Research in Microbiology 161 (2010) 464e471 www.elsevier.com/locate/resmic
Fluorescent pseudomonads as biocontrol agents for sustainable agricultural systems Monica Ho¨fte a,*, Nora Altier b a
Laboratory of Phytopathology, Department of Crop Protection, Faculty of Bioscience Engineering, Ghent University, Coupure Links 653, Gent, Belgium Plant Protection, National Institute for Agricultural Research, Instituto Nacional de Investigacio´n Agropecuaria, INIA Las Brujas, Canelones, Uruguay
b
Received 1 November 2009; accepted 12 April 2010 Available online 10 May 2010
Abstract The highly diverse genus Pseudomonas contains very effective biocontrol agents that can increase plant growth and improve plant health. Biocontrol characteristics, however, are strain-dependent and cannot be clearly linked to phylogenetic variation. Isolate screening remains essential to find suitable strains, which can be done by testing large local collections for disease suppression and plant-growth promotion exemplified in a case study on forage legumes in Uruguay or by targeted screening for Pseudomonas spp. which produce desirable secondary metabolites, as demonstrated in a case study on cocoyam in Cameroon. In both case studies, access to reference strains from public and private collections was essential for identification, phylogenetic studies and metabolite characterization. Ó 2010 Elsevier Masson SAS. All rights reserved. Keywords: Alfalfa; Biosurfactants; Birdsfoot trefoil; Cocoyam; Cyclic lipopeptides; 2,4-Diacetylphloroglucinol; Fluorescent pseudomonas; Hydrogen cyanide; Lotus corniculatus; Medicago sativa; Phenazines; Pyoluteorin; Pyrrolnitrin; Xanthosoma sagittifolium
1. Introduction 1.1. Biological control Biological control of plant diseases, in its widest sense, is any means of controlling disease or reducing the amount or effect of pathogens that relies on biological mechanisms or organisms other than man (Campbell, 1989). Within this definition, it also includes cultural practices such as crop rotation and soil amendments that affect pathogenic microorganisms. A more narrow approach is to restrict biological control to the artificial introduction of living microorganisms into the environment to control the pathogen. As such, these biopesticides are an alternative or supplementary way of reducing the use of chemical pesticides in agriculture. There is a genuine commercial interest in biopesticides, since they can * Corresponding author. Tel.: þ32 9 2646017; fax: þ32 9 2646238. E-mail addresses:
[email protected] (M. Ho¨fte),
[email protected]. uy (N. Altier). 0923-2508/$ - see front matter Ó 2010 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.resmic.2010.04.007
be used in rotation with chemical pesticides to reduce the development of pathogen resistance. Biocontrol can also be used in situations where no control is currently available, where conventional pesticides cannot be used due to re-entry or residue concerns or where the product must be certified organic (Fravel, 2005). The paper of Fravel (2005) lists 14 bacteria and 12 fungi registered with the United States Environmental Protection Agency for control of plant diseases. Among the 14 registered bacterial biocontrol agents, six are based on Bacillus, five on Pseudomonas, two on Agrobacterium and one on Streptomyces. Pseudomonas spp. are particularly suited as biocontrol agent because they can use many exudates as nutrient source, they are abundantly present in natural soils, especially in the rhizosphere, they have a high growth rate, they can be directly plant-growth-promoting and they have the ability to control diseases by a variety of mechanisms. They are also the most extensively studied group of bacterial biocontrol agents, since Pseudomonas bacteria are amenable to mutation and modification using genetic tools (Chin-A-Woeng et al., 2003).
