INTRODUCTION

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The search for alternatives to chemical control of plant pathogens, such as biological control, has gained momentum in recent years. Emergence of fungicide-resistant pathogens, health concerns for producer and consumer, and the phasing out of chemicals such as methyl bromide have prompted research into viable alternative practices to achieve more sustainable levels of agricultural production. Since the 1980s rhizobacteria and other microorganisms have been investigated as possible replacements for chemicals used to control a broad range of plant diseases. Isolates from the genus Pseudomonas have been tested due to their widespread distribution in soil, ability to colonize the rhizospheres of host plants, and ability to produce a range of compounds antagonistic to a number of serious plant pathogens (3, 13, 20, 24, 30).

One of the difficulties in developing microorganisms as viable alternatives to chemical control is that many biological control agents are found to be active only in certain soil types. The physical and chemical nature of soils varies greatly between agricultural regions; factors such as soil texture, organic matter, pH, water and oxygen availability, and competition for nutrients with indigenous microflora may significantly dampen the biological activity of introduced inocula. It has previously been demonstrated that an effective biological control strain isolated from one region may not perform in other soil and/or climatic conditions (5, 10, 16, 31). For this reason, one of the important factors to be considered when screening new isolates is their activity in the range of environments in which they would be expected to be used, in particular different soil types. New species of plant-associated rhizobacteria may provide potentially new biological control agents with novel mechanisms of disease suppression active in a range of environments.

Two new species of Pseudomonas isolated from soil samples in France have recently been described in the literature (1). Pseudomonas brassicacearum was isolated from the rhizospheres of plants belonging to the family Brassicaceae, while Pseudomonas thivervalensis was isolated from the rhizosphere of Arabidopsis in the Thiverval-Grignon region (1). Isolation of these new species from soil samples was undertaken by immunotrapping utilizing a polyclonal antibody raised against P. brassicacearum or by plating on selective media (1). P. brassicacearum and P. thivervalensis are discriminated from other Pseudomonas species based on unique restriction profiles generated by amplified 16S ribosomal DNA (rDNA) restriction analysis (ARDRA). These two new species are subsequently distinguished from each other based on isoelectric focusing of pyoverdines (1). Pseudomonas corrugata, a species closely related to the newly described pseudomonads, may also be isolated by immunotrapping with the same polyclonal antibody (2). Despite this cross-reaction, previous results have demonstrated that ARDRA profiles generated by TaqI digestion were able to distinguish between P. corrugata and both P. brassicacearum and P. thivervalensis (8).

In vitro trials with these isolates have demonstrated that strains of the two new species were able to antagonize a range of pathogenic fungi (8), including Gaeumannomyces graminis (Sacc.) von Arx and Olivier var. tritici the causative agent of take-all, a serious disease of wheat and barley. These results suggested that these new species might be useful as biological control agents against take-all if in vitro antagonism can be matched with disease control in soil.

The purpose of this study was to isolate P. brassicacearum and P. thivervalensis from wheat field soils in South Australia using a polyclonal antibody raised against P. brassicacearum (1). Soils from two sites where wheat monoculture has been practiced for many years, including a calcareous take-all suppressive soil, were selected for sampling. A take-all suppressive soil was included in the sampling, as potential biological control agents are often isolated from soils where the pathogen is present but disease symptoms are not observed on susceptible crops (7). The phenomenon of take-all decline in wheat monoculture, where the incidence of disease diminishes over time, has been attributed principally to biological factors, predominantly indigenous microflora suppressing the pathogen and thus limiting the incidence of disease (23). Isolates obtained by immunotrapping and plating on a semiselective medium were taxonomically characterized by ARDRA and siderophore typing as well as BIOLOG and gas chromatography-fatty acid methyl ester (GC-FAME) analyses. These isolates were then tested for in vitro antagonism of G. graminis var. tritici, and selected strains were subsequently tested for their ability to suppress take-all symptoms on wheat in two contrasting soil types.

	MATERIALS AND METHODS

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Strains and culture conditions. The type strain for P. corrugata, ATCC 29736 (27), was utilized in this study. Two novel pseudomonads, P. brassicacearum CFBP 11699 and P. thivervalensis CFBP 11261, were described previously (1). P. corrugata strain 2140 was isolated from wheat field soil in New South Wales and has previously been demonstrated to be a biological control agent against take-all of wheat (25). Pseudomonas fluorescens Pf5 was obtained from C. Howell, College Station, Tex. Bacterial field isolates obtained in this study that were subsequently selected for the pot trial are described in Table 1. G. graminis var. tritici isolate 8 was collected by H. McDonald from Avon, South Australia, in 1979. 

