Seeds

The seeds evaluated in the present study were those ecotypes of San Judas (SJ), Indio Yumi (IY) and Elena-Mora Property (EMP), which were collected from the Sierra de Sonora-Chihuaha (29° 05’ north latitude 110° 57’ west longitude) in the state of Sonora.

Halobacteria nitrogen fixers

Halobacteria nitrogen fixer

The total counts of nitrogen-fixing bacteria (BFN) are shown in Table 1. It could be seen that the isolates on control agar plates (N-free OAB agar medium without NaCl) were 8.6 to 12.6×10⁶ forming units of colonies (UFC g^-1 and from 7.6 to 10.6×10⁶ UFC g^-1 for seeds with and without seed coat, respectively.) Isolates in the SJ and EMP ecotypes decreased significantly (p≤ 0.05) in agar plates supplemented with NaCl 0.5 and 0.75 M. In the case of control seeds in the SJ ecotype (8.3-9.3×10⁶ CFU g^-1), the counts showed a NaCl tolerance of 45-54% (3.7-5×10⁶ CFU g^-1 seeds) and 29-33% (2.4-3.1×10⁶ CFU g^-1 seeds) of total culturable BFN at 0.5 and 0.75 M NaCl, respectively.

Similarly, counts in the EMP ecotype showed a salt tolerance of 28-41% (3.6-4.4×10⁶ CFU g^-1 seeds) and 18-22% (2.3×10⁶ CFU g^-1 seeds) of BFN total arable to 0.5 and 0.75 M NaCl, while in the control the values were 10.6-12.6×10⁶ CFU g^-1.

In relation to the capacity of N fixation (acetylene reduction), the strain encoded as RP22 had the highest acetylene reduction activity (6.97 ±1.1 nmol culture h^-1), which is similar to reference A. halopraeferens AU10 (7.83 ±1.3 nmol of culture h^-1). This result shows that growth improved by 28 and 43% with bacteria isolated at NaCl concentrations between 0.5 M and 0.75 M NaCl.

Analogously, ^{Rueda et al. (2003)} proposed that 5% of the halotolerant strains of saline-flat areas reduced acetylene ~ 6.26 ±0.56 nmol of culture h^-1 (Klebsiella pneumoniae) with the control A. halopraeferens, which produced a value of 7.1 ±1.7 nmol^-1 h^-1. This could be attributed to the codependency of other bacteria, a phenomenon that is quite common among microorganisms (^{Diby and Harshad, 2015}). The genetic identification of the bacterial isolate in the present study, based on the 16S rRNA gene sequence, showed a high similarity (99%) with the plant growth promoting bacterium Bacillus amyloliquefaciens (Figure 1).

Figure 1 Phylogenetic tree showing the similarity with the isolate Bacillus amyloliquefaciens RP22 (□) and the close relationship with Bacillus amyloliquefaciens (○) and its relationship with the halotolerant (●) fixative of N (▲) and endophyte (■) desert bacteria. Alignment and close relationship with the tree was done using MEGA6 (http://www.megasoftware.net/). The bars indicate 10% divergence; a bootstrap analysis was developed with 1000 trials. KJ433614, Bacillus amyloliquefaciens strain IARI-AR25; KM652480, Bacillus amyloliquefaciens strain RP22; HM854239, Bacillus safensis strain KLH-14; KF646682, Bacillus tequilensis strain DH-10; AB513629, Bacillus megaterium; KJ628451, Bacillus subtilis strain HPCAQRKSM106; JN119340, Halobacillus sp. KLBMP 2429; EU867381, Bacillus pumilus strain CCGE2028; KF831020, Bacillus firmus strain L-4; KF933708, Bacillus baekryungensis strain QD56; KF444385, Bacillus aryabhattai strain HYR8(1); FJ032017, Bacillus pumilus strain ES4; FN796812, Bacillus cereus strain GP17; HM151905, Bacillus subtilis strain DAZ26; AY373358.1, Bacillus megaterium strain c5; KC178613, Bacillus flexus strain DNEB39; KC790325, Bacillus methylotrophicus strain SY33; JX515571, Bacillus subtilis strain H171. 

The phylogenetic analysis based on the 16S rRNA gene isolated from the NFB halotolerant showed 99% similarity with a B. amyloliquefaciens (accession number KJ433614), a bacterium isolated from the cold desert of the northwest Himalayas by PGPR analyzes. As far as is known, the species B. amyloliquefaciens has never been reported for P. acutifolius, which is indicative of the association of beneficial bacteria and the plant under study. Other species of the genus Bacillus have been isolated and identified in other leguminous plants (B. pumilus, B. cepacia, B. japonicum, B. vallismortis, B. mojavensis, B. atrophaeus, B. megaterium and B. Stearothermophilus) (^{Bashan et al., 1993}; ^{Dobbelaere and Okon, 2007a b}).

