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Applied and Environmental Microbiology, June 2002, p. 2943-2949, Vol. 68, No. 6
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.6.2943-2949.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Identification and Isolation of Genes Involved in Poly(ß-Hydroxybutyrate) Biosynthesis in Azospirillum brasilense and Characterization of a phbC Mutant

Daniel Kadouri, Saul Burdman, Edouard Jurkevitch, and Yaacov Okon^*

Department of Plant Pathology and Microbiology and The Otto Warburg Center for Agricultural Biotechnology, Faculty of Agricultural, Food and Environmental Quality Sciences, The Hebrew University of Jerusalem, Rehovot 76100, Israel

Received 27 November 2001/ Accepted 29 March 2002

Top Abstract Introduction Materials and Methods Results Discussion References

 
Like many other prokaryotes, rhizobacteria of the genus Azospirillum produce high levels of poly(ß-hydroxybutyrate) (PHB) under suboptimal growth conditions. Utilization of PHB by bacteria under stress has been proposed as a mechanism that favors their compatible establishment in competitive environments, thus showing great potential for the improvement of bacterial inoculants for plants and soils. The three genes that are considered to be essential in the PHB biosynthetic pathway, phbA (ß-ketothiolase), phbB (acetoacetyl coenzyme A reductase), and phbC (PHB synthase), were identified in Azospirillum brasilense strain Sp7, cloned, and sequenced. The phbA, -B, and -C genes were found to be linked together and located on the chromosome. An A. brasilense phbC mutant was obtained by insertion of a kanamycin resistance cassette within the phbC gene. No PHB production was detected in this mutant. The capability of the wild-type strain to endure starvation conditions was higher than that of the mutant strain. However, motility, cell aggregation, root adhesion, and exopolysaccharide (EPS) and capsular polysaccharide (CPS) production were higher in the phbC mutant strain than in the wild type.

Top Abstract Introduction Materials and Methods Results Discussion References

 
A wide variety of microorganisms are known to produce intracellular energy and carbon storage compounds known as poly(ß-hydroxybutyrate) (PHB) or polyhydroxyalkanoates (PHA) (44). Knowledge of the structure and organization of the PHA biosynthetic genes from a wide range of bacteria has increased as a result of intensive research for industrial applications. Various pathways have been found among different bacteria (16).

PHA biosynthetic genes and other related genes in the PHA metabolism are often clustered together within bacterial genomes, as was revealed by nucleotide sequence mapping analyses (26, 27, 28, 34).

The gram-negative nitrogen-fixing rhizobacterium Azospirillum brasilense lives in close association with plant roots, where it exerts beneficial effects on plant growth and yield of many crops of agronomic importance (22, 23). Enzymes involved in the synthesis, accumulation, and degradation of PHAs in A. brasilense have been examined in detail (36, 37, 38). It was shown that in contrast to other bacterial species, A. brasilense does not produce copolymers of hydroxyalkanoates but rather only homopolymers of PHB (14).

It has been suggested for diverse ecological systems that the accumulation, degradation, and utilization of PHAs by several bacteria under stress is a mechanism that favors their establishment, proliferation, survival, and competition, especially in competitive environments where carbon and energy sources are limiting, such as those encountered in the soil and rhizosphere (24). Understanding the role that PHAs play as internal storage polymers is of fundamental importance in microbial ecology.

The role played by PHB in the survival, proliferation, and plant root colonization of Azospirillum spp. is not yet fully known due to the lack of mutants impaired in their ability to synthesize PHB. To gain insight into the possible influences of PHB in the free-living state and in the rhizosphere, we isolated and sequenced the phbA, -B, and -C genes from A. brasilense strain Sp7. We also report on the construction and characterization of a phbC mutant strain of A. brasilense unable to synthesize PHB.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Bacterial strains, plasmid, and growth conditions.
The bacterial strains and plasmids used in this work are listed in Table 1. For Escherichia coli growth, Luria Bertani (LB) (19) media was used. A medium with a high carbon-to-nitrogen (C:N) ratio (4, 25) was used to induce the accumulation of PHB in A. brasilense. Azospirillum transconjugants were selected on minimal medium for A. brasilense (MMAB) (42).

