Characterization of bacterial diversity between two coastal regions with heterogeneous soil texture
Soil sampling and determination of soil physical properties and synoptic data
Soil samples were taken from two coastal deserts in northern and southern Iran. Details of their geographical distribution and eco-physiological characterization are presented in Table 1. A total of 2 kg of soil samples were collected from 2 separate sampling locations ranging from 0 to 30 cm in depth , and the samples were room-dried for 3 days. temperature and in the dark before sieving. Soil samples were sieved using a 2 mm sieve to remove stones and other inert material before being stored in zip lock bags. Table 1 lists the physical characteristics of soil samples, including soil texture (sand 2–0.02 mm; silt 0.02–0.002 mm; clay 0.002 mm), pH, and proportions of clay, silt and sand. Synoptic data for the past 10 years (2009-2019), including average annual temperature, maximum temperature, minimum temperature, average precipitation, average annual wind speed and maximum wind speed, were obtained from from the IROF Iran Meteor (http://www.irimo.ir/far/index.php).
Bacterial isolation and the effect of manure medium on their growth
According to Chen et al. 2005, soil bacteria were isolated using the direct propagation method. For this species, soil samples were treated with a series of dilutions. The 1 g soil sample mixture was vortexed for 1 min after being suspended in 2 ml sterile saline (0.9% w/v NaCl). The mixture was then serially diluted (usually 10-1 at 10-seven), and the 100 μl level of the diluted soil samples were dispersed on the surface of the solidified plates using glass spreaders. The samples were then incubated for 1-3 days at 30°C in an inverted position without light. For bacterial isolation, we used eleven culture media including Nutrient Agar (NA), Nutrient Agar plus MnSO4 (NA + MnSO4), LB, Moller Hinton agar (MHA), Acidithiobacillus (APH), Violet Red Bile Lactose (VRB) agar medium, GYM Streptomyces medium, medium DPM, Azospiril medium, Azotobacteria medium and manure medium (MB).
To prepare MB medium, dry animal manure and distilled water (1:6 w/v) were combined to create MB medium, which was then allowed to stand at room temperature for 16 h. The resulting mixture was then centrifuged at 5000 rcf for 30 min after being filtered twice. The next step was to add Hoagland salts (10% w/v) to the final extract, adjusting the pH of the medium to 5.8.±0.02 and autoclaving for 20 min at 121°C and 1.5 kPa. Prior to sterilization, bacteriological agar (1.5 w/v) was used as a gelling agent to solidify the medium.
After bacterial isolation on NA, NA+ MnSO4, LB, MHA, APH, VRB, GYM, DPM and Azospibrillum, growth of all isolates was assessed on MB medium. To study the biomass of isolates in the same state, we eluted MB medium. At first, the bacteria were cultured in liquid form of NA, NA+ MnSO4LB, MHA, APH, VRB, GYM, DPM and Azospiril and Azotobacteria medium at 30°C for 48 h, then 103 cells from each isolate were transferred to 48-well plates containing MB medium and the plates were incubated at 30°C for 10 h. Then, the growth of bacteria was read at an optical density (OD) of 630 nm 10 h after inoculation, the experiment was carried out with three repetitions. In the next step, the equivalent CFU/ml of each OD was obtained by inoculating the uniform amount of liquid culture of the isolates on the solid form of the MB medium at 30°C for 16 h.
Phenotypic characterization and biochemical identification of bacterial isolates
Morphological analysis of cell shape, colony (i.e. shape, color and size) and biochemical tests were used to identify bacterial isolates. Biochemical characterization was performed using Gram stain, KOH27, oxidase and catalase. For this elixir, following Barthélemy’s method28, the Gram stain of the bacteria was studied 48 h after inoculation on MHA, and the KOH method without staining was used to confirm the results. Using 0.5 ml of a 10% hydrogen peroxide solution, a catalase test was performed and the generation of gas bubbles was monitored. Using biochemical oxidase discs, the oxidative activity of 27 isolates was studied.
