Kitazin-pea interaction: understanding the fungicide induced nodule alteration, cytotoxicity, oxidative damage and toxicity alleviation by Rhizobium leguminosarum

Realizing the severity of fungicidal toxicity to legumes and importance of fungicide tolerant rhizobia in legume production, kitazin tolerant (2400 mu g mL(-1)) strain RP1 was recovered from pea nodules and was identified as Rhizobium leguminosarum (accession no. KY940047). R. leguminosarum produced indole acetic acid (80.5 +/- 2.5 mL(-1)), siderophores: salicylic acid (54 +/- 7.3 mu g mL(-1)) and 2,3-dihydoxybenzoic acid (31.9 +/- 2.7 mu g mL(-1)), alpha-ketobutyrate (51 +/- 3.2 per mg per protein per hour), solubilized insoluble phosphate (29.5 +/- 1.8 mu g mL(-1)) and secreted 29.5 + 2.6 mu g mL(-1) exopolysaccharides, which, however, decreased consistently with gradually increasing kitazin concentrations. Beyond the tolerance level, kitazin caused structural damage and altered membrane integrity of RP1, as revealed under scanning (SEM) and confocal (CLSM) electron microscopy. Phytotoxicity of kitazin to peas was obvious under both in vitro and in vivo conditions. A significant reduction of 23, 68, 57 and 50% in germination, seedling vigor index, plumule length and radicle length was found at 2x kitazin compared to the control. Cellular damage and cytotoxicity induced by kitazin in membrane altered root cells was detected with acridine orange/propidium iodide (AO/PI) and Evans blue dye. A maximum increase of 1.72, 5.2, 9.3 and 1.72, 5.2, 9.3-fold in red and blue fluorescence was quantified at 1x, 2x, and 3x doses of kitazin, respectively. In contrast, application of R. leguminosarum RP1 alleviated toxicity and enhanced the length of plant organs, dry biomass, symbiotic attributes, photosynthetic pigments, nutrient uptake and grain features of peas comparatively uninoculated and fungicide-treated plants. Additionally, strain RP1 expressively reduced the antioxidant enzymes peroxidase, ascorbate peroxidase, guaiacol peroxidase, catalase and malondialdehyde contents by 10, 2.2, 11, 20 and 4% compared to stressed plants raised at 192 mu g kg(-1) soil. Moreover, a decline of 19, 21 and 20% in proline content extracted from roots, shoots and grains, respectively was recorded for R. leguminosarum inoculated pea plants grown with 96 mu g kg(-1) kitazin. Also, the SEM and CLSM of roots revealed the bacterial colonization. In conclusion, R. leguminosarum tolerated a higher level of kitazin, secreted plant growth promoting (PGP) bioactive molecules even under fungicide stress and significantly increased the performance of peas while reducing the levels of proline and antioxidant enzymes. So, it can safely be suggested to legume growers that RP1 strain could inexpensively be explored as an efficient biofertilizer for enhancing the production of legumes especially peas while growing even under fungicide (kitazin) enriched soils.