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TABLE 1. Bacterial strains and plasmids used in this study

DNA manipulations, sequencing, and analysis.
Subcloning, transformation, and DNA extractions were carried out according to standard methods (31). DNA sequencing was carried out with an ABI Prism 377 DNA sequencer (Applied Biosystem Inc., Foster City, Calif.). Sequence data were analyzed with the University of Wisconsin Genetic Computer Group software. Homology searches were performed using the Blast network service (1). Sequence alignments were done with the Clustal W program (40) and edited using GeneDoc (K. B. Nicholas and J. B. Nicholas, Jr., GeneDoc [http://www.cris.com/~Ketchup/genedoc.shtml]).

Synthesis of oligonucleotides and PCR.
Oligonucleotide primers were synthesized (General Biotechnology, Rehovot, Israel) by the phosphoramidate method, using a Pharmacia 4-Primer Gene Assembler, according to the codon frequency for A. brasilense extracted from the Codon Usage Database compiled from the codon usage tabulated from GenBank (21). Based on known phbC sequences, two degenerate primers were designed to amplify the putative phbC fragment from total DNA of A. brasilense Sp7: phbC-R, 5'-ATC-AAC-AAG-TTC-TAY-ATC-3', and phbC-L, 5'-GTT-CCA-GTA-SAG-SAG-GTC-GAA-3' (S = G or C; Y = C or T). The primers anneal with phbC of Rhodospirillum rubrum, Azorhizobium caulinodans, and Sinorhizobium meliloti in positions 780 to 1308, 744 to 1269, and 831 to 1356, respectively. PCR was performed with an automated PCR thermoblock (Mastercycler gradient; Eppendorf, Netheler, Hamburg). The PCR product was labeled with digoxigenin (DIG)-dUTP using the PCR-DIG probe synthesis kit (Roche Diagnostics Corp.).

Southern blot analysis and hybridization.
Total DNA from A. brasilense, Azospirillum lipoferum, Azospirillum amazonense, Azospirillum halopraeferens, Azospirillum irakense, and Azospirillum doebereinerae was isolated, digested with restriction enzymes, electrophoresed, and blotted onto MSI nylon transfer membranes (Roche Diagnostics Corp.) by standard methods (31). Prehybridization and hybridization were carried out at 68°C and detection was performed with the DIG luminescent detection kit (Roche Diagnostics Corp.)

Construction of A. brasilense Sp7 phbC::Km mutant.
The Tn5-derived 1.4-kb HincII kanamycin (Km) resistance cassette of pUCA800 was inserted in the unique SmaI restriction site of pPEB1.2 to yield pPEBKm2.6. The 2.6-kb phbC::Km EcoRI fragment of pPEBKm2.6 was subsequently cloned into the EcoRI site of the suicide vector pSUP202 to yield pSPEAKm. E. coli S17.1 was transformed with pSPEAKm, which was further mobilized to A. brasilense Sp7 through biparental mating. Azospirillum transconjugants were selected on MMAB supplemented with Km. Km-resistant clones were isolated. Southern blot hybridization, DNA sequencing, and PCR screening with specific oligonucleotides against either the 1.2-kb phbC fragment of pPEB1.2 or the 1.4-kb HincII Km resistance cassette of pUCA800, showed the correct genetic configuration. One of these A. brasilense Sp7 phbC::Km mutants was used for further analysis.

PHB production.
PHB conccntration was assayed by gas chromatography (2), using PHB from Alcaligenes (Sigma) as a standard and wild-type Sp7 as a control.

Transmission electron microscopy.
Bacteria were embedded in a low-viscosity epoxy resin as described by Burdman et al. (3). Thin sections were cut with a diamond knife on a 8800 ultramicrotome (LKB, Stockholm, Sweden) and examined on a JEM 100-CX transmission electron microscope (JEOL, Tokyo, Japan).

Starvation experiments.
Overnight cultures of A. brasilense (2-ml aliquots) were used to inoculate flasks containing 25 ml of high-C:N-ratio medium. After 24 h of growth, bacteria were collected and washed twice by centrifugation at 4,000 x g for 10 min in 0.06 M potassium phosphate buffer (pH 6.8). Bacteria were then resuspended in the same buffer and incubated on a shaker at 170 rpm for 12 days under starvation conditions (38). Bacterial density (CFU/milliliter) was further determined by dilution plating.