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1.2. Role of culture collections in biocontrol research Culture collections play a very important role in research as a source of authenticated biological material. They accept deposits subject to publication to enable confirmation of results and further studies, and offer safe, confidential patent deposit services (Smith, 2003). In relation to agriculture, specific collections are available mainly dealing with plant pathogenic microorganisms (Kang et al., 2006) and nitrogenfixing bacteria such as Rhizobium (World Federation for Culture Collections, 2010), but to our knowledge, there are no culture collections dedicated to bacterial biocontrol agents. In biocontrol research, screening is a critical step in the development of biocontrol agents, and the ultimate success of biocontrol depends on how well the searching and screening process is done (Fravel, 2005). The places to look for potential control agents must be selected carefully and the control agent eventually selected must be able to survive and grow in the environment in which it is expected to operate (Campbell, 1989). As stated by Campbell (1989), isolates from culture collections rarely produce useful organisms for the field because they are usually adapted to the high nutrient levels in common media. However, culture collections are important in biocontrol research because reference strains are needed in taxonomic and phylogenetic studies to identify the newly isolated biocontrol agents and to study their genotypic and phenotypic diversity. In this review, we focus on Pseudomonas as a case study of biological control agents in relation to culture collections because they are widely used and studied and highly diverse, which poses challenges to taxonomy and phylogeny. After a brief general overview about Pseudomonas taxonomy, plantassociated pseudomonads and the diversity of Pseudomonas biocontrol agents, two case studies will focus on the isolation and screening of effective Pseudomonas biocontrol agents in Uruguay and Cameroon. Microorganisms from Africa and South America are typically underrepresented in culture collections. Of the 574 culture collections registered with the World Data Center for Microorganisms (World Federation for Culture Collections, 2010), 11 collections are located in Africa and they contain only 0.81% of the microorganisms in these culture collections. A case study on the holdings of the environmental prokaryotes available at the American Type Culture Collection (Floyd et al., 2005) has revealed that only 2.8% of the accessions come from Africa and only 1.8% from South America. One question concerns whether isolation and screening of Pseudomonas biocontrol agents in Uruguay and Cameroon will deliver new Pseudomonas spp. with undescribed mode of actions. 2. Fluorescent pseudomonads as biocontrol agents 2.1. Pseudomonas taxonomy Pseudomonas is one of the most ubiquitous bacterial genera in the world and different species have been isolated
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from very diverse ecological niches. Since its discovery, the genus Pseudomonas has undergone numerous taxonomic changes and a detailed history of Pseudomonas taxonomy can be found in a recent paper by Peix et al. (2009). Currently, only the representatives of rRNA group I Palleroni et al. (1973) are included in the genus Pseudomonas and up to now, 128 species have been validly described for this genus, including the fluorescent pseudomonads that have the capacity to produce fluorescent pyoverdine-type siderophores under low-iron conditions. Most species are saprophytes that are commonly found in water and soil; 23 species are pathogenic to plants, including Pseudomonas syringae with 36 pathovars affecting different plants. In addition, 16 species are associated with diseases in humans and animals (Peix et al., 2009). Some species, such as Pseudomonas aeruginosa, are ubiquitous and can be associated with both plants and animals. Some of the saprophytic species have interesting characteristics and are used in biotechnological applications to improve plant growth and plant health, but also in water and soil bioremediation. 2.2. Plant-associated Pseudomonas spp Plant-associated Pseudomonas include both beneficial and pathogenic isolates (Ho¨fte and De Vos, 2006), which colonize the same ecological niches and possess similar mechanisms for plant colonization (Preston, 2004). Pathogenic, saprophytic and plant-growth-promoting strains are often found within the same species. Strains that improve plant growth and plant health, also called plant-growth-promoting bacteria (Kloepper et al., 1980) or plant-probiotic fluorescent pseudomonads in analogy with probiotic bacteria and yeasts in the gastrointestinal tract (Haas and Keel, 2003; Picard and Bosco, 2008), are commonly found within Pseudomonas fluorescens, Pseudomonas putida and Pseudomonas chlororaphis (¼Pseudomonas aureofaciens), but also isolates of P. aeruginosa and Pseudomonas syringae have been identified as efficient biocontrol agents (Anjaiah et al., 1998; Audenaert et al., 2002; Janisiewicz and Marchi, 1992). The most commonly reported mechanisms of biocontrol by fluorescent Pseudomonas spp. include production of antibiotics, hydrogen cyanide, lytic exoenzymes (Thomashow and Weller, 1996), cyclic lipopeptides (Raaijmakers et al., 2006), competition for nutrients and niches (Kamilova et al., 2005), competition for iron mediated by siderophores, competition for carbon (Thomashow and Weller, 1996) and induced systemic resistance (De Vleesschauwer and Ho¨fte, 2009). The antibiotics 2,4-diacetylphloroglucinol (DAPG), pyoluteorin, pyrrolnitrin and different phenazine derivatives have been described in biocontrol Pseudomonas spp. as the main cause of their antagonistic activity (De La Fuente et al., 2004; Thomashow and Weller, 1996; Weller et al., 2007). Additionally, many rhizospheric fluorescent Pseudomonas spp. have the capacity to stimulate plant growth, by increasing the availability and uptake of mineral nutrients via phosphate-solubilizing enzymes or by enhancing root growth and morphology via the production of phytohormones such as auxin (Vessey, 2003).
