Innovative Culturomic Approaches and Predictive Functional Metagenomic Analysis: The Isolation of Hydrocarbonoclastic Bacteria with Plant Growth Promoting Capacity

Innovative culturomic approaches were adopted to isolate hydrocarbonoclastic bacteria capable of degrading diesel oil, bitumen and a selection of polycyclic aromatic hydrocarbons (PAH), e.g., pyrene, anthracene, and dibenzothiophene, from a soil historically contaminated by total petroleum hydrocarbons (TPH) (10,347 ± 98 mg TPH/kg). The culturomic approach focussed on the isolation of saprophytic microorganisms and specialist bacteria utilising the contaminants as sole carbon sources. Bacterial isolates belonging to Pseudomonas, Arthrobacter, Achromobacter, Bacillus, Lysinibacillus, Microbacterium sps. were isolated for their capacity to utilise diesel oil, bitumen, pyrene, anthracene, dibenzothiphene, and their mixture as sole carbon sources. Pseudomonas, Arthrobacter, Achromobacter and Microbacterium sps. showed plant growth promoting activity, producing indole-3-acetic acid and expressing 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase activity. In parallel to the culturomic approach, in the microbial community of interest, bacterial community metabarcoding and predictive functional metagenomic analysis were adopted to confirm the potentiality of the isolates in terms of their functional representativeness. The combination of isolation and molecular approaches for the characterisation of a TPH contaminated soil microbial community is proposed as an instrument for the construction of an artificial hydrocarbonoclastic microbiota for environmental restoration.

Plant Growth Promoting Microorganism Selection and Activity Test for Reforestation of Topsoil Restoration Site

Objectives : This study was conducted to investigate the applicability of plant growth promoting microorganisms during restoration through re-vegetation of damaged topsoil.Methods : As the vegetation to be applied to the restoration site, Weigela subsessilis, Spiraea prunifolia, Pine densiflora, Pennisetum alopecuroides were selected. An attempt was made to isolate plant growth promoting microorganisms from the root zone of plants of the same species inhabiting domestic park sites and hiking trails. Plant growth promoting activities such as phosphate solubilization ability, siderophore production ability, IAA production ability, and ACC deaminase production ability were examined, and the species to be finally applied was selected and then identified. Among the strains whose plant growth promoting activity was confirmed, Arthrobacter sp. 1B2 and Paraburkholderia terrae 1P2 were applied to the genitalia and pine, respectively, and a pot experiment was conducted to confirm the activity.Results and Discussion : Forty-five strains were isolated from Weigela subsessilis, Spiraea prunifolia, Pine densiflora, Pennisetum alopecuroides and the IAA-producing ability and ACC deaminase-producing ability were confirmed for 16 strains whose phosphate solubilizing ability and siderophore-producing ability were confirmed. After selecting and identifying strains with excellent plant growth promoting ability, strains such as Cupriavidus sp, Arthrobacter sp., Pseudomonas fluorescens, Pseudomonas sp., Paraburkholderia terrae were obtained. Among them, Arthrobacter sp. 1B2 and Paraburkholderia terrae 1P2 strains were applied to genitalia and pine, respectively, and it was confirmed that plant growth was promoted.Conclusions : Bioassay experiments and field applications using plant growth promoting microorganisms have been mainly studied for herbaceous species (Grandaceae, corn, oats, etc.). However, in this study, the applied plants are shrubs class, which do not significantly grow in length, targeting damaged areas with high subsoil content, which are poor in environment and insufficient in organic and inorganic matter. Therefore, it is meaningful in that the activity of plant growth promoting microorganisms focused on absorption of inorganic substances, such as phosphate solubilization activity and siderophore ability, was investigated and the activity was confirmed by performing a bioassay.

Metagenomic insights into plant growth promoting genes inherent in bacterial endophytes of Emilia sonchifolia (Linn.) DC

Studies on the genome of endophytes reveal the metabolic potential of endophytic microbiome including both culturable and unculturable fractions. The metagenome analysis through the Illumina HiSeq platform gives access to the genetic data encrypted for the molecular machinery, which takes part in plant growth promotion activity of the endophyte in various aspects including production of plant growth hormones and enhancing nutrient availability for the host plant. The present work was undertaken to identify the genes involved in plant growth promotion activities from the endophytes of Emilia sonchifolia(Linn.) DC. through metagenome analysis. Metagenomic studies include the analysis of functional annotations which aid in the detection of biocatalysts taking part in the metabolic pathway of host plants. The annotations of expressed genes in different databases like NCBI Nr, KEGG, eggnog and CAZy resulted in enlisting the vast array of information on the genetic diversity of the endophytic microbiome. The metagenome analysis of endophytic bacteria from the medicinal plant E.sonchifolia unveiled characteristic functional genes involved in plant growth promotion such as nitrogen metabolism (nif) and siderophore production (enterobactin category), ipdC and tnaA (IAA producing), ACC deaminase coding genes (regulation of elevated ethylene levels in host tissues), Mo-Nitrogenase, nitrous-oxide reductase (nosZ), nitrate reductase (narG, napA), nitrite reductase (nirD) (nutrient assimilation and absorption) enterobactin siderophore synthetase components F and D and acid phosphatase genes. This clearly explains the effective plant-microbe relationship and the role of bacterial endophytic microbes in regulating the growth of host plants.

