Bioprospecting desert plant Bacillus endophytic strains for their potential to enhance plant stress tolerance

Abstract

Plant growth-promoting bacteria (PGPB) are known to increase plant tolerance to several abiotic stresses, specifically those from dry and salty environments. In this study, we examined the endophyte bacterial community of five plant species growing in the Thar desert of Pakistan. Among a total of 368 culturable isolates, 58 Bacillus strains were identified from which the 16 most divergent strains were characterized for salt and heat stress resilience as well as antimicrobial and plant growth-promoting (PGP) activities. When the 16 Bacillus strains were tested on the non-host plant Arabidopsis thaliana, B. cereus PK6-15, B. subtilis PK5-26 and B. circulans PK3-109 significantly enhanced plant growth under salt stress conditions, doubling fresh weight levels when compared to uninoculated plants. B. circulans PK3-15 and PK3-109 did not promote plant growth under normal conditions, but increased plant fresh weight by more than 50% when compared to uninoculated plants under salt stress conditions, suggesting that these salt tolerant Bacillus strains exhibit PGP traits only in the presence of salt. Our data indicate that the collection of 58 plant endophytic Bacillus strains represents an important genomic resource to decipher plant growth promotion at the molecular level.

Figure 1

Un-rooted phylogenetic tree based on 16S rRNA gene sequence showing the diversity of bacteria isolated from all plants, at both the phylum and genus level. The numbers in parentheses represent the total count of unique isolates at each level.

Figure 2

Bar graph showing the distribution and diversity of bacteria cultivated from each plant at genus level. Genera represented by one isolate are not shown.

Figure 3

Phylogenetic tree showing the relationship of the 58 newly isolated Bacillus strains, based on their 16S rRNA gene sequences. The strains used in this screen are marked with an asterisk (*). P. typhae [LN867175] was used as an out-group for the phylogenetic tree. Bacillus type strains used to construct the tree, include B. subtilis [KY206830], B. subtilis [NR_104873], B. badius [KT382256], B. circulans [KR055041], B. endophyticus [AF295302.1], B. megaterium [CP018874], B. cereus [CP023245], B. licheniformis [AY052767], B. mojavensis [NR_112725], B. axarquiensis [DQ993671.1], B. halotolerans [NR_115063], B. tequilensis [NR_104919], and Staphylococcus gallinarum [DQ350835]. Branches with support less than 50% were collapsed. Bootstrap values are shown in the tree branches.

Figure 4

Screening assay of A. thaliana inoculated with (A) Bacillus cereus PK6_15 and (B) Bacillus subtilis PK5_26 in non-salt (1/2 MS, 12 days) and in salt (1/2 MS + 100 mM NaCl, 15 days) conditions. The total fresh weight of Arabidopsis is presented as the mean of three biological experiments. Asterisks indicate statistical differences compared to the control samples under the same conditions based on Mann-Whitney U Test (*P < 0.05, **P < 0.01, ***P < 0.001). Abbreviations: ½ MS: ½ strength Murashige and Skoog (MS) basal salt macronutrient solution for plant medium; ½ MS + 100 mM NaCl: ½ strength Murashige and Skoog (MS) basal salt macronutrient solution with 100 mM NaCl salt added for plant stress medium; Col: Arabidopsis thaliana Columbia strain (Control).