fungi (Cook and Baker, 1983), there could be large differences in the saprophytic competence of the antagonists within the NSA groups. Most studies have utilized streptomycetes as potential biological control agents of soil-borne fungal plant pathogens (Broadbent et al., 1971; Turhan, 1981; Tahvonen, 1982; Tahvonen and Avikainen, 1987; Tahvonen et al., 1995; Yuan and Crawford, 1995; Berg et al., 2001; Xiao et al., 2002), and information on the potential of other genera from the order Actinomycetales is still relatively low (Table 2) compared to the application of streptomycete actinomycetes. 6. Methods of application and rhizosphere competence Choice of methods of application of NSA, as with other biocontrol agents, are determined by the nature of the pathogens and the ability of the antagonists applied to establish in soil and rhizosphere and the type of pathogen targeted. NSA have been tested as potential antagonists, in the presence of a host and a pathogen, as a seed coating (Sutherland and Lockwood, 1984; Filonow and Lockwood, 1985; Coombs et al., 2004), in soil mixes (Filonow and Lockwood, 1985; Sutherland and Papavizas, 1991; El-Tarabily et al., 1997, 2000), as a root dip (Smith, 1957; El-Tarabily, 2003) and as impregnated clay granules, vermiculite, perlite or rice hulls (Khan et al., 1997). As seeds and roots are the main tissues that require protection from soil-borne fungal plant pathogens, it is convenient that the antagonists introduced onto the seeds migrate to and colonize the roots. For this reason, the importance of rhizosphere competence has been recognized, especially after the work of Ahmad and Baker (1987) on Trichoderma strains. In relation to NSA, rhizosphere competence has been a focus in the work of El-Tarabily et al. (2000) where M. carbonacea was used to control S. minor. That study reported that a strain of M. carbonacea was a competent colonizer of lettuce roots and the rhizosphere for up to 4 cm of the root system. Experimentally, rhizosphere competence has been determined in two stages. Initially, an indicator root colonization plate assay (Kortemaa et al., 1994) carried out in vitro can determine whether the root exudates, acting as the sole nutrient source, could support the growth and activity of the antagonists. Subsequently, promising isolates from this initial assay can then be subjected to a rhizosphere competence assay using the non-sterile sand tube method described by Ahmad and Baker (1987) using antibioticresistant mutants. In a subsequent study, El-Tarabily (2006) found that strains of A. philippinensis M. chalcea and M. rosea, all competent as a root and rhizosphere occupants in the sand-tube assay, were able to rapidly colonized 12 cm of cucumber roots from the base of the stem 21 days after sowing the seeds, with the densities

signicantly greater in the rst 8 cm of the root system compared to other root depths. Colonization frequency of the root segments and the rhizosphere soil was greater in plants treated with A. philippinensis than with M. chalcea or M. rosea (El-Tarabily, 2006). Rhizosphere competence is clearly an attribute necessary for seed inoculation of any plant growth promoting rhizobacteria (PGPR) (Benizri et al., 2001). Weller (1988) described a good root colonizing strain as one which is able to colonize the whole root system, and survive during several weeks in the presence of the natural microora. Ahmad and Baker (1987) used the concept of rhizosphere competence to describe colonization of the rhizosphere in terms of time and space; Trichoderma species that did not colonize the rhizosphere to a depth greater than 2 cm were not classied as rhizosphere competent (Ahmad and Baker, 1987). It is noteworthy, that although rhizosphere competence has been used as a criterion for selection of effective biocontrol agents in bacteria (Weller, 1988), streptomycete actinomycetes (Kortemaa et al., 1994; Yuan and Crawford, 1995; Tokala et al., 2002) and fungi (Ahmad and Baker, 1987) very few attempts have been made to screen NSA for rhizosphere competency (El-Tarabily et al., 2000; ElTarabily, 2006). Rhizosphere competence appears to be a prerequisite for successful biological control of root diseases, and failure to adequately colonize roots may explain the lack of reliable biological control observed in many studies (Weller, 1988). The methods of application therefore should be assessed or evaluated in relation to the ability of the antagonist not only to be a competent saprophyte but also for its rhizosphere competence. 6.1. Inoculum and food base The addition of food bases or substrates either into the soil or on seed coats, which enable the biocontrol agent to survive and proliferate in the spermosphere and the rhizosphere, is a suitable approach for biocontrol agents as suggested by Hoitink and Boehm (1999). The food base, however, is only infrequently considered as being important or critical for the activity of these organisms, especially those which have to be active in the soil and on seeds before they have access to root exudates. The value of the food base, such as wheat bran (El-Tarabily et al., 1997) or sh emulsion (El-Tarabily et al., 2003) as nutrient sources for soil bacteria and actinomycetes, has been evaluated. Wheat bran as a food base has been successfully used in several other studies including antagonistic Trichoderma spp. (Roiger and Jeffers, 1991). The high nutrient content of wheat bran should help with both vegetative growth and spore production of actinomycetes. Such a food base may also help actinomycetes to persist in the soil and enhance their antagonistic activities against pathogens. The response of streptomycete actinomycetes and bacteria to a food base such as sh emulsion (El-Tarabily et al., 2003) clearly established the importance of food base in the

