ABSTRACT
A mutant of 5-enopyruvylshikimate-3-phosphate synthase from Ochrobactrum anthropi was identified after four rounds of DNA shuffling and screening. Its ability to restore the growth of the mutant ER2799 cell on an M9 minimal medium containing 300 mM glyphosate led to its identification. The mutant had mutations in seven amino acids: E145G, N163H, N267S, P318R, M377V, M425T, and P438L. Among these mutations, N267S, P318R, and M425T have never been previously reported as important residues for glyphosate resistance. However, in the present study they were found by site-directed mutagenesis to collectively contribute to the improvement of glyphosate tolerance. Kinetic analyses of these three mutants demonstrated that the effectiveness of these three individual amino acid alterations on glyphosate tolerance was in the order P318R > M425T > N267S. The results of the kinetic analyses combined with a three-dimensional structure modeling of the location of P318R and M425T demonstrate that the lower hemisphere's upper surface is possibly another important region for glyphosate resistance. Furthermore, the transgenic Arabidopsis was obtained to confirm the potential of the mutant in developing glyphosate-resistant crops.
INTRODUCTION
Glyphosate is a popular broad-spectrum, nonselective herbicide used to control weeds. This herbicide inhibits 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS; EC 2.5.1.19), a key enzyme in the shikimate pathway (2, 12, 21, 37). However, glyphosate can also kill crops. Since the early 1980s, researchers have sought to identify glyphosate-insensitive EPSPSs that could be introduced to crops for herbicide resistance. Although a number of such EPSPSs have been examined and characterized (4, 9, 14, 29, 31, 39), several disadvantages limit their use in developing glyphosate-tolerant crops. One serious disadvantage is the low affinity for phosphoenolpyruvate (PEP).
In vitro-directed evolution through DNA shuffling has been routinely applied to evolve enzymes for basic research or to improve proteins of industrial significance, such as improved enzyme kinetics (16, 18, 41, 42, 50) and altered substrate or product specificities (17, 28, 33, 48), as well as vaccine and pharmaceutical development (26, 43). In the case of directed evolution targeting the glyphosate resistance of EPSPS, both He et al. and Zhou et al. also obtained mutant EPSPS with increased glyphosate resistance and enzyme activity (13, 50). Therefore, mutant EPSPSs with improved enzymatic parameters such as higher Ki (glyphosate) and normal Km (PEP) values can be obtained by DNA shuffling.
However, only two patents have thus far been reported to produce glyphosate-tolerant crop plants except EPSPS from Agrobacterium tumefaciens strain CP4, which is used to generate currently available commercial glyphosate-resistant crops (6). One is the mutant maize EPSPS with Thr102Ile and Pro106Ser substitutions (35), and the other is the mutated EPSPS from Salmonella enterica serovar Typhimurium strain CT7 with glyphosate resistance in bacterial and plant cells (B. C. Raleigh, E. H. Cary, and H. Rougemont, U.S. patent application 2007/0169218 A1). Although the two patents have been successfully applied in glyphosate-tolerant crops, the identity of the novel mutant with altered amino acid residues different from known PEP/glyphosate-binding residues needs to be clarified for developing glyphosate-tolerant crops. Therefore, obtaining novel and valid glyphosate tolerance genes with multisite mutations at new amino acid residues is also important.
The main objective of our study was to identify an EPSPS or EPSPS mutant with properties appropriate for the development of transgenic glyphosate-tolerant plants. Recently, a new gene encoding glyphosate-resistant EPSPS was identified in Ochrobactrum anthropi (40). In the present study, we performed DNA shuffling on the new aroA gene from O. anthropi under selective pressure caused by high glyphosate concentrations. One mutant with a high Ki and 50% inhibitory concentration (IC50) was identified after four rounds of DNA shuffling and screening. To further evaluate its potential, the aroA mutant gene (aroAmutant) was introduced into Arabidopsis via the floral dip method. The transgenic Arabidopsis expressed significant glyphosate resistance.