Isolation of field strains. Wheat field soils collected from Avon (34°14'S,138°19'E) and Kapunda (34°21'S,138°54'E) were selected for isolating Pseudomonas strains from both bulk soil and wheat rhizosphere. Avon soil is a calcareous sandy loam classified as a fine mixed thermic Calcic Palexeralf (29), pH 8.4 (CaCl₂). Kapunda soil is a red-brown earth classified as a fine mixed thermic Natrixeralf (29), pH 5.5 (CaCl₂). Soil samples were obtained from random sites at both field sites. Wheat seeds (cv. Stiletto) were surface sterilized sequentially with 2% calcium hypochlorite and 10% H₂O₂ and sown into the soil samples in 300-ml pots. After 4 weeks of incubation at 15°C, plants were harvested and the roots were separated from shoots. Bulk soil and rhizosphere soil (soil loosely clinging to roots) samples were serially diluted in sterile distilled water. Rhizoplane isolates were obtained by macerating washed roots with a mortar and pestle prior to serial dilution as described above. Bacterial isolates were obtained either by plating samples on RCS semiselective agar medium (2) or by immunotrapping in 96-well microtiter plates, utilizing a polyclonal antibody against P. brassicacearum by methods previously described (2).

ARDRA. 16S rDNA primers rD1 and fD1 (32) were used to amplify a 1.6-kb internal region of the 16S rRNA gene. Genomic DNA was obtained by freezing 1.5 ml of an overnight culture grown in LB broth in liquid nitrogen and then rapid thawing in a 50°C water bath prior to phenol-chloroform extraction and washing in ice-cold 70% ethanol, drying, and subsequent suspension in 50 µl of TE8 (26). The PCR solution comprised, per 50-µl reaction mixture, 1 U of Taq polymerase (Promega), 5.0 µl of reaction buffer (Promega), 0.3 µg each of primers rD1 and fD1, 1.0 µl of genomic DNA, final concentrations of 1.25 mM MgCl₂ (Promega), and 0.25 mM deoxynucleoside triphosphates (Promega); the volume was taken to 50 µl with autoclaved ultrapure water. The cycles used were as follows: 1 cycle at 94°C for 4 min; 35 cycles at 94°C for 1 min, 55°C for 2 min and 72°C for 2 min; and one cycle at 72°C for 3 min. Amplification was undertaken in a PTC-100 Thermal Controller (M.J. Research, Watertown, Mass.). This amplified 16S rDNA was digested with six different restriction endonucleases, AluI, CfoI, HaeIII, HinfI, RsaI, and TaqI (Boehringer Mannheim), as per the manufacturer's instructions. Profiles of digested 16S rDNA were separated by gel electrophoresis using a 3.0% agarose gel (Nusieve; FMC BioProducts) in 0.5× Tris-borate-EDTA buffer at 5.0 V cm¹ (26).

BIOLOG analysis. Field isolates were assessed for their ability to metabolize 95 carbon substrates using the BIOLOG GN Microtiter system. Isolates grown in LB broth were washed and resuspended in 0.85% (wt/vol) NaCl solution. The A₅₅₀ was adjusted to 0.25, 150 µl was added to each microtiter well, and the plates were incubated at 28°C for 24 h. Color development was assessed using the associated Microlog 2 computer software (BIOLOG Inc., Hayward, Calif.).

GC-FAME. Bacterial isolates were subcultured twice on tryptic soy agar (Difco) for 24 h at 28°C prior to fatty acid extraction and methylation according to the Microbial Identification System procedure (MIDI Inc, Newark, Del.). Extracted samples were analyzed with a Hewlett-Packard S5890 Series II gas chromatograph. Fatty acid peaks were identified by the Microbial Identification System version 4, and profiles were compared to the Sherlock TSBA Library version 3.80 (Microbial ID; MIDI Inc.).

Fluorescence on King's B agar medium. The ability of isolates to produce fluorescent siderophores was tested by plating bacteria on King's medium B (18) and incubating for 2 days at 25°C. Plates were then inspected under 366-nm UV light, and fluorescence was compared visually to those of P. fluorescens Pf5 (15) and P. corrugata strain 2140 as positive and negative controls respectively.

Isoelectric focusing of pyoverdines. Strains were grown in CAA medium, comprising, per liter, Bacto Casamino Acids (Difco) (5 g), K₂HPO₄ · 3H₂O (1.54 g), and MgSO₄ · 7H₂O (0.25 g) at 25°C for 48 h. After centrifugation to pellet cells, the cell-free supernatants were then concentrated 20-fold by lyophilization. Isoelectric focusing of pyoverdines was undertaken by the method of Koedam et al. (19) with 1.5 µl of concentrated supernatant. Bands corresponding to specific pyoverdines were visualized under UV light (1).