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K.A. El-Tarabily, K. Sivasithamparam / Soil Biology & Biochemistry 38 (2006) 15051520 Table 2 Reports of biological control of diseases caused by soil-borne fungal plant pathogens using non-streptomycete actinomycetes Antagonist Actinoplanes spp. A. missouriensis A. missouriensis A. missouriensis A. philippinensis A. philippinensis A. utahensis Actinomadura sp. A. rubra Amorphosporangium auranticolor Microbispora sp. Microbispora rosea Micromonospora sp. Micromonospora sp. Micromonospora sp. M. carbonacea M. carbonacea M. carbonacea Micromonospora chalcea M. globosa Nocardia globerula Nocardioides sp. Nocardioides sp. Streptosporangium albidum Streptoverticillium netropsis Pathogen Pythium ultimum Phytophthora megasperma f. sp. glycinea P. megasperma f. sp. glycinea Plectosporium tabacinum Pythium coloratum Pythium aphanidermatum P. megasperma f. sp. glycinea Phytophthora cinnamomi P. coloratum P. megasperma f. sp. glycinea Gaeumannomyces graminis var. tritici P. aphanidermatum Fusarium oxysporum f. sp. lycopersici P. megasperma f. sp. glycinea G. graminis var. tritici P. cinnamomi P. coloratum Sclerotinia minor P. aphanidermatum Fusarium udum Helminthosporium solani Phytophthora fragariae var. rubi G. graminis var. tritici P. coloratum P. coloratum Plant Table beet Soybean Soybean Lupin Carrots Cucumber Soybean Snapdragon Carrots Soybean Wheat Cucumber Tomato Soybean Wheat Banksia sp. Carrots Lettuce Cucumber Pigeon-peas Potato Raspberry Wheat Carrots Carrots Disease Damping-off Root rot Root rot Root rot Cavity spot Damping-off Root rot Root rot Cavity spot Root rot Take all Damping-off Wilt Root rot Take all Root rot Cavity spot Basal drop Damping-off Wilt Silver scurf Root rot Take all Cavity spot Cavity spot Reference Khan et al. (1997) Sutherland and Lockwood (1984) Filonow and Lockwood (1985) El-Tarabily (2003) El-Tarabily et al. (1997) El-Tarabily (2006) Filonow and Lockwood (1985) You et al. (1996) El-Tarabily et al. (1997) Filonow and Lockwood (1985) Coombs et al. (2004) El-Tarabily (2006) Smith (1957) Filonow and Lockwood (1985) Coombs et al. (2004) El-Tarabily et al. (1996b) El-Tarabily et al. (1997) El-Tarabily et al. (2000) El-Tarabily (2006) Upadhyay and Rai (1987) Elson et al. (1997) Valois et al. (1996) Coombs et al. (2004) El-Tarabily et al. (1997) El-Tarabily et al. (1997) 1513

application of inoculants in soil. Smith (1957) used culture ltrate as the food base in which he dipped the uninjured tomato roots in the culture ltrate, for 12 days before planting them in soil infested with F. oxysporum f. lycopersici, to facilitate colonization of roots by the endophytic isolate of Micromonospora sp. This aspect of inoculum application technique clearly warrants investigation in relation to NSA. Khan et al. (1997) compared different carriers such as vermiculite, perlite and rice hulls and reported that while Actinoplanes spp. did not sporulate or sporulated poorly on these carriers, sporulation was abundant on montmorillonite clay granules. When Actinoplanes spp. were applied as clay granules at (5% w/w) to eld soil infested with oospores of P. ultimum, emergence was increased and root rot reduced in table beets. Khan et al. (1997) also reported that the inoculum viability on clay granules declined 100-fold during 2 months of storage at 535 1C, but thereafter remained stable for another 4 months. Actinoplanes strain 25844 on 6-month-old clay granules retained a high degree of hyperparasitic activity towards oospores of P. ultimum. Khan et al. (1997) also reported that augmentation of eld soil with sporangia of Actinoplanes spp. is a promising approach for biological control of Pythium damping-off in table beet. This is the only published study to date on inoculum survival and shelf life of NSA as biocontrol agents. The food base itself can be included as an amendment preceding application of the inoculum to soil. Amendment of soil with cellulose could provide the cellulase-producing

