1 Sino-German Joint Research Institute, Nanchang University, Nanchang 330047, China; 2 Institute of Biotechnology, Fujian Academy of Agricultural Sciences, Fuzhou 350003, China
Abstract：Aquatic plant Azolla is a mutualistic association among fern, Cyanobacterium and bacteria. At vegetative growth phase of Azolla, cyanobiont and bactbiont reside in the leaf cavity and symbiotically associate with the host plant. During the sexual reproduction of Azolla, cyanobiont was found to migrate into the sporocarp of Azolla. The presentt study aimed to explore the morphological and structural characteristics of the endo-bacteria during the sporocarp formation based on fluorescent microspcopic and electron microscopic observations using ultrathin sectioning, fluorescent in situ hybridazation and immno-gold labeling techniques. The results showed that the bacteria migrated into the developing sporocarp with cyanobiont and were positioned in the top area of pre-mature megasporocap. About 43% of the bacteria secreted numerous outer membrane vesicles. Some of them released DNA and fiberous polysaccharide-like substance, which became the main components of the matrix for the biofilm-like structure formed in the indusium of the megasporocarp. Around 25% bacterial cells were coated by the capsule, 17% of them accumulated poly-β-hydroxybutyrate granules in their cytoplasms, 9% of the bacteira appeared autophage-like or necrosis-like cell death. As megasporocarp matured completely, most of the bacteria were found to be inhabited in the biofilm-like matrix. These results illustrated that during the sporocarp formation of Azolla, the morphological and structural characteristics and cellular response of the endo-bactria might benefit them to keep the rhythm of the synchronous development with the host, then transfered to next symbiotic generation. Present study provides a new idea for artificial construction of crop-microbe symbiotic N2-fixing system.
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