Biological control of take-all in planta. Field isolates were tested for in vitro antagonism of G. graminis var. tritici on half-strength potato dextrose agar at 25°C. Measurements of zones of inhibition between the edge of the bacterial colony and a growing hyphal mat of G. graminis var. tritici were taken after 3 to 4 days of incubation, and the average was obtained from three replicates per isolate.

	RESULTS

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Isolation of pseudomonads from field soils. Two hundred isolates were obtained from Avon soil and 118 isolates were obtained from Kapunda soil by immunotrapping or semiselective plating. Approximately 50% of the isolates were obtained by each method. Isolates were obtained from bulk soil as well as the rhizoplanes and rhizospheres of wheat seedlings grown in soil taken from both sites. More than 95% of the field strains obtained were isolated from either the rhizospheres or rhizoplanes of the wheat plants. Isolates were designated with a strain number and an alphabetical prefix denoting the site from which it was obtained (A for Avon and K for Kapunda).

Phenetic characterization. Forty-two field isolates selected at random but representing each field site and environmental niche (source location) were analyzed by ARDRA (Table 1). Digestion of amplified 16S rDNA with six endonucleases revealed eight different groups of ARDRA patterns after hierarchical cluster analysis by Nei's genetic similarity statistic (22) (Fig. 1; Table 2). Six of these groups contained more than one representative isolate. P. corrugata type strain ATCC 29736 was distinguished from the type strains of the other two species by digesting the 16S rDNA PCR product with TaqI, as reported by Degraeve (8). P. corrugata strain 2140 was found to be different from the P. corrugata type strain based on DNA fragment profiles obtained from HaeIII- and TaqI-digested 16S rDNA.

View larger version (39K): [in this window] [in a new window]   FIG. 1.   (A) ARDRA banding patterns of amplified 16S rDNA. A total of 47 strains, including 42 isolates obtained from Avon and Kapunda soils, were analyzed. Molecular marker X (Boehringer Mannheim) was used to determine fragment lengths. Different banding patterns generated by single enzyme digestion are designated A, B, and C, except for RsaI, where only one pattern was observed for all isolates except A119 (see Table 2). (B) Results of cluster analysis of the ARDRA patterns.

View larger version (23K): [in this window] [in a new window]   FIG. 2.   Frequency of ARDRA groups at each sampling site. Frequencies were obtained by dividing the number of isolates from each site represented in a particular group by the total number of isolates tested from that site. Most isolates analyzed were found to be either identical to P. brassicacearum and P. thivervalensis (group 8) or closely related to P. corrugata (group 1). Four groups contained isolates from a single site only; the total number of isolates in each of these groups ranged from one (groups 6 and 10) to three (group 2) (see Table 1 for details of isolate numbers).

View larger version (13K): [in this window] [in a new window]   FIG. 3.   BIOLOG and GC-FAME dendrograms of Pseudomonas isolates from Avon and Kapunda soils. BIOLOG analysis revealed that most isolates were clustered in group K (32% of total isolates tested) or groups M1 and M2 (24 and 26%, respectively). When compared to GC-FAME data, 82% of BIOLOG group K isolates were clustered in GC-FAME group Q2, while 75% of BIOLOG group M1 and M2 isolates were clustered in GC-FAME group P2. The relationship of these groups to the ARDRA groups is included in Table 1.

Biological control. In vitro antagonism experiments with G. graminis var. tritici revealed that 86% of the 42 isolates tested demonstrated detectable antifungal activity. Eleven isolates, six from Avon and five from Kapunda, consistently induced reproducible zones of fungal inhibition on half-strength potato dextrose agar medium (Table 1); these were selected for the pot trial. Two ARDRA group 8 isolates from Avon, A134 and A526, were the most antagonistic against the take-all fungus, while the other isolates displayed various degrees of antifungal activity.

	DISCUSSION

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As P. corrugata is closely related to P. brassicacearum and a polyclonal antibody raised against the new species will cross-react with P. corrugata, it was expected that a proportion of the field isolates obtained by immunotrapping would be shown to be more closely related to the P. corrugata type strain than to P. brassicacearum or P. thivervalensis. In this study, almost 35% of the field isolates characterized by ARDRA were closely related to P. corrugata. Overall, more than 80% of the 42 isolates examined in this study showed >90% homology to either P. corrugata or P. brassicacearum-P. thivervalensis, and all 42 isolates showed >85% homology to the type strains.

In the original study describing P. brassicacearum and P. thivervalensis, it was found that these species were regularly isolated from the rhizospheres of Brassica napus and Arabidopsis thaliana in a range of soil types in France (1). This report shows that P. brassicacearum and P. thivervalensis are present in at least two distinct geographic locations and in dissimilar soils, implying that these new species may be widespread in Australia. Furthermore, the presence of these new species in soils subjected to wheat monoculture and their ability to be isolated from the rhizospheres of wheat seedlings suggest that they may be able to colonize a broad range of host plants, increasing the likelihood that they will be found in a range of environments.