MATERIALS AND METHODS
Bacterial strains, chemicals, and plant materials.Shikimate-3-phosphate (S3P), glyphosate (free-acid form), PEP, and Ni2+-NTA agarose affinity columns were purchased from Sigma Chemical Co. (St. Louis, MO). All other chemicals were analytical grade and purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, People's Republic of China). All primers were purchased from the Shanghai Sangon Biological Engineering Technology and Service Co., Ltd. (Shanghai, People's Republic of China). The RNA isolation kit was purchased from Fermentas International, Inc. (Burlington, Ontario, Canada) and a reverse transcription (RT) system was obtained from Promega (Madison, WI). Escherichia coli BL21(DE3) was obtained from Novagen, Inc. (Madison, WI), and E. coli ER2799 (3) (with the EPSP synthase gene deleted from its genome) was provided by Thomas C. Evans, Jr. (New England Biolabs). A. tumefaciens GV3101 and Arabidopsis thaliana (ecotype Columbia L.) plants were obtained from our laboratory.
DNA shuffling.DNA shuffling to generate improved glyphosate tolerance mutants was performed as described by Stemmer et al. (38) and Xiong et al. (44). The wild-type O. anthropi aroA gene (aroAO.anthropi) was fragmented with DNase I, and fragments from 50 to 100 bp were collected in a dialysis bag as they eluted from 10% (wt/vol) polyacrylamide electrophoresis gels. The fragments were subjected to PCR reassembly and amplification. The first step comprised 40 cycles of PCR without primers: denaturation at 94�C for 60 s, annealing at 54�C for 60 s, and elongation at 72�C for 60 s. The PCR products were then subjected to 25 cycles of PCR with the specific primers P1Z (5′-GAGAGAGGATCCATGTCCCATTCTGCATC-3′) and P1F (5′-GAGAGAGCTCTCATCGCGCGTCGCTCAGTTCGAT-3′) to amplify the aroAO.anthropi gene to full length. The conditions for the second PCR step were as follows: 94�C for 2 min, followed by 25 cycles of denaturation at 94�C for 30 s, annealing at 54�C for 30 s, and elongation at 72�C for 90 s. After the primerless PCR and primer PCR, a group of full-length aroA mutants were obtained and digested with BamHI and SacI enzymes. The isolated fragments were ligated into the prokaryotic expression vector pYPX251 (44). The mutant DNA library was translated into E. coli aroA mutant strain ER2799 by electroporation and plated on M9 agar plates supplemented with glyphosate at high concentrations. The aroA mutant was screened and identified by its ability to restore the growth of the mutant ER2799 cells on an M9 minimal medium containing glyphosate at high concentrations. The aroA mutants were pooled and used as templates to generate further mutants by DNA shuffling. The selected aroA mutants were sequenced and subsequently analyzed.
Site-directed mutagenesis.To determine the role of specific amino acid mutations in the mutant, site-directed mutagenesis was performed. The mutagenesis involved using an overlap extension PCR strategy to reverse each of the seven amino acid substitutions from mutant to wild type (30). The used primers were as follows: G145E, 5′-GGTGGAAGCAGCCGAAGGCGAC-3′ and 5′-CTGCTTCCACCACCTGAACG-3′; H163N, 5′-GGCCAATCCGATCACCTATVGA-3′ and 5′-ATCGGATTGGCCGTCTTGGGGCCGAT-3′; S267N, 5′-CCGCAACGTGCTGATGAACCCGA-3′ and 5′-GCACGTTGCGGATGGTAACATCCGA-3′; R318P, 5′-TCCGCCGGAACGCGCTCCGTCGA-3′ and 5′-GTTCCGGCGGAACGACAACGCCCTTC-3′; V377M, 5′-CGAGATGTCGCTGACGGTCCGTGGTC-3′ and 5′-GCGACATCTCGCCTTCGGTGCAATCGACG-3′; T425M, 5′-CACCATGATCGCCACGTCCTTCCCC-3′ and 5′-CGATCATGGTGCTGTCGTCAACCGTCAC-3′; and L438P, 5′-GATGCCGGGACTGGGCGCCAAGAT-3′ and 5′-GTCCCGGCATCATGTCCATGAATTCG-3′. The amino acids mutation that best contributed to glyphosate resistance were also determined. For this purpose, four amino acid mutation mutants were created using a PCR-based staggered extension process. Three had individual amino acid mutations (AroA267, AroA318, and AroA425), and one was mutated at all three of these amino acids (AroA267,318,425) (49).