Adhesion assay of bacteria to intact roots.
Seeds of sweet corn (cv. Jubilee) and wheat (cv. Atir) were surface-sterilized for 2 min in 95% ethyl alcohol, followed by 1 min in 1% sodium hypochlorite, and then washed five times with sterile distilled water. An A. brasilense overnight culture (1 ml) was used to inoculate flasks containing 0.4 or 0.2 g of freshly harvested 1-week-old roots of sweet corn and wheat seedlings, respectively, in 5 ml of 0.06 M potassium phosphate buffer (pH 6.8). After 2 h of incubation on a rotary shaker (170 rpm) at 30°C, the roots were washed three times by immersion in 0.06 M potassium phosphate buffer for 1 min without agitation. Buffer (6 ml) was added and the roots were washed intensively for 2 min by vortex. The supernatant was taken to determine the amount of bacteria attached to the roots. The percentage of adhesion was estimated as follows: % adhesion = [(CFU_t - CFU_s) x 100%]/CFU_t; with t and s being total and supernatant, respectively.

Extraction of extracellular polysaccharides.
Polysaccharides were extracted from a 48-h culture (3, 7). The amounts of sugar in the polysaccharide fractions were evaluated by the anthrone method, using glucose as the standard (9).

Bacterial dry weight.
Cell dry weight per milliliter of culture broth was determined by weighing dry cells with a microbalance (Mettler, Zurich, Switzerland) as described by Wang and Lee (43).

Nucleotide sequence accession number.
The sequences of A. brasilense corresponding to ß-ketothiolase, acetoacetyl-CoA reductase, PHB synthase, and acyl-CoA dehydrogenase genes were submitted to GenBank (accession no. AF353206, AY046923, AF353205, and AY046924, respectively).

Top Abstract Introduction Materials and Methods Results Discussion References

 
Cloning and sequence analysis of A. brasilense Sp7 phbABC.
Two degenerate primers were designed based on the phbC sequences of R. rubrum, A. caulinodans, and S. meliloti. Using total DNA of A. brasilense Sp7 as the template in PCR, a 500-bp product, homologous to known phbC genes, was obtained. Following sequencing, specific oligonucleotide primers were synthesized and used for PCR screening of an E. coli HB101 cosmid library containing partially EcoRI-restricted total DNA of A. brasilense strain Sp7 in pLAFR3. A clone containing a 20-kb A. brasilense DNA fragment including the region encoding PhbC was isolated. This DNA fragment was subcloned in pUC19 and pUC18, resulting in clones pP2EP5, pP13S2, and pP13S8.

The above subclones were sequenced by primer walking, and four reliable open reading frames (ORFs) were detected. The deduced amino acid sequence of the first ORF exhibited high similarity with an internal part of the PhbC proteins of R. rubrum (GenBank accession no. CAB65395; 69% identity, 83% similarity), A. caulinodans (GenBank accession no. CAA06928; 55% identity, 71% similarity), Mesorhizobium loti (GenBank accession no. BAB48418; 53% identity, 71% similarity), S. meliloti (GenBank accession no. AAC61899; 52% identity, 70% similarity), and R. eutropha (GenBank accession no. AAB24910; 44% identity, 69% similarity). Multiple alignment of the deduced amino acid sequence of the phbC from A. brasilense strain Sp7 with homologous phbC genes from other bacteria is shown in Fig. 1. The second ORF showed high similarity with an internal part of the PhbA proteins of Pseudomonas aeruginosa (GenBank accession no. AAG06328; 64% identity, 78% similarity), Bacillus halodurans (GenBank accession no. BAB07206; 49% identity, 64% similarity), and S. meliloti (GenBank accession no. CAC49901; 46% identity, 58% similarity). The third ORF showed similarity with an internal part of PhbB of Bacillus subtilis (GenBank accession no. CAB15273; 39% identity, 55% similarity) and Staphylococcus aureus (GenBank accession no. AAK51157; 30% identity, 48% similarity). Finally, the fourth ORF showed high similarity with an internal part of the acyl coenzyme (acyl-CoA) dehydrogenase proteins of P. aeruginosa (GenBank accession no. AAG03896; 57% identity, 70% similarity) and M. loti (GenBank accession no. BAB52037; 54% identity, 71% similarity). This protein is involved in fatty acid metabolism. The A. brasilense ORFs were therefore designated phbA, -B, and -C and fadE.

