Physiological and genetic study on mechanisms of growth promotion and salt tolerance improvement in rice seedlings by inoculation with biofertilizer microorganism Bacillus pumilus TUAT1 strain

概要

Rice is most broadly expended staple food everywhere throughout the world, particularly in Asia. It is very sensitive to salt stress at seedling stage. Yield is drastically reduced under salt stress. Mitigation of salt stress at the seedling stage could minimize the yield loss at later stages as the rice plant is reported to be the most susceptible to salinity at vegetative period than the reproductive period. Plant Grow出Promoting Rhizobacteria (PGPR) have been indicated as efficient overcome the salt stress at seedling stage. PGPR used in this study was Bacillus pumilus TUATl strain which used as an ingredient of commercialized biofertilizer "Kikuichi". Previous reports in rice and several other plants had suggested that applying "Kikuichi" or TUATl at the nursery stage enhances the grow出of seedlings as well as the yield. However, the mechanism by which inoculation with TUATl strain promotes the growth of rice seedlings has not been fully elucidated. In addition, it was not clarified whether inoculation with TUATl strain would increase the salt tolerance of rice seedlings. In this study, experiments were conducted to elucidate the mechanism by which TUATl strain inoculation promotes growth ofrice seedlings, to confirm salt tolerance of the seedlings is increased, and to identify genes involved in varietal differences in the increasement of salt tolerance.

The study was designed to track the colonization of TUATI strain as well as determination the effects of inoculation with spores and vegetative cells of TUATl strain. Moreover, elucidation the possible involvement of NO production and expression of root formation related genes in growth-promoting effect of TUATl strain on rice seedlings. The results showed that inoculation of spores at a density of lx107 colony-forming units (CFU) per mL yielded a significant increase in all traits compared to the control. In contrast, inoculation with vegetative cells did not yield significant effects in several traits, including the numbers of crown roots and lateral roots. CFUs of TUATl strain increased with time and maintained to a higher number in the root zone than at the stem base of rice seedlings. TUATl inoculation with spore led to increased accumulation of CRL5 transcripts and decreased accumulation of WOXJ 1 transcripts in the basal stem, where crown root initiation occurs, as well as involved to promoting the NO production. These results provide partial clarification of the mechanism of TUATI's growth promotion in rice.

Hypothesis that inoculation of spore of TUATI strain promotes the early growth of rice seedlings under salt stress condition by increasing salt tolerance was tested.

"Hitomebore" was used to clarified the effect of TUATI inoculation on the growth of seedlings under different salt treatments from different growth stages. Four varieties, "Kasalath", "Nipponbare", "Hitomebore", and "Koshihikari", were used to comprehensively investigate the differences in rice seedlings responsiveness to TUATl strains within and between varieties under salt stress. The results obtained in this chapter clearly indicates the growth of seedlings significantly decreased with the increase of salinity levels. It was also confirmed that the two varieties "Kasalath" and "Nipponbare" had low inoculation effect on increasing salt tolerance but other varieties had it. In addition, the inoculation effect was also different depending on the concentration and exposure time of salt stress.

Based on the hypothesis that the genetic variation is involved in varietal difference in the promoting effect of TUATl inoculation on salt tolerance, the seedlings of 70 rice varieties were inoculated with TUATl under salt treatment. The growth and salt tolerance of rice seedlings showed a wide range of varietal differences in both inoculation and non-inoculation of TUATl. Therefore, using these varietal differences, it was attempted to identify genes related to varietal differences in the inoculation effect of TUATl strain by GWAS. Although there was no significant SNPs obtained, the results obtained by GWAS suggested that genes containing the selected candidate SNPs play an important role in the enhancement of salinity tolerance by TUATl inoculation.

Furthermore, the detection method of causative genes in the experiment of this chapter was effective and provided meaningful clues for the future studies of PGPR involved in abiotic stress interaction.

In conclusion, this study provides new insights and hypotheses on the mechanism by which inoculation with B. pumilus TUATl promotes the growth of rice seedlings. These findings and further studies to test our hypotheses are expected not only to lead to the discovery of new mechanism(s) of plant growth promotion by PGPRs, but also to provide clues to the development of application methods for inducing consistent effects of biofertilization using this bacterium. On the other hand, the causative genes detection method by GWAS is effective and provides meaningful clues for the future studies of PGPR involved in abiotic stress interaction. Finally, it demonstrates that use of the inoculation with TUATl significantly improves the growth, root system of rice seedlings, thus allowing them to cope with imposed salinity stress.

They have shown their vast potential in remediation and productivity enhancement of agro-ecosystems suffering from problems of salinity in Mekong Delta.