Protein overexpression and purification.All mutants digested with BamHI and SacI were cloned into the expression vector pYM4087 (45). They were then expressed and introduced into the competent expression host, E. coli BL21(DE3). Bacteria were then plated on Luria-Bertani agar (1% [wt/vol] Bacto tryptone, 0.5% [wt/vol] yeast extract, and 1% [wt/vol] NaCl) supplemented with ampicillin (100 μg/ml) and X-Gal (5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside; 100 μg/ml). The culture was incubated at 37�C overnight. Several positive colonies were selected and inoculated into 50 ml of Luria-Bertani medium containing 50 mg of ampicillin/liter. They were grown at 37�C until the optical density at 600 nm reached 1.0. Cells were harvested and disrupted by sonication. The soluble fraction was loaded onto a Ni2+-NTA agarose affinity column at 4�C according to the manufacturer's instructions. The purified protein was analyzed by 12% (wt/vol) sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
Enzyme assay.EPSPS activity was measured by determining the amount of inorganic phosphate produced in the reaction using the malachite green dye assay method (23). Reaction mixtures (50 μl) contained 50 mM HEPES (pH 7.0), 1 mM S3P, 1 mM PEP, and 0.75 μg of purified enzyme. The reaction was allowed to proceed for 3 to 5 min before the addition of 800 μl of malachite green-ammonium molybdate colorimetric solution. Color development was stopped after 1 min by the addition of 800 μl of sodium citrate solution (34% [wt/vol]). After standing at room temperature for 30 min, the absorbance at 660 nm was measured. Reaction mixtures without S3P served as controls.
The Km values for PEP, the Ki values for glyphosate, and the IC50 value for glyphosate were measured as described by Tian et al. (40).
Plant expression vector construction and plant transformation.Construction of the plant expression vector and plant transformation were performed as described by Xu et al. (46). The method was modified by adding a chloroplast transit sequence to the constructs to ensure that the transgene would be targeted to the chloroplast because EPSPS in plants is located in the chloroplast (5). Accordingly, the fragment encoding the chloroplast transit peptide (TSP) of Arabidopsis was amplified from Arabidopsis genomic DNA using the primers TSPZ (5′-GTCGACATGGCGCAAGTTAGCAGAATC-3′) and TSPF (5′-GGATCCCTCCGCCGTGGAAACACAAGAC-3′) (22). The PCR fragment was then inserted downstream of the double CaMV35S promoter, and the final constructs D35S, TSP, aroA, and Nos were introduced into A. tumefaciens GV3101 by electroporation (Fig. 1 A). The constructs were introduced into the plants (A. thaliana ecotype Columbia) by a previously described floral dip method to generate transgenic plants (47).
(A) AroA expression vector for Arabidopsis transformation. Nos-Ter, terminator sequence of the nopaline synthase; SAR, scaffold attachment region; TSP, Arabidopsis transit peptide. (B) Expression of the AroAmutant or AroAO.anthropi cDNA in different genotypes. Each lane contained 4 μl of the RT-PCR products obtained using total RNA extracted from 3-week-old plants grown under normal conditions. WT, wild type; Oa, transgenic Arabidopsis lines with aroAO.anthropi gene; Mu, transgenic Arabidopsis lines with aroAmutant gene. The data shown are representative of three independent experiments.
Transgenic plant selection.Transgenic plants were obtained from plantlets grown on Murashige and Skoog medium containing 30 mg of hygromycin/liter. The transgenic nature of the plants was confirmed by PCR analysis of genomic DNA by using the specific primers P1Z (5′-GAGAGAGGATCCATGTCCCATTCTGCATC-3′) and P1F (5′-GAGAGAGCTCTCATCGCGCGTCGCTCAGTTCGAT-3′). RT-PCR was used to determine the levels of aroAmutant and aroAO.anthropi transcription. Total RNA was extracted from T2 generation seedlings by using an RNA isolation kit, and an RT system was used to synthesize first-strand cDNA in 20-μl reaction mixtures containing 5 μg of total RNA as the template. In order to improve the reliability of RT-PCR, the A. thaliana actin gene (GenBank accession no. U41998) was amplified with the primers TactZ (5′-GCACCCTGTTCTTCTTACCGAG-3′) and TactF (5′-AGTAAGGTCACGTCCAGCAAGG-3′) and used as an internal standard to normalize the amount of cDNA. Specific DNA fragments (∼1,300 bp) of the aroAmutant or aroAO.anthropi genes were then amplified from the transgenic plants using the same amounts of cDNA. PCR products were separated on 2% (wt/vol) agarose gels and quantified using a Model Gel Doc 1000 (Bio-Rad). The expression patterns of the aroAmutant or aroAO.anthropi genes were evaluated with a Shine Tech Gel analyzer (Shanghai Shine Science of Technology Co., Ltd., People's Republic of China).