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FIG. 1. Multiple alignment of the deduced amino acid sequence of phbC of A. brasilense (accession no. AF353205) with corresponding sequences of phbC from R. rubrum (accession no. CAB65395), A. caulinodans (accession no. CAA06928), S. meliloti (accession no. AAC61899), M. loti (accession no. BAB48418), and R. eutrophus (accession no. AAB24910). Three levels of similarity are shown according to the default settings of GeneDoc.

A physical map of the phbABC and fadE cluster was established (Fig. 2). The phbC ORF of A. brasilense is putatively 1,854-bp long and is transcribed in a direction opposite to the other three ORFs. The putative phbA gene is 1,161-bp long, followed by the putative 2,332-bp-long phbB and the putative 1,788-bp fadE genes. Purine-rich tracts were found to precede the putative start codons of the four ORFs, as characterized for prokaryotes. Typical Shine-Dalgarno sequences were found 10 nucleotides upstream from the putative start codon of fadE and phbB and 12 nucleotides upstream of that of phbA (not shown).

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FIG. 2. Physical map of A. brasilense Sp7 phbABC and fadE region. Open arrows indicate the locations and directions of transcription of phbC, phbA, phbB, and fadE. The triangle represents the location of the insertion of the kanamycin resistance cassette in the phbC mutant strain. E, EcoRI; B, BamHI; S, SmaI.

To establish whether phbABC is located in the chromosome or in one of the A. brasilense megaplasmids (either the 115-MDa or the 90-MDa plasmid), total DNA was extracted from A. brasilense Sp7, A. brasilense 7030 (a strain lacking the 115-MDa megaplasmid), and a pooled Sp7-p90 cosmid library. PCRs were performed, using a specific oligonucleotide for the phbABC cluster. Positive reactions were observed only with DNA from A. brasilense Sp7 and A. brasilense 7030 (data not shown), suggesting that phbABC is located in the chromosome and not in one of the megaplasmids.

Southern blot hybridization.
The original 500-bp PCR product corresponding to an internal region of phbC was DIG-labeled and used in Southern blot hybridization against total DNA from other Azospirillum species digested with EcoRI (Table 1). DNA from A. brasilense Sp7 and from E. coli DH5 were used as positive and negative controls, respectively. All the Azospirillum DNA tested yielded a positive reaction, hybridizing with the phbC probe (Fig. 3), suggesting the presence of the phbC gene in all of them.

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FIG. 3. Southern blot hybridization using a DIG-labeled 500-bp long PCR-generated probe representing an internal region of the A. brasilense Sp7 phbC gene. Total DNA from A. doebereinerae (lane 1), A. brasilense Sp7 (lane 2), A. lipoferum (lane 3), A. irakense (lane 4), A. amazonense (lane 5), A. halopraeferens (lane 6), and E. coli (lane 7). The DNA was digested with EcoRI. Arrows indicate the estimated position of the putative phbC bands.

Construction and characterization of A. brasilense Sp7 phbC::Km mutant.
An A. brasilense phbC mutant was isolated following the cloning of a Tn 5-derived Km resistance cassette into the unique SmaI restriction site of pPEB1.2 (Fig. 2) and double homologous recombination into the A. brasilense Sp7 phbC locus.

No substantial differences were found between Sp7 and the phbC mutant strain in their growth curves and generation time in LB media. A. brasilense Sp7 showed a generation time of 2.1 h, whereas for the phbC mutant, the generation time was 2.2 h (Fig. 4). A few phenotypic differences between the wild type and the mutant were detected: the motility exhibited by the mutant was considerably higher than that of the wild-type strain, as observed by contrast microscopy, and the mutant strain exhibited higher aggregation after 24 h of growth in high-C:N-ratio media (Fig. 5).

