Study On The Salt And Alkali Tolerance Of An Endophytic Bacterium From Cistanche Deserticola (Pseudalkalibacillus Sp. A8)

 

Abstract

Objective: To isolate endophytic bacteria from Cistanche deserticola and investigate the potential mechanisms underlying the salt-alkali tolerance of Pseudalkalibacillus sp. A8.
Methods: Endophytic bacteria were isolated using the dilution plating method. The classification of strain A8 was determined by morphological traits and 16S rRNA gene sequencing. Whole genome sequencing and metabolomic analyses were then conducted.
Results: Fifty endophytic bacterial strains were isolated from fresh Cistanche deserticola samples, covering 4 phyla, 8 classes, and 35 genera. Among them, strain A8 is the first Pseudalkalibacillus species isolated from plants, demonstrating exceptional salt-alkali tolerance. It was capable of growing in pH ranges from 6.0 to 13.0 and NaCl concentrations of up to 17%. Genome annotation revealed 10 genes associated with salt-alkali resistance. Metabolomic analysis under high-salinity and high-pH conditions identified 186 differential metabolites enriched in seven functional pathways related to salt-alkali stress adaptation.
Conclusion: This study provides valuable microbial resources for enhancing crop salt-alkali tolerance and offers theoretical support for saline-alkali soil improvement through microbial means. It also lays the groundwork for understanding the molecular mechanisms of salt-alkali-tolerant bacteria.

Keywords: Cistanche deserticola; endophytic bacteria; salt-alkali tolerance; whole genome sequencing; UPLC–MS/MS

 

 

 

Cistanche deserticol flower

Introduction

Soil salinization and alkalization are major global challenges, contributing significantly to land degradation and desertification. It is estimated that approximately 20% of the world's arable land is affected by salinity to varying degrees, and China alone possesses 9.9×10⁷ hectares of saline-alkali land-the largest in the world. Such soils typically exhibit poor permeability, low fertility, and hard texture, severely impairing crop growth and yield. Therefore, developing effective strategies for utilizing and improving saline-alkali soils remains a critical issue.

Recent studies suggest that microbial inoculants, particularly salt-alkali-tolerant strains, can enhance crop tolerance and improve soil properties in a cost-effective and environmentally friendly manner. Endophytic bacteria-those that reside within plant tissues without causing disease-share ecological and physiological traits with their host plants. Cistanche deserticola, a desert-dwelling parasitic plant known for its extreme salt-alkali resistance, is a promising source for isolating stress-tolerant endophytes.

This study aims to:

Isolate endophytic bacteria from Cistanche deserticola;

Screen for salt-alkali-tolerant strains;

Explore the stress-resistance mechanisms of the selected strain Pseudalkalibacillus sp. A8 through whole-genome and metabolomic analyses.

Cistanche deserticol flower

Materials and Methods

1.1 Materials

Plant Sample: Cistanche deserticola collected in March 2023 from Xiquan Town, Jingtaixian, Baiyin, Gansu Province, China. Identified by Prof. Guo Yehong, Gansu Agricultural University.

Media: TSB liquid medium; solidified using 20 g/L agar.

Reagents and Instruments: Analytical-grade NaClO, KH₂PO₄, K₂HPO₄, TAE buffer; Agilent QTOF 1290–6542 mass spectrometer, R-1001V rotary evaporator, Neofuge 15R centrifuge, SureCycler 8800 gradient PCR.

1.2 Isolation of Endophytic Bacteria

Fresh Cistanche tissue was surface-sterilized and homogenized. Serial dilutions were plated on R2A, TSB, ISP-2, and 2216E media. Colonies were isolated and purified through three subcultures, then stored in 20% glycerol at −80 °C.

1.3 16S rRNA Gene Identification

Genomic DNA was extracted and amplified using universal primers 27F/1492R. PCR products were sequenced and analyzed using EzBioCloud database.

 

Cistanche base 

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1.4 Phenotypic and Physiological Characterization

SEM: Cell morphology observed after gold-coating.

NaCl Tolerance: Growth assessed at 0–20% NaCl.

pH Tolerance: Growth measured at pH 4–13.

Temperature Range: Growth tested at 4–50 °C.

1.5 Whole Genome Sequencing

Strain A8 was sequenced using PacBio technology. Genome assembly and annotation were performed against six major databases (NR, Swiss-Prot, Pfam, EggNOG, GO, KEGG). Genes related to salt-alkali tolerance were screened.

1.6 UPLC-MS/MS Analysis

Samples were cultured under three conditions:

A: pH 7.2, NaCl 0.5%

B: pH 7.2, NaCl 14%

C: pH 13.0, NaCl 0.5%

Supernatants were dried, dissolved in methanol, and analyzed using UPLC-MS/MS (ZORBAX Eclipse Plus C18, positive ion mode).

Results and Analysis

2.1 Endophyte Isolation

Fifty strains were isolated from Cistanche deserticola, belonging to 4 phyla and 35 genera.

2.2 Identification of Strain A8

16S rRNA sequence showed 99.30% similarity with Pseudalkalibacillus salsuginis. Phylogenetic analysis confirmed it as a member of Pseudalkalibacillus. Morphologically, colonies were smooth, yellow-brown, rod-shaped (1–6 µm) with slight fuzz under SEM.

Growth range: 20–45 °C (optimal: 30 °C)

pH range: 6–13 (optimal: 9.0)

Salt range: 0–17% NaCl (optimal: 0–5%)

2.3 Genome Analysis

Genome Length: 6,022,064 bp

GC Content: 40.99%

Protein-coding genes: 5,825 (82.5%)

tRNA/rRNA: 76 tRNAs, 24 rRNAs

ANI Values: All <95%, indicating a potential novel species.

COG Annotation: 4,683 genes (80.39%)

Amino acid transport/metabolism (556), carbohydrate metabolism (501), transcription (464), lipid metabolism (440), coenzyme metabolism (298), defense mechanisms (100)

Salt-Alkali Tolerance Genes Identified:

Glycine/betaine transporter

Sodium symporter

Antiporters

F0F1 ATP synthase

2.4 Metabolomic Analysis

Metabolite Identification: 986 total metabolites detected

OPLS-DA Analysis: Clear separation between groups; R²X > 0.5, R²Y = 1, Q² > 0.85, P < 0.005

Differential Metabolites:

186 total:

Group B: 96 (13 upregulated, 83 downregulated)

Group C: 132 (41 upregulated, 91 downregulated)

49 upregulated in both B & C

196 uniquely expressed metabolites in B & C

246 total upregulated or unique metabolites

Pathway Enrichment (KEGG):

Sesquiterpenoid and triterpenoid biosynthesis

Purine metabolism

Lysine biosynthesis and degradation

Pantothenate and CoA biosynthesis

Arginine and proline metabolism

Steroid biosynthesis

These results suggest that strain A8 adapts to salt-alkali stress through both genetic and metabolic regulation.

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Conclusion

This study successfully isolated and identified Pseudalkalibacillus sp. A8 from Cistanche deserticola, a potential novel species exhibiting strong salt-alkali resistance. Through whole genome and metabolomic analyses, multiple genes and metabolic pathways have been identified as contributors to its stress adaptation. These findings provide a theoretical basis for developing microbial agents to improve crop tolerance and reclaim saline-alkali soils.

 

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