Glyphosate resistance assay.For the glyphosate resistance plate assay, T2-sterilized A. thaliana seeds were grown directly on Murashige and Skoog (MS) medium (27) containing various glyphosate concentrations (0, 100, 250, 500, and 750 μM) in petri dishes under a controlled-environment chamber (22�C, 16/8-h day/night cycle). To observe the germination process, photos were taken after 2 weeks of growth.
For the glyphosate spray treatment, plants were grown in pots (12 seedlings per pot) filled with 9:3:1 vermiculite-peat moss-perlite in a controlled-environment chamber (22�C, 16/8-h day/night cycle, about 120 μmol of photons m−2 s−1 light intensity). After 3 weeks, the seedlings in the pots were twice treated with 5, 15, and 20 mM glyphosate at 3-day intervals.
RESULTS
DNA shuffling and sequencing.Using the DNA shuffling system, the aroAO.anthropi gene was shuffled. Over 500,000 variant colonies were screened using the pYPX251 vector in each round of DNA shuffling and screening. One mutant, aroAmutant, was identified by its ability to restore growth in the mutant ER2799 cell on M9 minimal medium containing 300 mM glyphosate after four rounds of DNA shuffling and screening. The mutant had mutations in 10 nucleic acid sites, resulting in alterations in seven amino acids: E145G, N163H, N267S, P318R, M377V, M425T, and P438L.
Role of each altered amino acid in aroAmutant.Site-directed mutagenesis via an overlap extension PCR strategy was performed to determine the role of specific amino acid mutations in aroAmutant. The results show that the mutations N267S, P318R, and M425T collectively improved glyphosate tolerance because no mutant with a reverse mutation in either S267N, R318P, or T425M could grow on M9 minimal medium containing glyphosate (300 mM). In contrast, cells carrying the reverse mutations of G145E, H163N, V377M, and L438P could grow on M9 medium. To further verify whether the mutations at these three amino acids were additive or interactive in altering resistance, four mutants (AroA267, AroA318, AroA425, and AroA267,318,425) were created using the PCR-based staggered extension process. The three amino acid mutations proved to be additive in improving resistance because cells carrying only a single mutation in either AroA267, AroA318, or AroA425 could not grow on M9 medium containing glyphosate, whereas the triple mutant AroA267,318,425 grew well.
3D structure of AroAmutant.The three-dimensional (3D) structure of AroAmutant was modeled according to the Swiss-Model and was drawn with the Swiss-Pdb Viewer (11). The structure shows that N267S is located near the hinge between the two globular domains of EPSPS. Given the flexibility of the hinged region, amino acid substitution in this region is likely to cause conformational changes in EPSPS, consequently altering glyphosate resistance. P318R and M425T lie at the upper surface of the lower hemisphere in the interdomain cleft of the closed enzyme state, and at the “forming active site” when the globular domains are closed in a screw-like manner (Fig. 2).
Location of the seven mutated amino acids residues in the three-dimensional structure of AroAmutant.
