Microbial Diversity of Psychrotolerant Bacteria Isolated from Wild Flora of Andes Mountains and Patagonia of Chile towards the Selection of Plant Growth-Promoting Bacterial Consortia to Alleviate Cold Stress in Plants

Abstract

Cold stress decreases the growth and productivity of agricultural crops. Psychrotolerant plant growth-promoting bacteria (PGPB) may protect and promote plant growth at low temperatures. The aims of this study were to isolate and characterize psychrotolerant PGPB from wild flora of Andes Mountains and Patagonia of Chile and to formulate PGPB consortia. Psychrotolerant strains were isolated from 11 wild plants (rhizosphere and phyllosphere) during winter of 2015. For the first time, bacteria associated with Calycera, Orites, and Chusquea plant genera were reported. More than 50% of the 130 isolates showed ≥33% bacterial cell survival at temperatures below zero. Seventy strains of Pseudomonas, Curtobacterium, Janthinobacterium, Stenotrophomonas, Serratia, Brevundimonas, Xanthomonas, Frondihabitans, Arthrobacter, Pseudarthrobacter, Paenarthrobacter, Brachybacterium, Clavibacter, Sporosarcina, Bacillus, Solibacillus, Flavobacterium, and Pedobacter genera were identified by 16S rRNA gene sequence analyses. Ten strains were selected based on psychrotolerance, auxin production, phosphate solubilization, presence of nifH (nitrogenase reductase) and acdS (1-aminocyclopropane-1-carboxylate (ACC) deaminase) genes, and anti-phytopathogenic activities. Two of the three bacterial consortia formulated promoted tomato plant growth under normal and cold stress conditions. The bacterial consortium composed of Pseudomonas sp. TmR5a & Curtobacterium sp. BmP22c that possesses ACC deaminase and ice recrystallization inhibition activities is a promising candidate for future cold stress studies.

Keywords: Andes Mountain; Curtobacterium; Patagonia; Pseudomonas; bacterial consortium; cold stress; plant growth-promoting bacteria; psychrotolerant bacteria; tomato.

Figure 1

Location of sampling sites and sampled wild flora of Andean Mountains and Patagonia in Chile.

Figure 2

Biodiversity of the selected psychrotolerant bacterial strains from wild plants of Andean and Patagonia areas of Chile. The different genera were represented in solid colors for isolates from rhizosphere (R) and crosshatch colors for isolates from the phyllosphere (P). Circles next to the wild plant genera represent the regions of Chile. Red circles, Valparaíso Region; green circles, Libertador General Bernardo O’Higgins Region; blue circles, Ñuble Region; and purple circle, Magallanes and Chilean Antarctica Region.

Figure 3

Auxin production and bacterial cell survival percentage (BCS%) of bacterial strains. (a) Auxin production and BCS% of Proteobacteria (green bars). (b) Auxin production and BCS% of Actinobacteria (red bars). (c) Auxin production and BCS% of Firmicutes (yellow bars) and Bacteroidetes (purple bars). The blue dots (joined by dotted lines) represent BCS% of each isolate. The solid red line is the 50% BCS value. The dashed black line represents the value of [Auxin] (µg mL⁻¹)/Turbidity_600nm = 0.5 µg mL⁻¹. Small black arrows indicate selected strains. Each value is a mean ± SD of three independent trials.

Figure 4

Phylogenetic tree of the 10 selected psychrotolerant strains for the formation of potential bacterial consortia. The dendrogram was constructed using the Neighbor-Joining method based on the partial sequence of the 16S rRNA gene. The tree has arbitrarily been rooted by the Antarctic archaeon Halohasta litchfieldiae. Values of 1000 bootstraps are informed at the branching point. GenBank accession numbers of 16S rRNA sequences are indicated in parentheses. Scale bar represents 0.05 substitutions per nucleotide positions. The circles represent the regions of Chile. Red circles, Valparaíso Region; Green circles, Libertador General Bernardo O’Higgins Region; blue circles, Ñuble Region; and purple circle, Magallanes and Chilean Antarctica Region.

Figure 5

Selected bacterial consortia. The three bacterial consortia formulated with two bacteria are illustrated. Bacteria were grown in Tryptic Soy Agar (TSA) medium at room temperature. Abbreviation: BC, bacterial consortium.

Figure 6

Effects of selected bacterial consortia on tomato plant growth. Tomato seedlings were arranged in peat pots and inoculated every 2 weeks in roots and at the leaves. (a) Representative photograph of tomato plants assay. (b) Graphs of parameters evaluated for tomato plants growth. Each value is a mean ± SD of seven independent replicates. Significant differences were analyzed by one-way ANOVA followed by LSD Fisher test. Means with different letters indicate significant differences (p < 0.05). Abbreviations: NC, negative control; PC, positive control (Nutrisac); BC1-3, bacterial consortium 1, 2, and 3, respectively.

Figure 7

Effect of selected bacterial consortia on the growth of tomato seeds at 25 and 14 °C in the biopriming assay. (a) Representative photograph of tomato seed growth inoculated with selected consortia at 25 °C. (b) Graphs of evaluated parameters of tomato seeds growth at 25 °C. (c) Representative photograph of tomato seed growth inoculated with selected consortia at 14 °C. (d) Graphs of evaluated parameters of tomato seeds growth at 14 °C. Each value is a mean ± SD of three independent replicates, with a total of 30 seeds per treatment. Significant differences were analyzed by the Kruskal–Wallis test followed by all pairwise multiple comparisons. Means with different letters indicate significant differences (p < 0.05). Abbreviations: NC, negative control; PC, positive control (Nutrisac); BC1-3, bacterial consortium 1, 2, and 3, respectively.

Figure 8

IRI activity of bacterial strains. The protein extracts were exposed to −6 °C for 48 h to measure their absorbance at 500 nm. Each value is a mean ± SD of 6 independent replicates. Significant differences were analyzed by one-way ANOVA followed by the LSD Fisher test. Means with different letters indicate significant differences (p < 0.05). Abbreviations: NC, negative control (E. coli JM109); PC, positive control (Type III AFP); IRI, ice recrystallization inhibition.

Figure 9

Growth in ACC and ACC deaminase activity of bacterial strains. (a) Growth in DF medium with ACC addition as sole carbon and nitrogen source. (b) ACC deaminase activity in µmol α-KB mg protein⁻¹ h⁻¹ of strains with positive growth. Each value is a mean ± SD of three independent replicates. Abbreviations: NC, negative control; PC, positive control (Achromobacter sp. 188); BC, bacterial consortium; ACC, 1-aminocyclopropane-1-carboxylic acid; α-KB, α-ketobutyrate.