Infection And Colonization Of Rice Seedlings By The Plant Growth-Promoting Endophytic Diazotroph HAUM10

Using the endophytic diazotrophs to provide nitrogen for the rice can alleviate a range of hazards caused by the excessive usage of chemical fertilizers in environment. In this study, a diazotrophic endophyte HAUM10 was originally isolated from surface-sterilized Echinochloa crusgalli, which showed a 99% match with Pantoea sp. RL32.2 in 16S rDNA sequence. The isolate was identified as Pantoea sp on the basis of 16S rDNA sequence and morphology, physiological and biochemical characteristics. Diazotrophy of HAUM10 was confirmed by PCR amplification of nifH gene and acetylene reduction assay. The nitrogenase activity of HAUM10 was tested about 0.824μmol C2H4·mL-1·h-1 in modified Do semi-solid medium. HAUM10, through the liquid chromatography method, its IAA production capability was detected up to 15.44μg·mL-1, when cultured in CCM medium supplemented with tryptophan(0.2 g/L).To examine its interaction with rice, strain HAUM10 was marked with gfp gene by the triparental bacterial mating, and the marked strain was inoculated onto rice seedlings under axenic conditions. Comparing with wild-type HAUM10, this gfp-tagged HAUM10 was proved getting no significant change in growth and stability. Light and laser scanning confocal microscope were conducted to examine gfp-tagged HAUM10's infection and colonization of the rice Zhonghua 11 seedlings. A large number of gfp-tagged bacteria attached to the rice root hair zone at 3 DAI (days after inoculation), indicating this space offered the best way to get in, and cellulase and pectinase test also proved its high infection capability. A small amount of HAUM 10-gfp were observed in epidermal and cortical cells at 5 DAI and multiplied at 7 DAI, HAUM10-gfp were observed mainly localized in cortical,phloem cells and the large cells that were destined to die and become the lysigenous aerenchyma. HAUM10-gfp were also observed in transverse and longitudinal sections of the leaf sheath, localizing in phloem and parenchyma cells. In general, the bacteria rarely penetrated into the vascular system. No bacteria were observed in uninoculated control plant tissues.Pot experiments comprised five levels of inoculation viz. (1) control pots treated with uninoculated bacteria (CK); (2) HAUM10, (3) a combination of HMLR8 and HAUM10, (4) HAUM10 and HMLR13, (5) a combination of all three strains, Population dynamics of endophytes within rice different parts and the promotion to it were surveyed. There was no significant differences of colonization density among the treatments. The bacterial population denisty in root, stem and leaf raised up to 108 CFU/g fresh weight, 106 CFU/g fw,103 CFU/g fw at 7 DAI; and then decreased gradually. Endophytic bacteria in rice migrated from the root to the stem and the leaves, and the amount of bacteria was as follows:root>stem>leaf. Pot experiments showed that different endophyte inoculation systems can increase chlorophyll a and chlorophyll content to promote the photosynthesis of rice; nitrogen and phosphorus content in above and below ground rice tissues were increased compared to uninoculated rice plants at 45 DAI. The nitrogen and phosphorus content of leaves endophyte inoculated rice was found lower than the control, it is indicated that HAUM10 possibly transport the nitrogen and phosphorus from leaf to spike in the late growth stage. In addition, a combination of HAUM10 and HMLR13 mixture system promote the rice booting and earing than others. There was no symptoms of pathogenic disease in rice growth period in this study, and no endophyte was seen in inoculated surfaced-sterilized rice seeds.