Kinetic properties of mutants.All mutants were overexpressed, extracted, and purified in E. coli. The obtained kinetic constants are listed and shown in Table 1 and Fig. 3. AroAmutant had a Ki 2-fold higher and an IC50 0.5-fold greater than that of AroAO.anthropi while retaining a high affinity for the PEP substrate. To determine the role of specific amino acids mutations in the mutant, additional kinetic analyses were performed. The values for Km, which is a measure of the affinity for substrate, were 0.38, 0.32, 0.31, and 0.25 mM for AroA267, AroA318, AroA425, and AroA267,318,425, respectively, similar to the wild-type except for AroA267. The IC50 for AroA267,318,425 was 0.91 mM, which is greater than the IC50 values for AroA267, AroA318, and AroA425. The Ki values for glyphosate of AroA267, AroA318, AroA425, and AroA267,318,425 were 0.08, 0.091, 0.11, and 0.15 mM, respectively. The values for Kcat, which is the rate constant of the catalytic reaction, were 1.9 � 103, 2.1 � 103, 2.6 � 103, and 3.1 � 103 min−1 for AroA267, AroA318, AroA425, and AroA267,318,425, respectively. Also, the Kcat/Km ratio, a measure of enzyme efficiency and specificity, was determined. Compared to that of AroAO.anthropi, the Kcat/Km ratios for AroA318 and AroA267,318,425 were obviously higher. Thus, AroA318 and AroA267,318,425 with higher efficiency and increased affinity for PEP were more suitable to developing herbicide-tolerant plants than AroAO.anthropi. All of the kinetic analyses indicated that the effectiveness of the three individual amino acids alterations on glyphosate tolerance is in the following order: P318R > M425T > N267S. Although this trend reveals that P318R has the greatest effect on glyphosate resistance, the results also imply that all three mutations must be concurrently present to confer improved glyphosate resistance.
Kinetic properties of AroAO.anthropi (wild type) and O. anthropi AroA mutants
The IC50 values of glyphosate inhibition were determined by fitting the data to the equation v = Vmin + (Vmax − Vmin)/[1 + ([I]/IC50)s], where [I] is the glyphosate concentration, s is the slope of the curve at the IC50, and v was determined at 1 mM PEP and 1 mM S3P with glyphosate concentrations ranging from 0.0001 to 100 mM. The data were fit to v = Vmin + (Vmax − Vmin)/[1 + ([I]/IC50)s], yielding IC50s for AroAO.anthropi, AroAmutant, AroA267, AroA425, AroA318, and AroA267,318,425 of 0.61, 0.92, 0.69, 0.73, 0.81, and 0.91 mM, respectively.
Transgenic plant selection.Transgenic Arabidopsis was used to evaluate the potential application of AroAmutant in developing glyphosate-resistant crops. Three AroAO.anthropi (Oa2, Oa4, and Oa5) and two AroAmutant (Mu4 and Mu8) transgenic lines were analyzed for gene expression using RT-PCR analysis, as previously described (46). First, the A. thaliana actin genes (GenBank accession no. U41998) from the five transgenic lines (Oa2, Oa4, Oa5, Mu4, and Mu8) and the wild type were amplified with the two primers TactZ and TactF in order to ensure that the same amount of cDNA was used to amplify the target genes in the transgenic lines. Then, specific aroAmutant and aroAO.anthropi DNA fragments of ∼1,300 bp were amplified from the transgenic lines. Agarose gel electrophoresis showed that the DNA intensity of aroAmutant and aroAO.anthropi in the five different transgenic lines was the same (Fig. 1B), confirming that the levels of transcription of aroAmutant and aroAO.anthropi in the two transformants were equal. The RT-PCR results showed that the inserted genes were actively and stably transcribed in the transgenic plants. The AroAO.anthropi-transgenic line Oa4 and the AroAmutant transgenic line Mu8 were chosen for further experiments.
Assay for the glyphosate resistance of transgenic Arabidopsis.Glyphosate can affect seed germination. Seeds are usually poorly developed under glyphosate stress (7). Figure 4 shows that AroAmutant-transgenic plants grew well in 750 μM glyphosate, whereas AroAO.anthropi-transgenic plants did not grow at 500 μM. The control plants grew only at 250 μM.
Comparative germination images of transgenic seeds on MS medium containing various glyphosate concentrations (0, 100, 250, 500, and 750 μM) in petri dishes.
A previous study showed that sublethal concentrations of glyphosate caused chlorotic symptoms on leaves (50). This indicates that the phenomenon of yellow leaves is a distinguishing characteristic of plants exposed to sublethal glyphosate concentrations. After 3 weeks, the seedlings in pots were twice treated with 5, 15, and 20 mM glyphosate at 3-day intervals. Figure 5 shows that the three genotypes were not damage by 5 mM glyphosate, whereas all died at 20 mM glyphosate. Three days after the first application using 15 mM glyphosate, the leaves of the control turned yellow, and minor damage appeared in the AroAO.anthropi-transgenic plants (Fig. 5A). After the leaves were sprayed for the second time, most of the control leaves turned severely yellow; the AroAO.anthropi-transgenic plants also turned yellow, but the AroAmutant plants grew well, with normal morphology (Fig. 5B). These results also indicate that AroAmutant-transgenic plants are more resistant to 15 mM glyphosate exposure than are AroAO.anthropi-transgenic and control plants.