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2.3. Pseudomonas diversity Plant-probiotic Pseudomonas strains have been isolated from different soils and plant hosts worldwide. However, there is a general lack of association between biocontrol characteristics and chromosomal phylogeny at the species level and it is at present unclear whether biocontrol can be associated directly with phylogenetically distinct subspecies (McSpadden Gardener, 2007). A large number of biocontrol strains are presented as P. fluorescens in the literature, but complete genome sequences of a number of P. fluorescens strains have revealed that genomes within the species are extremely variable. Comparison of three P. fluorescens genomes (the biocontrol agents SWB25 and Pf-5 and the soil inhabitant Pfo1) revealed that they share just 61% of their genes, suggesting that they are a species complex rather than a single species (Silby et al., 2009). On the other hand, biosynthetic loci for antibiotic production are highly conserved and this has been most intensively studied for DAPG-producing strains (Keel et al., 1996; Frapolli et al., 2007). The biosynthetic locus for DAPG is conserved among all known DAPG-producing fluorescent Pseudomonas spp. including isolates from the US, Europe, South America, Asia and Africa (Weller et al., 2007). Multilocus sequencing revealed six main groups of DAPGproducing Pseudomonas spp. within the P. fluorescens complex. Interestingly, strains from the same phylogenetic cluster could originate from very distant geographic locations (Frapolli et al., 2007) and there are indications that some DAPG-producing genotypes have a preference for the roots of certain crop species (Weller et al., 2007). Likewise, it was recently shown that a superior inorganic phosphate solubilization potential was linked to a single phylogenetic lineage within the P. fluorescens complex (Browne et al., 2009), which emphasizes the importance of taxonomic and phylogenetic studies. Besides crop species, other environmental factors such as plant tissue, soil type, structure and fertility, soil temperature and moisture content have an effect on the relative abundance, diversity and activity of plant-associated Pseudomonas spp. (McSpadden Gardener, 2007). As stated by Picard and Bosco (2008), knowledge of the factors affecting the diversity of plant-associated Pseudomonas populations is far from complete, and much more research is needed for it to be effectively applicable in agriculture. This research is hampered by the fact that, until now, different sets of strains have been used in different studies, which themselves were isolated from different soils and different plants and assessed by different techniques in different research laboratories (Picard and Bosco, 2008). 2.4. Culture collections and Pseudomonas For both pathogenic and beneficial plant-associated Pseudomonas it is important to have access to strains for various reasons. Reference strains are needed in taxonomic and phylogenetic studies to identify new Pseudomonas isolates and to study their genotypic and phenotypic diversity. Reference strains are also essential to test the specificity of detection
techniques aimed at identifying pathogenic Pseudomonas bacteria in various ecosystems. For pathogenicity assays, it is important to have access to strains with known virulence. Pseudomonas reference strains for taxonomic and phylogenetic purposes and plant pathogenic Pseudomonas strains can usually be found in general culture collections and in collections dedicated to plant pathogenic bacteria. In the field of plant beneficial strains, reference strains that are known producers of certain antibiotics or other important secondary metabolites are needed to include as positive controls in assays to identify these compounds in newly isolated strains. As stated above, the capacity to produce metabolites important for biocontrol is isolate-dependent and effective biocontrol strains are usually not included in public culture collections. Some of the biocontrol strains are commercialized; others are in private collections in research labs and can usually be obtained by signing a material transfer agreement. Some biocontrol strains have been intensively studied, such as the DAPG, pyoluteorin, pyrrolnitrin and hydrogen cyanide-producing strains P. fluorescens Pf-5, for which a whole genome sequence is available (Paulsen et al., 2005) and the closely related strain P. fluorescens CHA0 (Haas and De´fago, 2005); the phenazine-producing strains P. fluorescens 2-79 (Weller and Cook, 1983), P. chlororaphis PCL1391 (Chin-A-Woeng et al., 1998) and P. aureofaciens (¼P. chlororaphis) 30-84 (Pierson and Thomashow, 1992); and resistance inducers P. fluorescens WCS365, P. fluorescens WCS374 and P. putida WCS417 (Bakker et al., 2007). In addition, many local strains have been isolated and studied in varying detail. 