evaluation of the role of salt tolerant bacteria in developing resistance of plants to salt ‎stress conditions

Abstract

Salt tolerant bacteria were isolated from the rhizosphere of Scirpus maritimus (RS-1, RS-2, RS-‎‎3), Imperata sp. (RI-1, RI-2, RI-4, RI-5), Equisetum debile (RE-1, RE-3) and Heleochloa ‎shoenoides (RH-4) growing in the area of salt range, Kallar Kahar and from saline soil samples ‎‎(SP-1, SP-2, SP-3, SP-4, SP-5, SP-6, SP-7, SP-8) collected from saline patches around Kasoor ‎area. Twenty two salt tolerant bacterial strains were also isolated from rhizoplane, histoplane of ‎roots and phylloplane ofAchyranthus aspera (RAal, RAa2,HAal, HAa2, HAa3, PAal, PAa2, ‎PAa3, PAa4, PAa5, PAa6), Euphorbia heliscopia (REhl,REh2, HEhl, HEh2, PEhl, PEh2) and ‎Malvestrum tricuspidatum (RMtl, HMtl, HMt2,PMtl, PMt2) growing in the area of salt range, ‎Kallar Kahar, Pakistan. Excluding HAa2 (Gram-positive) RI-1, PAa1, PAa5 (Gram variable) ‎majority of the isolates were Gram-negative rods (except RH-4, SP-1, RAa1, HAa3, PMt1 which ‎were cocci). The isolates SP-5, PAa2, PAa3, PAa4, PAa5, RMt1 were strictly aerobic, while rest ‎of the isolates were facultative anaerobes. They had catalase and cytochrome oxidase enzymes. ‎Isolates exhibited variable results for other biochemical reactions. On this basis the isolates could ‎be affiliated with family Enterobacteriaceae (RS-I, RI-5, SP-2, SP-3, SP-4, SP-6, SP-7, SP-8, ‎RAa2, HAaI, PAa6, REhl, REh2, HEhl, HEh2, PEhl, PEh2, HMtl, HMt2, PMt2), ‎Pseudomonadaceae (SP-5, PAa2, PAa3, PAa4, RMt1), Vibrionaceae (RS-2, RS-3, RI-2, RI-4, ‎RE-I, RE-3), Nesseriaceae (PMtl) and Bacillaceae (HAa2), while RI-1, RH-4, SP-I, RAal, HAa3, ‎PAal and PAa5 remained uncertain. The isolates could tolerate 2.5 - 3.5 M NaCl in the solid ‎medium as well as in the liquid medium. Maximum Na+ uptake by the bacterial cells was ‎detected between 1.0-3.0M NaCl. Optimum growth temperature was 32°, 37° or 42°C while ‎optimum pH ranged from 7-10 in NaCl free (without extra salt) and 6-9 in NaCl supplemented ‎medium. The isolates had multiple antibiotic and heavy metal resistances. Single plasmid band ‎‎(excluding RS-2 where two plasmid bands were observed) was detected in all the isolates. ‎Plasmids residing in these strains could not be transformed to E. coli strains through conjugation ‎and transformation (except a few cases) experiment.‎