antagonists with a substrate selectively promoting these antagonists over other organisms in soils that are incapable of producing cellulases. Such provision of selective advantages in the soil environment was reported by ElTarabily (2006) where improved reduction of damping-off caused by P. aphanidermatum by genera of NSA was enhanced in the presence of cellulose amendments. Kundu and Nandi (1985) also found that the addition of commercial cellulose and cellulosic waste products (rice stubble or water hyacinth biomass) resulted in the reduction of cauliower damping-off caused by Rhizoctonia solani and they ascribed this to the increased numbers of unidentied antagonistic actinomycetes and bacteria encouraged by the presence of soil amendments. Mature compost amendment containing chitin residues have shown suppressive properties against several plant pathogens (Labrie et al., 2001). In these suppressive composts, the microbial population is characterized by a proliferation of Gram-positive bacteria belonging mainly to actinomycetes (Labrie et al., 2001). Biological control studies with NSA using either soil treated with aerated steam (El-Tarabily et al., 1997) or non-sterile soils (Smith, 1957; Valois et al., 1996; ElTarabily et al., 2000; El-Tarabily, 2003) have been conducted under controlled greenhouse or under eld conditions. Coombs et al. (2004), however, tested the endophytic isolates of Microbispora, Micromonospora and Nocardioides in both steamed and natural soil and reported that the reduction of root rot caused by G. graminis var.

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tritici was greater in the eld soil than in the steamed soil. This suggests benets from general antagonism expressed by the resident microora of the eld soil. Many workers have introduced the inocula of NSA and the pathogen together at seeding or planting (Sutherland and Lockwood, 1984; El-Tarabily et al., 1996b; Valois et al., 1996; Coombs et al., 2004), whilst others incorporated the antagonist(s) and incubated the soil for a period of time prior to infesting the soil with the pathogen and planting (Smith, 1957; El-Tarabily et al., 1997, 2000). The pre-incubation of NSA inoculum in the soils for a period of time before the addition of the pathogen during greenhouse studies may help the antagonist(s) to establish in the soil prior to exposure to the pathogen and/or the plant. 6.2. Endophytic NSA Although the activity of antagonistic endophytic NSA was reported nearly 50 years ago by Smith (1957), it is only recently that interest has been shown in the behavior and activity of these antagonists as endophytes. Application of antagonistic endophytic NSA as biocontrol agents is highly desirable because the antagonist applications targeting the rhizosphere have to be large for effective establishment and operation in the competitive environment of the rhizosphere and the spermosphere. In addition, these endophytes are able to occupy the cortical tissues of roots and have been very effective in the defences against infection processes of invading pathogens (Sivasithamparam, 1998). The cortex or the root tissues occupied by the antagonistic endophytes clearly confers protection to these antagonists from the harsh environment of the bulk soil and the rhizosphere where not only the biotic but also the abiotic environment can vary considerably (Sivasithamparam, 2002). Endophytic NSA (e.g. Actinoplanes, Micromonospora, Microbispora, Nocardia, Nocardiopsis, Nocardioides and Streptosporangium) (Smith, 1957; Sardi et al., 1992; de Araujo et al., 2000; Stamford et al., 2001; Kunoh, 2002; Stamford et al., 2002; Coombs and Franco, 2003; El-Tarabily, 2003; Coombs et al., 2004) have been isolated from inside live tissues of various plant species. Endophytic NSA have been shown to protect plants against different soil-borne fungal plant pathogens including F. oxysporum (Smith, 1957), P. tabacinum (El-Tarabily, 2003); G. graminis var. tritici and R. solani (Coombs et al., 2004). Endophytic NSA have been reported to inhibit the growth of soil-borne fungal plant pathogens through the production of inhibitory antifungal metabolites (Smith, 1957; Taechowisan et al., 2003; Coombs et al., 2004; Tian et al., 2004), and cell wall degrading enzymes such as chitinase (El-Tarabily, 2003). Inoculation of endophytic microorganisms can be achieved by many methods in relation to bacteria (Musson et al., 1995). For example, with NSA, Smith (1957) and ElTarabily (2003) used the pruned root dip method, while Coombs et al. (2004) employed the seed coating technique.