3. Case study: Pseudomonas bacteria for growth promotion and disease management of forage legumes 3.1. Importance of seedling diseases of forage legumes Forage legumes are essential for efficient animal-based agriculture worldwide. Besides providing high quality feed for livestock, they are a key component in the sustainability of crop-pasture rotations. Their value lies essentially in their ability to fix nitrogen (N2) in symbiosis with root nodule soil bacteria, collectively called rhizobia. In Uruguay, improved pastures integrated by forage legumes are the foundation of agriculture, dairy and livestock production (Rebuffo et al., 2006). The perennial strategy of most temperate forage legumes like alfalfa (Medicago sativa L.) and birdsfoot trefoil (Lotus corniculatus L.) relies on the success of stand establishment and early development of healthy root systems to achieve optimal productivity. Seedling diseases caused by soilborne pathogens, primarily Pythium species, are one of the main constraints for legume establishment (Altier and Thies, 1995). Favorable environmental conditions for disease development are low soil temperatures and high soil moisture, which slow down germination rate and reduce seedling emergence (Altier and Thies, 1995; Martin and Loper, 1999; Pe´rez et al., 2000). Effective management of soilborne plant pathogens requires
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integrated strategies which can include the use of rhizospheric antagonistic microorganisms (Martin and Loper, 1999; Weller et al., 2007, 2002). 3.2. Use of rhizosphere bacteria as biocontrol agents Microbial-based strategies that improve forage legume establishment and optimize N2 fixation have been deployed worldwide through rhizobial inoculant technology (Catroux et al., 2001). However, the study of rhizospheric bacteria for plant-growth promotion remains a challenge (Handelsman et al., 1990; Jones and Samac, 1996; Villacieros et al., 2003; Xiao et al., 2002). Fluorescent Pseudomonas spp. have been extensively reported as effective biocontrol agents (McSpadden Gardener, 2007; Weller et al., 2007) and suggested control of Pythium seedling diseases in other crops (Loper, 1988; Martin and Loper, 1999). In addition, the use of Bacillus spp. (Handelsman et al., 1990) and Streptomyces spp. (Jones and Samac, 1996; Xiao et al., 2002) has been explored to control alfalfa seedling damping-off. 3.3. Research on biocontrol agents in Uruguay: a long journey from the lab to the field Since 1995, research has been done to explore the biocontrol of Pythium seedling diseases using native fluorescent Pseudomonas isolated from Uruguayan soils (Bagnasco et al., 1998; Bajsa et al., 2005; De La Fuente et al., 2002; De La Fuente et al., 2004; Pe´rez et al., 2000; Quagliotto et al., 2009; Yanes et al., 2005). Several strains with enhanced disease-suppressing and plant-growth-promoting abilities have been selected to develop bacterial inoculants (De La Fuente et al., 2002; De La Fuente et al., 2004; Quagliotto et al., 2009; Yanes et al., 2005) and commercial registration is currently undertaken. 3.4. Field isolations and phenotypic characterization under controlled conditions Initially, a collection of P. fluorescens with 700 bacterial strains was established. They were isolated from the rhizosphere of field-grown birdsfoot trefoil plants, collected from different agro-ecological regions in Uruguay. In vitro assessment of antagonism against plant pathogens and screening for antimicrobial compounds were performed. The presence of genes for antibiotic biosynthesis was also investigated (Bagnasco et al., 1998; De La Fuente et al., 2004). Three selected P. fluorescens strains, UP61, UP143 and UP148, demonstrated in vitro antagonism and were able to protect birdsfoot trefoil from the infection caused by Pythium ultimum and Rhizoctonia solani in vivo under controlled conditions (Bagnasco et al., 1998). Hydrogen cyanide and fluorescent siderophore production were detected among the factors possibly involved in their biocontrol activity (Bagnasco et al., 1998). P. fluorescens UP61 produces the antibiotics DAPG, pyoluteorin and pyrrolnitrin and appears to be very similar to the well-known biocontrol strains P. fluorescens CHA0, isolated from tobacco roots in Switzerland and P. fluorescens
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Pf-5, isolated from cotton roots in the US (De La Fuente et al., 2004), whereas P. fluorescens UP148 produces a phenazinederivative antifungal compound not previously described (Bajsa et al., 2005). The interaction of P. fluorescens UP61, UP143 or UP148 with rhizobial strains used locally as commercial inoculants was also assessed. In growth chamber conditions, birdsfoot trefoil and alfalfa seed inoculation with Pseudomonas strains did not affect different parameters of host-rhizobium symbiosis, as observed in plant dry weight, nodulation rate, biological N2 fixation efficiency and rhizosphere colonization (Bagnasco et al., 1998; De La Fuente et al., 2002). A second collection of 702 native P. fluorescens strains, isolated from the rhizosphere of field-grown alfalfa plants, was established later on. A growth chamber in vivo assay was