3.1. Isolation and identification of cultivable bacteria
A total of 567 strains belonging to four phyla, six classes, 25 orders, 36 families, and 66 genera were isolated and purified from the plant and soil samples (root and shoot, and rhizosphere and bulk soil) of three halophytes (Supplementary Table S1). At the phylum level (Fig. 1A), Actinomycetota (50.62%) was the dominant bacterial community in three halophytes, followed by Bacillota (24.87%), Pseudomonadota (23.99%), and Bacteroidota (0.53%). At the class level (Fig. 1B), Actinomycetia (50.62%) was the dominant group. The remaining isolates were Bacilli, Gammaproteobacteria, Alphaproteobacteria, Flavobacteriia, and Betaproteobacteria, with 24.87%, 19.75%, 3.88%, 0.53%, and 0.35%, respectively. The predominant genera were Streptomyces, Halomonas, and Bacillus, accounting for 15.70%, 12.35%, and 12.17%, respectively (Fig. 1C).
Three halophytes' associated cultivable microorganisms showed various levels of diversity. (Table 2). About 23 strains were isolated and purified from P1PE, belonging to three phyla, four classes, six orders, six families, and seven genera. P2PE harbored three phyla, five classes, 11 orders, 16 families, 24 genera, and 67 strains; P3PE harbored three phyla, four classes, 16 orders, 18 families, 25 genera, and 101 strains; P1RR harbored three phyla, four classes, 11 orders, 13 families, 16 genera, and 53 strains; P2RR harbored three phyla, four classes, 15 orders, 17 families, 21 genera, and 77 strains; P3RR harbored four phyla, five classes, 13 orders, 15 families, 20 genera, and 80 strains; P1RS harbored three phyla, four classes, nine orders, 11 families, 18 genera, and 48 strains; P2RS harbored four phyla, five classes, 12 orders, 14 families, 22 genera, and 60 strains; and P3RS harbored three phyla, three classes, 11 orders, 12 families, 16 genera, and 60 strains.
Table 2
Diversity of cultivable bacteria isolated from different sites
Index | P1PE | P2PE | P3PE | P1RR | P2RR | P3RR | P1RS | P2RS | P3RS |
Phylum | 3 | 3 | 3 | 3 | 3 | 4 | 3 | 4 | 3 |
Class | 4 | 5 | 4 | 4 | 4 | 5 | 4 | 5 | 3 |
Order | 6 | 11 | 16 | 11 | 15 | 13 | 9 | 12 | 11 |
Family | 6 | 16 | 18 | 13 | 17 | 15 | 11 | 14 | 12 |
Genus | 7 | 24 | 25 | 16 | 21 | 20 | 18 | 22 | 16 |
Isolates | 23 | 67 | 101 | 53 | 77 | 80 | 48 | 60 | 58 |
Richness | 13.00 | 36.00 | 47.00 | 28.00 | 42.00 | 37.00 | 27.00 | 35.00 | 27.00 |
Shannon | 2.36 | 3.39 | 3.57 | 3.02 | 3.47 | 3.40 | 3.04 | 3.29 | 2.98 |
Simpson | 0.88 | 0.96 | 0.96 | 0.93 | 0.96 | 0.96 | 0.94 | 0.95 | 0.93 |
Pielou | 0.92 | 0.93 | 0.93 | 0.91 | 0.93 | 0.94 | 0.92 | 0.92 | 0.90 |
The biodiversity profiles of the cultivable bacterial communities with different parts of three halophytes were compared based on the diversity indices computed by the R package vegan, including species richness and α-diversity indices (Shannon and Simpson) and Pielou evenness. Our findings showed that the species richness and Shannon diversity index of P3PE was the highest, and P1PE was the lowest. The Simpson diversity index of P2PE, P3PE, P2RR, and P3RR were the highest, and P1PE was the lowest. The Pielou evenness of P3RR was the highest, and P3RS was the lowest. There was no significant difference in the diversity of the PE (endophytic bacteria), RR (rhizosphere bacteria), and RS (bulk soil bacteria) cultivable bacteria.
The analysis of the dominant species at the genus level (top 20 dominant genera) of cultivable bacteria in different sites showed the high relative abundance of Streptomyces in P2RR, P3RR, and P2RS, Halomonas in P2RS and P3RS, Bacillus in P1PE, P3PE, and P1RS, Nocardiopsis in P1RR, and Brevibacterium in P2PE (Fig. 2A).
Similarities and differences in the appearance of cultivable bacterial communities were reflected in the number of shared and exclusive species displayed throughout nine sections of the Flower plot. (Fig. 2B). Only one species, Bacillus swezeyi, was present in all nine samples, representing that the cultivable bacterial community composition was not very similar. In PE, the unique species of P3 was 23, P2 was 20, and P1 was 7. In RR, the unique species of P1, P2, and P3 were 5, 9, and 7, respectively. RS's amazing P1, P2, and P3 species were 10, 11, and 8 (Supplementary Table S2). When comparing PE, RR, and RS, the cultivable bacterial composition of PE indicated more variability.
3.2. Potential Novel Bacterial Strains of Three Halophytes Cultivable Bacteria
Based on 16S rRNA gene identification, the 16S rRNA gene similarity of 147 strains was less than 98.65% belonging to 29 genera and 57 novel species, accounting for 25.93% of the total isolated strains. At the genus level, the predominant genera of the potential novel strains were Streptomyces (37.41%), followed by Alkalihalobacillus (13.61%), and Microbacterium (7.48%) (Fig. 3).