Pseudomonas 1-aminocyclopropane-1-carboxylate (ACC) Deaminase and Its Role in Beneficial Plant-Microbe Interactions

The expression of the enzyme 1-aminocylopropane-1-carboxylate (ACC) deaminase, and the consequent modulation of plant ACC and ethylene concentrations, is one of the most important features of plant-associated bacteria. By decreasing plant ACC and ethylene concentrations, ACC deaminase-producing bacteria can overcome some of the deleterious effects of inhibitory levels of ACC and ethylene in various aspects of plant-microbe interactions, as well as plant growth and development (especially under stressful conditions). As a result, the acdS gene, encoding ACC deaminase, is often prevalent and positively selected in the microbiome of plants. Several members of the genus Pseudomonas are widely prevalent in the microbiome of plants worldwide. Due to its adaptation to a plant-associated lifestyle many Pseudomonas strains are of great interest for the development of novel sustainable agricultural and biotechnological solutions, especially those presenting ACC deaminase activity. This manuscript discusses several aspects of ACC deaminase and its role in the increased plant growth promotion, plant protection against abiotic and biotic stress and promotion of the rhizobial nodulation process by Pseudomonas. Knowledge regarding the properties and actions of ACC deaminase-producing Pseudomonas is key for a better understanding of plant-microbe interactions and the selection of highly effective strains for various applications in agriculture and biotechnology.

ACC Deaminase-Positive Halophilic Bacterial Isolates With Multiple Plant Growth-Promoting Traits Improve the Growth and Yield of Chickpea (Cicer arietinum L.) Under Salinity Stress

In this study, a total of 50 halophilic bacterial isolates were screened for 1-aminocyclopropane-1-carboxylate (ACC) deaminase activity, of these six with the highest ACC deaminase activity were selected for an increase in chickpea yield under salinity. The ACC deaminase activity among the isolates was ranged between 0.12 and 3.56 mM α-KB mg−1 min−1. These six isolates and one reference strain from the Agricultural College, Raichur, were used in the microcosm experiment during the rabi season of 2018. After 60 days of sowing, decreased rhizosphere pH and electrical conductivity (EC) from 8.4 to 7.6 and 4.3 to 3.4 dS m−1, respectively, were reported in chickpea. Among the treatments, Bacillus safensis (B. safensis)-inoculated plants showed a higher number of flowers (71 flowers/plant), pods (49.3 pods/plant), branches (33.3 branches/plant), and enhanced fresh weight (17.2 g/plant) and dry weight (8.1 g/plant). They were corroborated by improved nitrogen and phosphorus absorption of 71.5 and 43.5%, respectively, in B. safensis-treated plants. Based on the microcosm experimental findings, three cultures improving biometric and yield attributes were chosen for the field investigation. The field study was carried out at the Agricultural Research Station, Ganagavathi, during Kharif 2019. The chickpea plants treated with the consortium [B. safensis, Pseudomonas stutzeri, and Staphylococcus xylosus] increased the superoxide dismutase and catalase activity of plants by 258 and 196%, respectively. In addition, an increase in ascorbate peroxidase activity (0.41 μmol of ascorbate oxidized s−1 g−1 fresh weight) in the leaves and proline content was also recorded. The consortium (B. safensis, P. stutzeri, and S. xylosus) significantly increased nutrient uptake (N and P), the number of flowers, number of pods, and yield by 63.26, 39.03, 110, 59.96, and 17.56%, respectively, in chickpeas. Finally, inoculation with a mixture of three isolates is an effective method for increasing chickpea production under osmotic stress.

Salinity Stress: Toward Sustainable Plant Strategies and Using Plant Growth-Promoting Rhizobacteria Encapsulation for Reducing It

Salinity is one of the most important abiotic stresses that influences plant growth and productivity worldwide. Salinity affects plant growth by ionic toxicity, osmotic stress, hormonal imbalance, nutrient mobilization reduction, and reactive oxygen species (ROS). To survive in saline soils, plants have developed various physiological and biochemical strategies such as ion exchange, activation of antioxidant enzymes, and hormonal stimulation. In addition to plant adaption mechanisms, plant growth-promoting rhizobacteria (PGPR) can enhance salt tolerance in plants via ion homeostasis, production of antioxidants, ACC deaminase, phytohormones, extracellular polymeric substance (EPS), volatile organic compounds, accumulation of osmolytes, activation of plant antioxidative enzymes, and improvement of nutrients uptake. One of the important issues in microbial biotechnology is establishing a link between the beneficial strains screened in the laboratory with industry and the consumer. Therefore, in the development of biocontrol agents, it is necessary to study the optimization of conditions for mass reproduction and the selection of a suitable carrier for their final formulation. Toward sustainable agriculture, the use of appropriate formulations of bacterial agents as high-performance biofertilizers, including microbial biocapsules, is necessary to improve salt tolerance and crop productivity.