developed to screen the fluorescent Pseudomonas isolates for their ability to suppress disease and promote plant growth in the alfalfa-Pythium pathosystem, under controlled conditions (Yanes et al., 2005). When challenged with Pythium debarianum, a wide response in terms of disease suppression ability was found among Pseudomonas isolates. Twelve percent of the screened isolates protected alfalfa plants, showing an emergence of over 60% as compared to 33% emergence recorded in the non-inoculated control treatment (Yanes et al., 2005). A similar procedure in the absence of the pathogen was used to evaluate alfalfa growth-promoting ability of selected Pseudomonas strains as shown by biomass weight. Five P. fluorescens strains, aC119, aP271, aP388, aT633 and aT688, which showed the ability to suppress disease and promote plant growth, were selected to be further tested under field conditions (Yanes et al., 2005). 3.5. Evaluation of control efficacy in field trials Over the past several years, experiments have been conducted under field conditions to evaluate the ability of P. fluorescens UP61, UP143 and UP148 to suppress seedling diseases on alfalfa and birdsfoot trefoil (Bajsa et al., 2005; Pe´rez et al., 2000; Quagliotto et al., 2009). Combinations of different years, locations and sowing dates resulted in twenty environments for each crop. The P. fluorescens strains successfully colonized alfalfa and birdsfoot trefoil roots at adequate densities for biocontrol activity. Results demonstrated that bacterial seed inoculation provided a 10e13% increase in the number of alfalfa plants established relative to the control, while in birdsfoot trefoil the increase ranged from 6 to 10% (Quagliotto et al., 2009). In the presence of biocontrol strains, the above-ground biomass was increased by 15e18% and 6e10% in alfalfa and birdsfoot trefoil, respectively. Our results confirmed that an adequate stand of plants is initially required to forward the productive potential of the pasture (Quagliotto et al., 2009). 3.6. Development of bacterial inoculants Laboratory assays were performed to identify culture media for adequate biomass production of P. fluorescens on an
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industrial scale using commercially available carbon and nitrogen sources. Sterile peat was assessed as a carrier for formulating the bacterial inoculant following rhizobial inoculant technology. Thus, P. fluorescens and rhizobia strains survived at 109 and 1010 CFU/g, respectively, in sterile peat inoculated with each bacterial species, when stored at 4 C during 1 year (Bagnasco et al., 1998; De La Fuente et al., 2002). Based on the strengths of already developed rhizobial inoculant technology, research has focused on the commercial development and agronomic performance of biological control agents. The key to the success of the Uruguayan biological N2 fixation system has been implementation of a national government-supported strategy where regulatory authorities are sustained by appropriate legislation on inoculant registration, quality control and usage (Brockwell and Bottomley, 1995; Lupwayi et al., 2000). High quality standards of rhizobial inoculants are achieved using sterile peat carrier as well as liquid formulations, with mandatory high numbers of viable bacteria in the packages (2 109 rhizobia/g peat) (Lupwayi et al., 2000). As a result of research and extension policies, farmers have adopted the inoculation technology to an outstanding extent of 100%. 3.7. Final remarks The attainment of well-structured scientific knowledge for developing biocontrol strategies has been demonstrated worldwide. However, scale up, formulation, commercial production, quality control issues and agronomical use remain a challenge. Some actions must be strengthened on a global scale to recognize the ecology of forage legume microbes as a key tool for developing sustainable agricultural systems: (i) establishment of regulatory legislation for registration and use of biocontrol agents, (ii) risk assessment for human health and environment, (iii) investment in research facilities, (iv) recruitment and training of human resources, (v) support of technological ventures between the public and private sector, (vi) strengthening of international cooperation for collaborative research, and (vii) education and extension policies for use by farmers. 4. Case study: biological control of cocoyam root rot disease 4.1. Cocoyam root rot disease Cocoyam (Xanthosoma sagittifolium) is a member of the Araceae family and a staple food for more than 200 million people in the tropics and subtropics. This subsistence crop is widely cultivated in West Africa, Puerto Rico, The Dominican Republic, Cuba, Oceania, and Southeast Asia. It is mainly grown for its tubers, but the leaves can also be eaten. Production of cocoyam is greatly impaired by cocoyam root rot disease, which has been reported in Central America, West Africa and Asia. Symptoms include rotting of the cormels, stunting of the plant and yellowing of the leaves.