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FIG. 4. Growth curves of A. brasilense Sp7 () and of the phbC mutant (•) in LB media. Bacteria were grown in a shaker (170 rpm at 30°C). Each value represents the mean of three replicates from one representative experiment. Each experiment was carried out three times, each yielding similar results.

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FIG. 5. Aggregation of A. brasilense Sp7 (A) and of the phbC mutant (B) after 24 h of growth in medium with a high C:N ratio. Bacteria were transferred to Petri dishes before the photograph was taken.

No apparent PHB was detected in the phbC mutant grown for 24 h on high-C:N-ratio medium (not shown), indicating that the phbC locus is required for synthesis of PHB. This result was confirmed by transmission electron microscopy, which showed large PHB granules in the wild type (Fig. 6A) but none in the phbC mutant (Fig. 6B).

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FIG. 6. Electron micrographs of thin sections of A. brasilense (A) and of the phbC mutant (B), grown for 24 h in medium with a high C:N ratio. The arrow indicates PHB granules. Bars, 1 µm. At least 10 sections of each strain were examined.

Starvation experiments.
Upon starvation in a phosphate buffer suspension, without exogenous carbon or energy sources in the medium, A. brasilense Sp7 survived longer than the phbC mutant strain (Fig. 7). The density of viable cells of the phbC mutant decreased from the onset of the starvation period. In contrast, strain Sp7 showed a density increase at the beginning of the starvation period, followed by a moderate decrease during the time course of the experiment.

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FIG. 7. The effect of PHB on survival capability of starved bacteria. Cells of A. brasilense Sp7 () and phbC mutant (•) were grown on medium with a high C:N ratio for 24 h and transferred to phosphate buffer, where they were incubated for 12 days. Bacterial density was determined using dilution plating. Each value represents the mean of three replicates from one representative experiment. Each experiment was carried out three times, each yielding similar results.

Adhesion assay of bacteria to intact roots.
Bacteria were grown for 24 h in high-C:N-ratio medium and used in the adhesion assay. The percentage of adhering phbC mutant cells to roots of sweet corn and wheat was five to six times higher than that of A. brasilense Sp7 (Table 2).

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TABLE 2. Adhesion of A. brasilense Sp7 and phbC mutant to roots of sweet corn and wheata

Extraction of extracellular polysaccharides.
The amount of exopolysaccharide (EPS) and capsular polysaccharide (CPS) extracted after 48 h of growth in medium with a high C:N ratio was considerably higher in the phbC mutant than in strain Sp7 (Table 3).

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TABLE 3. Extraction of extracellular polysaccharides from A. brasilense Sp7 and phbC mutant after 48 h of growth in high C:N-ratio mediuma

Top Abstract Introduction Materials and Methods Results Discussion References

 
In this study, we cloned and sequenced three genes from A. brasilense that are traditionally considered to be necessary in the PHB biosynthetic pathway of a wide variety of microorganisms. A mutant strain impaired in PHB synthesis was also constructed and characterized. The PHB biosynthetic pathway described for R. eutrophus is probably present in most bacteria able to accumulate PHB. The pathway begins with the condensation of two acetyl-CoA molecules catalyzed by ß-ketothiolase. An NADPH-dependent acetoacyl-CoA reductase catalyzes the conversion of acetoacyl-CoA to ß-hydroxybutyryl-CoA. Finally, the resulting ß-hydroxybutyryl-CoA is polymerized by a PHB synthase (35).

The PHB synthase gene of A. brasilense (phbC) was identified by means of PCR, using two degenerate primers designed on the basis of the PhbC sequences of other bacteria. Analysis of the deduced amino acid sequence of the A. brasilense phbC gene showed a high degree of similarity with other PHB synthases. Disruption of this gene suppressed PHB accumulation, as evidenced by gas chromatography and electron microscopy.

Additional sequencing from the phbC region and homology analysis of the identified putative ORFs suggested the presence of other PHA biosynthetic genes, phaA and phaB, coding for ß-ketothiolase and NADPH-dependent acetoacetyl-CoA reductase, respectively. An additional ORF was detected downstream of the phbB gene. Sequence homology analysis suggested that this ORF encodes acyl-CoA dehydrogenase (FadE), a protein that is known to play a role in fatty acid metabolism (18).