Effect of abiotic stresses on bacterial isolates
Determine the effect of abiotic stresses on alkaline isolates (MH medium at pH 10), salinity (MH medium supplemented with the final concentration of 100 mM NaCl), osmotic [MH medium supplemented with 25% polyethylene glycol (PEG) Mn6000], and heat stress (MH medium incubated at 15°C for cold stress and 60°C for heat stress) were examined. For all experiments, the incubation period was 15 h and the plates were kept in the dark.
MALDI-TOF MS isolate identification
Soil bacterial isolates were subcultured twice on MHA and incubated at 30°C for 24 h before MALDI-TOF MS measurement. So ∼0.1 µg of cellular material was directly transferred from a bacterial colony or a smear of colonies to a MALDI target spot. After drying at room temperature, the sample spots were covered with 1 μl of matrix solution (10 mg/mL a-cyano-4-hydroxycinnamic acid in 50% acetonitrile and 2.5% d trifluoroacetic acid) and each measurement was carried out in triplicate (technical replicates). MS analysis was performed on an Autoflex MALDI-TOF mass spectrometer (Bruker Daltonics, Germany) using Flex Control 3.4 software (Bruker Daltonics, Germany). Calibration was performed using the bacterial test standard (Bruker Daltonics, Germany). Soil isolates with a valid MALDI-TOF MS score of 2 were undoubtedly assigned to the genus/species level. For bacterial classification and identification, BioTyper 3.1 software (Bruker Daltonics, Germany) equipped with MBT 6903 MPS library (released April 2016), MALDI Biotyper standard pretreatment method and MALDI Biotyper MSP identification standard method adjusted by the manufacturer (Bruker Daltonics, Germany) were used. Only the highest score value of all mass spectra belonging to individual cultures (biological and technical replicates) was recorded25. The score between 2.3 and 3.00 indicates a highly probable identification to the species level and between 2.0 and 2.29 represents identification to the genus level and the probable level of identification to the species. A score between 1.7 and 1.99 indicates likely gender identification.29.
Effects of bacterial isolates on plant growth
Karaj Seed and Plant Improvement Institute (Karaj, Iran; http://www.spii.ir/homepage.aspx?site=DouranPortal&tabid=1&lang=faIR) provided corn, canola and wheat seeds (Zea mays. Var Kosha; Brassica napus Var Nima; Triticum aestivum Var Kalate). In greenhouse trials, 2 × 103 cells/seeds of soil isolates grown in manure-based medium were inoculated into maize, rapeseed and wheat plants. During the studies, acid-washed and autoclaved sand was used for planting. For three weeks, the seedlings were maintained under a day/night photoperiod of 16/8 h at a temperature of 25°C. Three replicates of a complete randomized block design were used for the colonization experiment treatments. Within the framework of the bacterial treatments, measurements were carried out on the growth parameters of the plants, in particular the dry biomass of the shoots (mg), the dry biomass of the roots (mg), the length of the shoots (cm), the length of the roots (cm), shoot density (mg/cm), root density (mg/cm) and shoot/root weight (mg). Samples were dried at 60°C for three days to measure dry biomass.
Statistical analysis was performed using R software (version 4.1.3). One-way analysis of variance (ANOVA) was used to determine the significance of the experiment, and Fisher’s protected least significant difference (LSD) test with a P value of 0.01 was performed to separate the means. Additionally, a PCA analysis was performed based on the Clustvis package and the SVD imputation approach.
Ethical approval and consent to participate
All authors accept the ethics and consent to participate in this article and declare that this submission follows the policies of Scientific reports. Accordingly, the material is the original work of the author, which has not previously been published elsewhere. The article is not considered for publication elsewhere. All authors have been personally and actively involved in substantial work leading to the article and will bear public responsibility for its content.
Plant Research Ethics
All authors confirmed that the experimental research and field studies on the plants, including the receipt of seeds from the Karaj Seed and Plant Improvement Institute, complied with institutional, national and international guidelines and legislation. relevant. Furthermore, the methods were conducted in accordance with the relevant guidelines and regulations.