Yield reductions up to 90% have been reported in some plantations in Cameroon (Adiobo et al., 2007). The causal agent of cocoyam root rot disease has been identified as Pythium myriotylum, but isolates of P. myriotylum that infect cocoyam are distinct from P. myriotylum isolates from other crops and have developed a certain degree of host adaptation (Perneel et al., 2006). The name P. myriotylum var. araceae has been proposed to distinguish these isolates (Tambong et al., 2006). In Cameroon, there are two types of cultivated cocoyam varieties. The white cocoyam is the main cultivated variety, because of its early maturation and high yield, but is very susceptible to cocoyam root rot disease. The red cocoyam has a certain degree of field tolerance against cocoyam root rot disease, but is less frequently grown because of its long maturation process. In addition, there is a wild yellow variety which is resistant to the disease, but does not produce tubers. Control of cocoyam root rot disease is difficult because fungicides are not fully effective, and breeding for resistance is hampered because of ploidy incompatibility between the resistant yellow cocoyam variety and susceptible varieties. That is why alternative control strategies were studied, such as the use of suppressive soils (Adiobo et al., 2007), locally produced composts (Adiobo et al., unpublished) and biological control with fluorescent pseudomonads (Perneel et al., 2007). 4.2. P. aeruginosa is an effective biocontrol strain against P. myriotylum P. aeruginosa PNA1 was isolated from chickpea roots in India and appeared to be an effective biocontrol agent against Fusarium wilt on chickpeas and pigeonpeas and Pythium splendens damping-off on beans (Anjaiah et al., 1998, 2003). P. aeruginosa PNA1 also effectively controlled P. myriotylum root rot on cocoyam (Tambong and Ho¨fte, 2001). P. aeruginosa PNA1 produces the phenazine antibiotics phenazine-1carboxylate (PCA) and phenazine-1-carboxamide (PCN) and rhamnolipid biosurfactants. Perneel et al. (2008) showed that these phenazine antibiotics and rhamnolipids act synergistically in biocontrol of P. myriotylum on cocoyam and P. splendens on beans. A rhamnolipid-deficient mutant and a phenazine-deficient mutant were used either separately or jointly in plant experiments. When the mutants were used separately, no disease suppressive effect was observed. When the mutants were concurrently introduced into the soil, biocontrol activity was completely restored and even exceeded the biocontrol efficacy of the parental strain PNA1. The rootcolonizing capacity of the rhamnolipid mutant was impaired in comparison with the wild type strain or the phenazine mutant. Microscopic analysis revealed substantial vacuolization and leakage of P. myriotylum hyphae after incubation in liquid medium amended with both rhamnolipids and phenazines. Neither phenazines nor rhamnolipids alone were able to induce such significant damage to the hyphae. It remains to be investigated whether the production of biosurfactants can increase the biocontrol efficacy of phenazines in other biocontrol Pseudomonas strains.