NSA were recoverable from surface-sterilized roots 8 weeks after inoculation of lupin seedlings (El-Tarabily, 2003). 7. Plant growth promotion and production of plant growth regulators (PGRs) While soil and rhizosphere NSA have been shown to promote growth of many plant species, there are no records in the literature on the effects of endophytic NSA on plant growth promotion. Rhizosphere isolates of A. missouriensis and A. utahensis, have been reported to increase root and shoot weight of soybean in a soil naturally infested with P. megasperma f.sp. glycinea (Filonow and Lockwood, 1985). Mishra et al. (1987) tested the culture ltrates of Micromonospora, Nocardia, Rhodococcus, Streptosporangium and Oerskovia from rhizosphere for plant growth regulatory properties and found that these isolates caused dry-weight increases in corn, soybeans, cucumbers, tomatoes and sorghum. El-Tarabily et al. (1996b) reported that the application of cellulase-producing M. carbonacea in the presence or absence of P. cinnamomi, the causal agent of root rot of B. grandis, signicantly increased both root and shoot weights. The application of Streptoverticillium netropsis, Actinomadura rubra, A. philippinensis, M. carbonacea and Streptosporangium albidum isolated from carrot rhizosphere, in the presence or absence of P. coloratum, increased mean root weight of carrot (except A. rubra in the presence or absence of the pathogen and M. carbonacea in the presence of the pathogen only) (El-Tarabily et al., 1997). In these studies it is noteworthy that the presence of the pathogen did not diminish the growth promotion effect evident in the plants exposed only to the NSA isolates. This suggests that the parasitic activity of these pathogens could be independent of certain biological activities of NSA and is an indication that the NSA were producing PGRs, active in the rhizosphere, rather than competing for the same sites as the pathogen. It may also indicate the involvement of induced resistance in plants inoculated with NSA. Enhancement of plant growth following inoculation with plant growth promoters can result from wide varieties of direct and indirect activities (Glick, 1995). It could be the direct action of the introduced isolates in making available soil nutrients for plant growth or by the production of plant growth regulators (PGRs) in planta or in the rhizosphere by the introduced microorganisms. Indirect effects are those related to the production of metabolites, such as antibiotics which increase plant growth by decreasing the activities of pathogens or deleterious microorganisms (Glick, 1995). Plant growth enhancement resulting from increased availability of nutrients was evident in our recent work (El-Tarabily,K.A., Nassar,A.H., Sivasithamparam,K., unpublished data) where a rock phosphate-solubilizing, rhizosphere-competent isolate of Micromonospora endolithica was found to increase availability of phosphorus and promote plant growth, while a strain of Micromonospora

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olivasterospora incapable of solubilizing rock phosphate failed to promote growth. Several reports exist detailing the production of PGRs such as auxins, gibberellins and cytokinins by NSA in vitro, usually in media containing precursors of PGRs such as L-tryptophan. Auxins have been reported to be produced by Actinomyces spp. (Kaunat, 1969), Nocardia sp. (Brown, 1972) and Frankia sp. (Wheeler et al., 1984). Gibberellin-like substances have been reported to be produced by Actinomyces spp. (Panosyan et al., 1963) and a Nocardia spp. (Katznelson and Cole, 1965; Brown, 1972). A Clavibacter sp. has been reported to produce cytokinins (Armstrong et al., 1976). Unfortunately no attempt to date has been made to assay for in planta PGRs in plants treated with these NSA, as was done in the case of Streptomyces griseoavus, S. rimosus and S. diastaticus in radish (El-Tarabily et al., 2003). Streptomyces griseoluteus has also been shown to promote growth of bean through the production of polyamines including putrescine, spermidine and spermine (Nassar et al., 2003). Infestation of soil with S. griseoluteus resulted in a signicant increase in the levels of endogenous putrescine, spermidine and spermine, and certain endogenous PGRs including indole-acetic acid and gibberellic acid. The involvement of polyamines in plant growth promotion was clearly established in the study by Nassar et al. (2003) where a polyamine non-producing mutant strain that failed to produce polyamines did not promote plant growth. Selections of NSA for growth promotion could be facilitated by the inclusion of polyamine production as a component of routine screens. It should be noted that not all metabolites of NSA promote plant growth. For instance, Mishra et al. (1988) found that the metabolites from Nocardiopsis, Actinoplanes, Actinomadura, Micromonospora, Micropolyspora, Streptosporangium, and Streptoverticillium to be toxic to cress seeds. 8. Conclusion Evaluation of literature showed that NSA have great potential as candidates for the biocontrol of soil-borne fungal plant pathogens and also as plant growth promoters. One of the reasons why NSA have received less attention is the difculty in the isolation of NSA in routine isolations of soil- and plant-associated actinomycetes. This can be overcome by utilizing the available methods for selective isolation of NSA. Many of the techniques developed for the assessment of mechanisms of action and methods of applications currently used for bacteria and streptomycete actinomycetes, could easily be adapted for NSA. With better understanding and screening of NSA, successful candidates from among NSA for biocontrol and plant growth promotion could be sourced, that could match, and in certain situations, out perform other products (e.g. Mycostop, Actinovate and Actino-Iron) with Streptomyces spp. (Tahvonen and Avikainen, 1987; Cross and Polonenko, 1996; Crawford et al., 2005).

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