Photographs of the transgenic plants sprayed with various glyphosate concentrations (5, 15, and 20 mM). (A) Photograph taken 3 days after the first spray treatment. (B) Photograph taken 3 days after the second spray treatment. The same results were obtained in three independent experiments and are represented by the effects shown here.
DISCUSSION
In the present study, we focused on identifying the roles of specific amino acids mutations in a new mutant of EPSPS from O. anthropi. The highly glyphosate-resistant mutant with seven amino acid variations was isolated after four rounds of DNA shuffling and screening. Although there are seven amino acids substitutions on this mutant, only three residue changes were identified by site-directed mutagenesis to be essential and additive in altering resistance.
Numerous amino acid residues that affect EPSPS glyphosate resistance have already been identified (1, 8, 10, 13, 15, 19, 20, 24, 25, 31, 35, 40, 50). Notably, these residues are mostly distributed in two regions according to their 3D structure. One region is at the hinge between the two large globular domains of EPSPS. A typical example in this region is the T42M substitution of bacterial EPSPS that generates an enzyme with an increased resistance to glyphosate (13). The other is the third helix of the core of the N-terminal domain. Given that this helix is a universal mutation hot spot for glyphosate resistance, the Thr97Ala Pro101Ser double mutation in class I EPSPS from E. coli produced the first commercial varieties of glyphosate-tolerant maize (10). Three new residues—N267S, P318R, and M425T—that also affect glyphosate resistance have been identified in our study. Among them, N267S is located near the hinge between the two EPSPS globular domains (13). However, the P318R and M425T residues are not located in these two regions, but at the upper surface of the lower hemisphere in the interdomain cleft of the closed enzyme state. When S3P triggers the two globular domains to move toward each other and cause its transition from the open to the closed state (34), the active site around S3P/PEP located in the cleft interface between the two hemispheres is formed. Therefore, although both P318R and M425T are not involved in the PEP/glyphosate binding, considering that they are within the “emerging active site” of inner cleft, any mutation in this active site may indirectly induce structural adjustments in the hydrogen bonding networks. These adjustments result in positional shifts of the adjacent and nearby residues around S3P/PEP (36). Thus, these changes in P318R and M425T improved glyphosate tolerance. Hence, we propose that the upper surface of the lower hemisphere is also important for glyphosate resistance. In addition, the effectiveness of three concurrent amino acid alterations on glyphosate tolerance is more pronounced than any individual amino acid mutation. This phenomenon also implies that multisite mutations, rather than single-site mutations, may be suitable for the generation of glyphosate-resistant crops.
In summary, we used DNA shuffling to develop a new mutant of EPSPS from O. anthropi with improved resistance to glyphosate. Remarkably, the mutant exhibits higher tolerance to glyphosate than AroAO.anthropi while retaining a high affinity for the PEP substrate. The present study also represents a successful generation of class II EPSPS transgenic plants. Our results indicate that the Arabidopsis transformation with the new class II EPSPS, as well as with this mutant, is stable and heritable in transgenic plants, which is confirmed by a glyphosate resistance assay. The resistance level of the AroAmutant-transgenic plants is higher than the agricultural application level recommended by most manufacturers. In addition, compared to the reported results that the glyphosate resistance levels of transgenic tobacco, rapeseed, and rice are 18, 10, and 18 mM, respectively (7, 19, 50), the 15 mM glyphosate resistance of transgenic Arabidopsis is almost equal to them. Also, the assay of transgenic plants corroborates the view that transgenic plants carrying the aroAmutant gene exhibit significant tolerance to glyphosate.
ACKNOWLEDGMENTS
The research was supported financially by the Shanghai National Science Foundation (11ZR14324000), the Key Project Fund of the Shanghai Municipal Committee of Agriculture (2008-7-5, 2009-6-4), and the Youth Fund of Shanghai Academy of Agricultural Sciences (2009-19).
FOOTNOTES
- Received 26 April 2011.
- Accepted 19 September 2011.
- Accepted manuscript posted online 23 September 2011.
- Copyright � 2011, American Society for Microbiology. All Rights Reserved.
