The endophytic bacteria in the seeds of rice plants (Oryza sativa, cultivar Kinuhikari) cultivated on an experimental plot adjacent to a paddy field were studied as the seeds matured by comparing them with the bacteria on the surface of the seeds. Endophytic and surface bacteria were isolated using a nutrient broth and a diluted nutrient broth agar medium. The isolates were identified based on 16S rRNA gene sequences. Three genera (Paenibacillus, Acidovorax and Pantoea) and 2 genera (Stenotrophomonas and Rhizobium) were specific to the inside and to the surface of the seeds, respectively. Six genera (Bacillus, Curtobacterium, Methylobacterium, Sphingomonas, Xanthomonas and Micrococcus) were common to both the inside and the surface. As the seed matured, the flora of culturable endophytic bacteria changed in a different manner from that of culturable surface bacteria. More isolates tolerant of high osmotic pressure were found among the endophytes than among the surface bacteria, especially at the later stages of the maturation process. An increasing number of endophytic isolates exhibited amylase activity at the later stages.

Endophytic bacteria are considered to originate from the external environment. To examine the hypothesis that rice (Oryza sativa, cultivar Kinuhikari) seeds are a source of endophytic bacteria, we isolated endophytic bacteria from the shoots, remains of the seeds, and roots of rice seedlings that were aseptically cultivated in vitro from surfacedisinfected seeds. Of the various bacterial strains isolated, the closest relatives, identified by 16S rRNA gene sequencing, were: Bacillus firmus, B. fusiformis, B. pumilus, Caulobacter crescentus, Kocuria palustris, Micrococcus luteus, Methylobacterium fujisawaense, Me. radiotolerans, and Pantoea ananatis. The latter three species have been detected frequently inside both rice seedlings and mature rice plants. These results indicate that rice seeds are an important source of endophytic bacteria. The bacteria that colonize the seed interior appear to infect the subsequent generation via rice seeds and become the dominant endophytic species in the mature plant.Key words: culturable endophytic bacteria, rice seed, infection from seed to shoot, 16S rRNA gene sequence Various types of microorganisms, including fungi, actinomycetes, and other bacteria, are found inside plants and designated endophytes. In general, microorganisms having visibly harmful effects on plants are not considered endophytes. Some endophytic bacteria have beneficial effects on the host, e.g., promoting plant growth, strengthening resistance to pathogens, and increasing the supply of fixed nitrogen to the plant (9). Studies are underway to exploit these beneficial features for agriculture. However, our knowledge of endophytic bacterial ecology, including the origins of these bacteria, remains incomplete.Endophytic bacteria are considered to originate from the external environment and to enter the plant through the stomata, lenticles, wounds (including broken trichomes), areas of emergence of lateral roots, and germinating radicles (12). The notion of seeds as a source of endophytic bacteria remains controversial (9). Sato (39) did not detect endophytic bacteria in rice seeds. Mundt and Hinkle (27) obtained endophytes only from the damaged seeds of 28 plant types other than rice. Endophytic bacteria were detected in cotton seeds at viable population densities that ranged from 1×10 3 to 1×105 CFU g −1 fresh weight, as well as in the seeds of sweet corn at viable population densities of <1×10 CFU g −1 fresh weight (26). Endophytic bacterial populations increased from nondetectable levels in cotton seeds to detectable levels in radicles 4 days after placing surface-disinfected cotton seeds on water agar (1).Rice (Oryza sativa) is the most important cereal crop in the world, feeding more than 50% of the global population (8). In our search for endophytic bacteria in rice plants, we detected culturable endophytic bacteria in rice seeds at viable population densities that ranged from 10 2 to 10 6 CFU g −1 fresh weight (22,31). Endophytes in seeds may be carried over to the subsequent generation. Thus, in the p...

The culturable endophytic bacteria in the leaves and roots of rice plants (Oryza sativa, cultivar Kinuhikari) cultivated on an experimental plot adjacent to a paddy field were studied during the maturation process, and a comparison with the bacteria inside rice seeds reported in a previous study was conducted. Culturable epiphytic bacteria were also isolated from the leaves and compared with the endophytic bacteria. The isolates were identified based on their 16S rRNA gene sequences. Many endophytes closely related to Bacillus, Curtobacterium, Methylobacterium, Sphingomonas and Pantoea from the leaves, and to Bacillus, Brevibacillus, Mycobacterium, Enterobacter, and Chryseobacterium from the roots were isolated. The endophytic bacterial flora of the leaves was similar to that of the seeds, but differed from that of the roots. As the rice plant matured, the ratio of Gramnegative strains among the isolates increased in the leaves (the isolates related to Curtobacterium decreased in number and those related to Methylobacterium and Pantoea increased), whereas the ratio of Gram-negative strains decreased in the roots (the isolates related to the order Rhizobiales decreased in number and were replaced by strains related to Bacillus and Brevibacillus capable of forming endospores). The isolates from the inside and surface of the leaf were closely related to 13 and 19 species, respectively. Among these isolates, the following 6 species related to Bacillus pumilus, Curtobacterium citreum (or C. flaccumfaciens, C. pusillum as the closest species), Methylobacterium aquaticum, Methylobacterium fujisawaense (or M. radiotolerans), Sphingomonas melonis and Sphingomonas yabuuchiae were common to both sites and constituted the majority at both sites.