P. brassicacearum and P. thivervalensis were originally isolated from the rhizospheres of rape and Arabidopsis (1). Taken together with the data from this study of wheat field soil isolates, the results suggest a close interaction between these new species of rhizobacteria and host plants. The high proportion of isolates obtained from the rhizospheres and rhizoplanes of wheat roots compared to bulk soil (95 and 97% for Avon and Kapunda soils, respectively) observed in this study provides further evidence of a possible plant-microbe relationship. It is yet to be established what the overall nature of these relationships is, i.e., whether these species are beneficial or benign or if there are plant-deleterious strains of these species. Studies with these new species have yet to establish whether they have evolved with and play a role in the growth of commercially important crops.

Comparison of BIOLOG results indicated a >90% homology between the P. brassicacearum and P. thivervalensis type strains from France and the field isolates obtained from Australian soils. It was interesting that the differences in carbon utilization between the French and Australian isolates were identical. This may be an evolutionary factor whereby geographically separate strains of the same species isolated from the rhizospheres of different host plants have acquired the capacity to metabolize different carbon compounds. It has been shown that root exudates and lysates from different host plants can influence the structure of rhizobacterial populations in the short term (12). Over a longer period, however, the influence of different root-derived carbon compounds may influence the genetic characteristics of individual species. Another explanation for this observation is that different root exudates from distinct host plants exert a selective pressure in the rhizosphere, favoring isolates with specific carbon utilization pathways that are able to exploit the differing ranges of exudates and lysates.

Results of in vitro antagonism assays have demonstrated that, like other biological control agents, P. brassicacearum and P. thivervalensis are capable of producing antifungal metabolites (reference 8 and this study). This study has further shown that some isolates of the new species are useful in controlling take-all in planta. A P. thivervalensis isolate obtained from Kapunda soil, K208, exhibited disease suppression in both Kapunda and Avon soils. This may be due to a number of factors, including in planta antifungal metabolite production or possible systemic induced resistance. Other mechanisms, such as rhizosphere exclusion or competition for nutrients, may also play a role in biological control of take-all.

An important factor to emerge from the pot trial was the detection of biological control rhizobacteria active in the two contrasting soil types. Take-all is a serious problem in alkaline soils (6), and the disease is still a threat to productivity in these soil types throughout southeastern Australia (4, 21). Previous attempts to isolate bacterial strains that can control take-all on wheat in calcareous sandy soils have been unsuccessful. A number of soil factors could contribute to this, including high pH, causing low survival and persistence of the inocula, and lack of expression of certain biocontrol-related genes in calcareous soil. In this study, strain K208 showed promise as a biological control agent. This strain reduced symptoms of take-all in both soil types, an important finding for the development of commercially viable biological control strains. Further tests in other soils, either as a single inoculant or in combination with other biocontrol isolates such as Bacillus and Trichoderma, need to be undertaken to ascertain the full potential of this isolate.

During the final 2 weeks of the pot trial, water consumption (as measured by changes in pot weight) was monitored. G. graminis var. tritici infests wheat through infection of the seminal roots, where thin-walled fungal microhyphae penetrate root cell walls through the production of cellulase and pectinase and subsequent colonization of vascular tissue (9, 33). Obstruction of the stele results in decreased nutrient flow in the plant, eventually leading to premature ripening (white heads with shriveled grain). Although measuring and rating roots and lesions still constitute the primary means for assessing disease severity, this is a time-consuming and generally destructive procedure that can be undertaken only after harvesting plants. Our results have shown a significant negative correlation between take-all-induced root lesions and the ability of plants to take up water (and associated soil nutrients). Measurement of water uptake by plants in a nondraining system may be a viable method for nondestructive monitoring of the state of the plants in an experiment. This can be used to indicate disease severity and plant health at any time point in the trial.

In conclusion, this study has shown the presence of two newly described species of pseudomonads, P. brassicacearum and P. thivervalensis, in Australian soils. ARDRA shows them to be readily identifiable and that although they are closely related to P. corrugata, they can be distinguished from this species. Further analysis with BIOLOG and GC-FAME indicated a degree of intraspecific variation, but this was not great enough to separate isolates into species-specific groups. Some isolates of these new species demonstrated a level of biological control of take-all of wheat comparable to those of previously described strains, suggesting that they have potential for use in the field and in a greater range of soil types. The full potential can be assessed only after field trials. Further studies to elucidate the genetic variability within this species, levels of host plant adaptation, whether particular genetic groups are more efficacious as biological control agents, and the mechanisms of biological control of these species may enable further progress towards commercial biological control of take-all, particularly in calcareous soils.