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4.3. Targeted screening for alternative pseudomonads Since P. aeruginosa is an opportunistic human pathogen, we started targeted screening for alternative pseudomonads producing both phenazines and biosurfactants that could replace P. aeruginosa PNA1. Fluorescent Pseudomonas strains were randomly isolated from the rhizosphere of healthy white and red cocoyam plants appearing in naturally infested fields in Cameroon. All selected Pseudomonas isolates were screened for in vitro antagonism against P. myriotylum in dual cultures. Bacterial colonies that were able to induce an inhibition zone on agar medium were considered to be producers of diffusible antibiotics and were selected for further characterization. Biosurfactant production was tested using the drop collapse technique, and phenazine production was evaluated by thinlayer chromatography and confirmed by HPLC using P. aeruginosa PNA1 as a positive control. SDS-PAGE profiles of cocoyam rhizosphere isolates were compared with profiles of well-characterized strains of different Pseudomonas species present in the database of the Laboratory of Microbiology at the Ghent University. The majority of the isolates of the red and white cocoyam belonged to the “Pseudomonas putida” species complex as defined by Anzai et al. (2000). Antagonistic strains could only be retrieved from the rhizosphere of the red fieldtolerant cocoyam variety. They showed weak similarity with validly described Pseudomonas species and BOX-PCR revealed that they were closely related, implying they may represent a novel Pseudomonas species. Antagonistic strains produced phenazine antibiotics (PCA, PCN or 1-hydroxyphenazine). Only PCN producers also produced biosurfactants which appeared to be cyclic lipopeptides. One of the antagonistic strains, Pseudomonas CMR5c, not only produced PCA and PCN, but also hydrogen cyanide, pyrrolnitrin and pyoluteorin. Pseudomonas CMR5c and Pseudomonas CMR12a, another phenazine and biosurfactant-producing strain, were further tested for their disease suppressiveness against cocoyam root rot disease and their effectiveness was compared with the biocontrol efficacy of P. aeruginosa PNA1. While P. aeruginosa reduced root rot severity by 48%, Pseudomonas CMR5c and CMR12a suppressed root rot severity by 53% and 60%, respectively (Perneel et al., 2007, 2008). Interestingly, the well-known phenazine-producing strain P. chlororaphis PCL1391 (Chin-A-Woeng et al., 1998) was also very effective in controlling cocoyam root rot disease (Perneel, 2006). We hypothesize that the red cocoyam may have evolved a strategy of stimulating and supporting specific groups of antagonistic microorganisms as a first line of defense against P. myriotylum. This hypothesis is supported by the observation that red cocoyam is as sensitive to cocoyam root rot disease as white cocoyam under sterile conditions (Perneel et al., 2007). 4.4. Role of phenazines and biosurfactants produced by Pseudomonas CMR12a in biocontrol of P. myriotylum on cocoyam Among the antagonistic strains, Pseudomonas CMR12a was selected because of its combined production of
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phenazines (PCA and PCN) and cyclic lipopeptide (CLP) biosurfactants and its excellent in vivo biocontrol of P. myriotylum. Site-specific mutants of CMR12a in phenazine and/or CLP biosynthesis were constructed and used in infection experiments with cocoyam. The effectiveness of the single mutants appeared to be substrate-dependent, indicating that soil texture can have a profound effect on metabolite production, while mutants impaired in both phenazine production and CLP lost the ability to suppress the cocoyam root rot in all substrates (Jolien D’aes, unpublished observations). 4.5. Practical use of biocontrol in Cameroon The conventional approach in which Pseudomonas CMR12a and CMR5c are developed into a commercial product will not be very useful for the Cameroon subsistence farmer who has no means to buy expensive (bio)pesticides. Instead, we emphasize developing a strategy based on indigenous resources. Field observations learned that the cocoyam root rot disease incidence is negatively correlated with the amount of organic matter in soil (Adiobo et al., 2007). In Cameroon, readily compostable plant residues are abundantly available from the various industrial plantations including banana, oil palm and sugar cane plantations. Incorporation of composts based on oil palm waste significantly reduced cocoyam root rot disease in the field (Adiobo et al., unpublished). In addition, red cocoyam alternately planted with white cocoyam may stimulate phenazine-producing bacteria in soil and may thus protect the white cocoyam from fungal attack (Perneel, 2006). 5. Conclusions Within the group of fluorescent pseudomonads, very effective biocontrol agents can be found that improve plant health and plant growth. Biocontrol capacities, however, are isolate-dependent and up to now cannot clearly be linked to taxonomic groups. Isolate screening remains important for identifying biocontrol strains that are effective on local crops under local environmental conditions. This can be done by testing large collections of preferentially local isolates for disease suppression or plant-growth-promoting or by targeted screening for organisms that produce desirable secondary metabolites. Isolate screening in Uruguay and Cameroon has revealed new Pseudomonas species which produce wellknown antibiotics, as is the case for Pseudomonas CMR5c and CMR12a from Cameroon; biocontrol agents very similar in phylogeny and metabolite production to well-known Pseudomonas biocontrol agents from other countries such as P. fluorescens UP61 from Uruguay; and Pseudomonas strains that produce new derivatives of well-known antibiotics such as phenazine-producing P. fluorescens strain UP148. In screening and isolation of these effective Pseudomonas biocontrol agents, access to reference strains from public and private collections has proven to be essential for identification, phylogenetic studies and metabolite characterization.
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