To evaluate the role of salt tolerant bacteria in improving plant growth and developing resistance ‎of plants to salt stress, mono and mixed cultures of bacteria from rhizosphere, rhizoplane, ‎histoplane of roots and phylloplane of plants were taken. Experiments were carried out in four ‎Phases. In Phase I monocultures of bacteria isolated from rhizosphere, rhizoplane, histoplane of ‎roots and phylloplane ofMazus sp., Launea nudicolus, and Astragalus sp. were used to inoculate ‎Triticum aestivum varieties (Inqlab91 and Rawal 87) under 0, 50 and 100 mM NaCl treatments. ‎In Phase II-A impact of mono and mixed cultures (from one source) of Mazus sp. and in Phase ‎II-B mono and mixed cultures (from different sources) ofAstragalus sp. on Triticum aestivum var ‎Inqlab91 under 0 and 100 mM NaCl was studied. In Phase III, mono and mixed cultures of salt ‎tolerant bacteria exhibiting maximum growth stimulation in Triticum aestivum (in Phase II-A & ‎B) were selected to evaluate their role in growth promotion of different plants (Vigna radiata var NM-92, Helianthus annuus var Slobal-1) under O and 100 mM NaCl stresses. Phase IV included ‎the inoculation effect of freshly isolated bacteria conferring resistance to 2.5 and above NaCl on ‎Triticum aestivum var Inqlab91 under salt (0, 100 mM NaCl) stress. NaCl stress exhibited ‎reduction in germination and growth parameters of different plants. Increases in dry weight ‎parameters (dry weight and dry weight per gram fresh weight), Na+ and K+ contents, auxin and ‎soluble protein contents and enzyme (peroxidase and acid phosphatase) activities, were observed ‎under NaCl stress. Generally mono and mixed culture bacterial inoculations promoted (in all the ‎four phases), over non-inoculated respective treatments, germination and length parameters ‎‎(shoot, root and seedling lengths) under salt stress. In Phase I although germination and growth ‎parameters ofTriticum aestivum variety Inqlab 91 were more severely affected by salt stress but ‎enhancement with bacterial inoculations was more pronouncedas compared to variety Rawal 87. ‎In Phase II-A, all mixed culture bacterial combinations (from one source) significantly stimulated ‎growth of Triticum aestivum var Inqlab 91. While in Phase II-B, although bacterial combinations ‎‎(from different sources) promoted seedling growth of Triticum aestivum var Inqlab 91, but ‎increases were not significant in most of the cases. These studies revealed that mixed culture ‎combinations of bacteria from one source promoted seedling growth relatively more as compared ‎to mixed culture combinations from different sources under salt stress. In Phase III inoculation ‎effect of mixed culture bacteria was more pronounced in Helianthus annuus as compared to ‎Vigna radiata. Some mixed culture bacterial combinations in Phase II-A (ST-3,4; ST-1,2,4; ST-‎‎1,3,4; ST-2,3,4; HT-1,2; HT-1,3; HT-2,3; HT-1,2,3; RT-1,3; PT-1,2; PT-1,3; PT-2,3; PT-1,2,3), II-‎B (2b; 3e) and III (ST-2,3,4 inoculation in Helianthus annuus and HT-1,2,3 inoculation in Vigna ‎radiata) had synergistic growth stimulatory effects on seedling growth relative to their respective ‎monoculture inoculations under salt stress. Some bacterial inoculations (mono and mixed ‎cultures) have slightly deleterious effects on shoot, root (most of the inoculations) and seedling ‎lengths (in all the four Phases) at 0 mM NaCl treatment relative to non-inoculated respective ‎treatment. Under salt stress some bacterial combinations also caused decrease in shoot, root and ‎seedling lengths. With bacterial inoculations increases in fresh weight and decreases in dry ‎weight accumulation (except Phase IV where both increases and decreases were recorded), over ‎non-inoculated respective treatment, were recorded at 100 mM NaCl treatment. Bacterial ‎inoculations caused reduction in Na+ uptake (except Triticum aestivum var Rawal 87 in Phase I ‎and Vigna radiata in Phase III at 100 mM NaCl) by the seedlings at 100 mM NaCl, relative to ‎non- inoculated respective treatment. Under salt stress, stimulation in auxin and soluble protein ‎content (except a few cases) was recorded with bacterial inoculations. Activity of enzymes ‎peroxidase and acid phosphatase (excluding some cases) decreased with mono and mixed culture ‎bacterial inoculations under salt stress. These results suggest that decreased dry weight ‎accumulation, Na+ content and enzyme (peroxidase and acid phosphatase) activities and ‎increased auxin and soluble protein contents might be involved in stimulating the growth of ‎plants under NaCl stress.‎