Inoculation tests

When the seeds were inoculated with bacterial strains, the negative effects of 2% NaOCl on germination of the seeds decreased (Table 2). Thus, higher germination rates were obtained under growth chamber conditions with inoculated seeds (53.3-100%) than with non-inoculated seeds (46.7 - 86.7%) in the three ecotypes of tepary beans treated with 0 M NaCl and 0.25 M. In addition, the germination rate was strongly affected when 0.50 M NaCl was applied, showing ranges of 0-13.3% and 6.7-26.7% for the non-inoculated and inoculated seeds, respectively (Table 2).

Regarding the parameters measured in growth chamber conditions, treatments of bean tepary in the variables of shoots and roots in seedlings, the maximum effects of bacterial inoculation with B. amyloliquefaciens RP22, as well as A. halopraeferens AU10 were for the ecotype SJ, showing significant results with p≤ 0.05 to 0 M NaCl (8.9 and 10.5 cm of plant^-1), between 6.9 and 8.7cm plant^-1 for B. amyloliquefaciens and A. halopraeferens when adding 0.25 M NaCl, in comparison with the non-inoculated seedlings that were lower: (7.8 and 8.5 and 5.8 and 6.7 cm plant^-1 for 0 and 0.25 M NaCl (Figure 2).

Figure 2 Effect of NaCl concentrations and bacterial inoculation on the height and dry weight of Phaseolus acutifolius seedlings under growth chamber conditions. The values represent the mean ± standard error (n= 3). Different letter indicates significant differences (p≤ 0.05, Tukey test) of the inoculated plants (Ba= Bacillus amyloliquefaciens, Ah= Azospirillium halopraeferens AU10) in relation to the non-inoculated controls (U). Ecotypes SJ= San Judas; IY= Indian Yumi; EMP= Elena-Mora Property. 

Root lengths showed significant statistical differences (p≤ 0.05) between the inoculated and non-inoculated seedlings in IY and EMP ecotypes, considering all the salinities, except the SJ ecotype where the plants were inoculated with B. amyloliquefaciens RP22, (4.4 and 2.7 cm plant^-1 to 0 and 2.5 M NaCl (p≤ 0.05) (Figure 2).

The production of dry biomass according to the effect of the treatments were statistically different (p≤ 0.05); the increases in dry biomass of the plants inoculated with respect to those not inoculated were 17 and 38%, respectively. Coincident with the growth chamber test, greenhouse test results (Figure 3) showed lower emergence seed rates and seedling biomass and growth values of the three ecotypes of tepary beans irrigated with 0.25 and 0.5 M NaCl with NaCl at 0 M, this assay also confirms the growth promoting effect of the plants on seeds and seedlings of both inoculated bacterial strains, which reaffirms the possibility of proposing beneficial microorganisms as biofertilizing prospects (^{Stefana et al., 2013}; ^{Diby and Harshad, 2015}).

Figure 3 Effect of NaCl concentrations and bacterial inoculation on the growth and dry weight of Phaseolus acutifolius seedlings under greenhouse conditions. The values represent the mean ± standard error (n= 3). Different letters indicate significant differences (p≤ 0.05) of the inoculated plants (Ba= Bacillus amyloliquefaciens, Ah= Azospirillium halopraeferens AU10) in relation to the non-inoculated controls (U). Ecotypes SJ= San Judas; IY= Indian Yumi; EMP= Elena-Mora Property. 

Similar results indicate that the positive effects of this type of bacteria apparently are due to the production of growth promoting substances as reported in other studies (^{Yadav, 2011}; ^{Rugheim et al., 2012}).

Regarding the variables of growth and biomass in greenhouse conditions, significant differences were observed (p≤ 0.05) in lengths of shoots in the SJ ecotype, considering the inoculation with B. amyloliquefaciens RP22 (70 cm plant^-1), as well as A. halopraeferens AU10 (58 cm plant^-1), in relation to the non-inoculated controls (U) (35 cm plant^-1) in NaCl M (Figure 3).

Regarding the dry weight, the maximum values were recorded in inoculated seedlings in the three ecotypes at 0, 0.25 and 0.5 M NaCl in relation to the non-inoculated controls (U). However, it should be mentioned that salinity reduces dry weight, while it increases in all treatments (Figure 3). In particular, the values of germination, emergence, growth and biomass were higher in the seedlings inoculated with B. amyloliquefaciens RP22 than in those inoculated with A. halopraeferens AU10, particularly in the ecotype IY treated with NaCl 0 and 0.25 M (Figures 2 and 3).

Likewise, the analysis of foliar chlorophyll and the contents of proteins, ashes and crude fiber confirm the effect of NaCl treatments and bacterial inoculation in relation to controls (not inoculated) on the growth and biomass of the seedlings (Cuadro 3).