Therefore, our findings indicate that the phbABC genes in A. brasilense are linked in a cluster similar to the ones found in Acinetobacter sp., Alcaligenes latus, Pseudomonas acidophila, and R. eutrophus and most recently in Azotobacter sp. (16, 28, 44). Southern blot hybridization suggested that all known species of Azospirillum contain at least one phbC gene similar to that of A. brasilense. In A. doebereinerae, two bands hybridized to the probe (Fig. 3). This bacterium was recently characterized and proposed to belong to the Azospirillum genus (10). Although it exhibits general characteristics of Azospirillum, A. doebereinerae differs from its nearest relatives in some physiological properties (10). The fact that two bands were observed for A. doebereinerae could be due to a nonspecific reaction or to the presence of an EcoRI site in the reacting fragment. However, we cannot discard the possibility that this bacterium carries a second copy of the gene, as reported for some other bacteria (41).

Starvation experiments showed a clear decrease in the survival ability of the phbC mutant compared to the wild-type strain Sp7. This is in accordance with previous results by Tal and Okon (38), in which bacteria containing high levels of PHB survived and proliferated to a higher extent than bacteria containing low levels of PHB. These findings demonstrate the role of PHB in A. brasilense as an intracellular energy and carbon storage compound, which can enhance survival when these sources are limited.

In A. brasilense, PHB oxidation involves a specific NADH-dependent dehydrogenase (36), which competes for tricarboxylic acid (TCA) cycle intermediates (for example, NADH, NADPH, ATP, and acetyl-CoA) in the electron transport system (37). When PHB accumulation is disrupted, more resources are accessible to the TCA cycle. The presence of intracellular PHB in the cells has been previously shown to prevent chemotaxis to any chemoattractant (13). Therefore, with no PHB being produced by the mutant, the bacteria may be in a constant chemotactic "spree," responding to chemoattractants present in the medium. This could explain the increased motility in the phbC mutant compared to that of the wild type, as seen by contrast microscopy.

The phbC mutant also showed more aggregation than the wild-type strain in a medium containing a relatively high C:N ratio. Azospirilla are known for their capacity to aggregate and flocculate under diverse stress conditions. Previous studies have suggested the involvement of extracellular polysaccharides in cell aggregation (5). The concentrations of EPS produced by four different strains of A. brasilense differing in their ability to aggregate increased with the extent of aggregation (3). Similarly, in our experiments, there was an association between the quantities of EPS and CPS and the extent of aggregation.

In a root adhesion assay, the phbC mutant strain exhibited increased ability to adhere to roots relative to the wild-type Sp7 (Table 2). As for cell aggregation, the differences in EPS and CPS contents between wild-type and mutant strains might explain the difference observed in root adhesion properties (8). In A. brasilense, chemotaxis and motility are probably involved in promoting root colonization (29). Therefore, in addition to differences in polysaccharides, the enhanced motility observed for the mutant strain could also contribute to stronger root adhesion than that of the wild type.

In conclusion, a cluster of genes involved in the biosynthesis of PHB was isolated from A. brasilense and a PhbC-defective mutant was shown to have decreased in survival while exhibiting increased motility, production of extracellular polysaccharides, and adhesion to cereal roots. With the commercial application of field inoculations using A. brasilense, there is increasing interest in the development of efficient and reliable inoculants. We are also testing the effects of PHB synthesis on the ability of A. brasilense to endure stress factors involved in inoculum efficiency and viability.

 
This research was supported by "The Israel Science Foundation" founded by "The Academy of Sciences and Humanities," and by the European Union-5th Framework contract QLK3-CT-2000-31759-ECO-SAFE.

 
* Corresponding author. Mailing address: Department of Plant Pathology and Microbiology, Faculty of Agricultural, Food and Environmental Quality Sciences, The Hebrew University of Jerusalem, P.O. Box 12, Rehovot 76100, Israel. Phone: 972 8 9489216. Fax: 972 8 9466794. E-mail: okon{at}agri.huji.ac.il.

Top Abstract Introduction Materials and Methods Results Discussion References

 

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Applied and Environmental Microbiology, June 2002, p. 2943-2949, Vol. 68, No. 6
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.6.2943-2949.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

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