Potential new taxa were isolated from each isolation site of three halophytes, and each isolation site showed different isolation effects (Fig. 4). The three halophytes in RR showed the most potential for isolating novel strains. Only novel Streptomyces species were isolated from P1PE, and only a few potentially novel strains were found in P3RS. For potentially novel strains, P2 showed the best isolation effect overall. In saline-alkali environments, microbial resources are rich and untapped for excavation and utilization.
3.3. Comparison of Isolation Effects between Two Media
The dominant phylum grown on both media was Actinomycetota. However, the proportion of Actinomycetota on M2 (Fig. 5B) was more significant than on M1 (Fig. 5A), indicating that M2 was more selective in isolating Actinomycetota species. In addition, Bacteroidota were isolated only on M1. On the other hand, compared to the two media at the genus level, the number of bacteria species isolated on M1 was more than on M2. While the quantity of strains obtained from M2 at P2RR was more significant than that obtained from M1, the number of genera isolated by M2 was still lower than that of M1.
Moreover, the number of isolated strains was more than 30 in P2PE, P2RR, P2RS, P3PE, and P3RR, and the number of genera was more than 18. The overall isolation effect of P1 was not as good as P2 and P3, especially since the number and species of strains isolated from the P1PE were the lowest. Only three endophytic bacteria distributed in two genera were isolated on M2. Therefore, the two media were unsuitable for the isolation of P1PE (Fig. 5C).
The 147 potential novel strains were isolated on M1 and M2 media (98 and 49). Different plants showed different effects on the potentially novel strain rate (Fig. 6). In P1, the rate of novel strains isolated on M1 media was 10%, whereas no novel strains were isolated on M2 media. In P3, M2 media showed better results than M1. Interestingly, the number of bacteria species isolated on M2 in P2PE, P3PE, P3RR, and P3RS was less than on M1, but the rate of novel strains was higher than on M1. It's also worth noting that M2 had a higher success rate in isolating new strains than M1.
3.4. in vitro Screening of Bacterial Strains for Plant Beneficial Traits
In the present study, 213 strains, where PE, RR, and RS had 72, 79, and 62, respectively, were screened to investigate multi-beneficial traits in vitro to determine the most promising bacterial isolates (Supplementary Table S3). For isolates with phosphate-solubilizing ability, their proportions in PE, RR, and RS bacterial populations were 40.28%, 53.16%, and 41.94% (Fig. 7), respectively. Only one endophytic bacterial isolate, EGI P1K047, showed vigorous phosphate-solubilizing activity and was identified as Advenella kashmirensis subsp. methylica (similarity: 99.72%) (Supplementary Table S1). The proportions of strains producing siderophores in PE, RR, and RS populations were 29.17%, 64.56%, and 61.29% (Fig. 7), respectively. Most of them were low-active strains. The proportions of strains producing protease in PE, RR, and RS populations were 38.89%, 39.3%, and 29.03% (Fig. 7), respectively. Six strains EGI P1B044, EGI P2B047, EGI P2K012, EGI P3B010, EGI P3B014, and EGI P3K010 demonstrated protease-producing solid activity. They were identified as Isoptericola halotolerans (similarity: 98.98%), Isoptericola halotolerans (similarity: 99.62%), Isoptericola halotolerans (similarity: 98.94%), Zhihengliuella halotolerans (similarity: 99.41%), Alkalihalobacillus berkeleyi (similarity: 98.40%), and Halomonas elongata (similarity: 99.78%), respectively (Supplementary Table S1). The proportions of cellulase-producing strains in PE, RR, and RS were 25.00%, 31.65%, and 25.81%, respectively. Highly active cellulase-producing strains in RR bacterial populations were 16.46%, which was higher than in PE (8.33%) and RS (8.06%) (Fig. 7). A total of 24 strains exhibited vigorous cellulase-producing activity, and these isolates included members of the genera Streptomyces, Bacillus, Isoptericola, Nocardiopsis, Arthrobacter, Halomonas, Microbulbifer, and Salininema (Supplementary Table S1).
The tested strains with nitrogenase activity in PE (68.06%), RR (46.84%), and RS (46.84%) were grown both on Ashby and NFC medium (Fig. 8). They included:Streptomyces, Bacillus, Nocardiopsis, Zhihengliuella, Paracoccus, Brevibacterium, Isoptericola, Microbacterium, Alkalihalobacillus, Brachybacterium, Cytobacillus, Kocuria, Nesterenkonia, Staphylococcus, Corynebacterium, Dietzia, Halomonas, Kushneria, Advenella, Alcanivorax, Amycolatopsis, Exiguobacterium, Gordonia, Mammaliicoccus, Marmoricola, Mycolicibacterium, Prauserella, Priestia, Pseudarthrobacter, and Rhizobium genera (Supplementary Table S1 and Table S3). The results of the tested strains from three halophytes showed high salt tolerance. All tested strains could resist 5% NaCl concentration, more than 80% could resist 10% NaCl concentration, about 50% could withstand 15% NaCl concentration, and still, some strains in PE (19.44%), RR (8.86%), and RS (12.9%) could resist 20% NaCl concentration (Fig. 8). The strains that could resist 20% NaCl concentration belonged to Halomonas, Kushneria, Staphylococcus, Cytobacillus, Georgenia, Gracilibacillus, Halobacillus, Bacillus, Exiguobacterium, Mammaliicoccus, Marinococcus, Nesterenkonia, and Oceanobacillus genera (Supplementary Tables S1 and S3).
Among all tested strains, about 20 were positive for five plant-beneficial traits in vitro, including phosphate-solubilization, nitrogen fixation, production of siderophores, protease, and cellulase, belonged to various species within Bacillus, Streptomyces, Isoptericola, and Nocardiopsis genera (Supplementary Table S1 and Table S3).