Culturable Surface and Endophytic Bacterial Flora of the Maturing Seeds of Rice Plants (Oryza Sativa) Cultivated in a Paddy Field

Microbes Environ. Vol. 21, No. 2, 86–100, 2006 http://wwwsoc.nii.ac.jp/jsme2/

Culturable Surface and Endophytic Bacterial Flora of the Maturing Seeds of Rice Plants (Oryza sativa) Cultivated in a Paddy Field

HIRONOBU MANO1, FUMIKO TANAKA1, ASUKA WATANABE1, HIROKO KAGA1, SUGURU OKUNISHI2 and HISAO MORISAKI1*

1 Department of Bioscience and Biotechnology, Faculty of Science and Engineering, Ritsumeikan University, 1–1–1 Noji-higashi, Kusatsu 525–8577, Japan 2 Center for Promotion of The COE Program, Ritsumeikan University, 1–1–1 Noji-higashi, Kusatsu 525–8577, Japan

(Received December 8, 2005—Accepted February 8, 2006)

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 (Paeniba- cillus, 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, Sphingomo- nas, 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 exhibit- ed amylase activity at the later stages.

Key words: rice seed, paddy field, culturable bacterial flora, 16S rRNA gene sequence, osmotic pressure

myxa, bacteria which have been isolated from sterilized rice Introduction seedlings and which protect the seedlings from pathogens23). Various kinds of microorganisms have been found inside All of these studies focused on the endophytes in the stem plants. These microbes (endophytes) include fungi8), and root of the plant. Little is known about the endophytic actinomycetes3) and bacteria27). Microbial endophytes are bacteria inside rice seeds. defined as microorganisms detected inside surface-sterilized Most plants regenerate by producing seeds, and in the plants14). Endophytes can be either pathogenic or nonpatho- case of rice, almost half of its life is spent in the production genic to their host. of seeds. The seeds must maintain the ability to germinate Although rice is one of the most important crops agricul- properly for a long period of time. The endophytes may turally and economically, the study of endophytic bacteria have an important effect on the formation and germination in rice plants has been limited to the endophytes colonizing of seeds. The endophytes have been regarded to be derived the intercellular spaces of the leaves6), Herbaspirillum sp. from vegetative plant material, rhizosphere soil, and phyl- which fixes nitrogen as an endophyte in the young seedlings loplane in addition to seeds. Thus the investigation of seed of wild rice5), and Enterobacter cloacae and Bacillus poly- endophytes seems to be important for further understanding endophyte ecology. * Corresponding author; E-mail: [email protected], Tel: A rice plant usually takes about 60 days to mature after +81–77–561–2767, Fax: +81–77–561–2659 flowering. During the maturation process, the amounts and Culturable Bacterial Flora in a Paddy Field Rice-Seed 87 concentrations of water and sugar in the seed change days after being planted as a seedling. Although, the rice greatly15). The endophytes seem to be affected by the drastic seed matures ca. 60 days after flowering, in paddy fields the changes taking place inside the seed. Previously25), we clari- rice seeds are harvested 30–40 days after flowering. We fied the nature of culturable bacterial flora in the seed dur- sampled rice seeds aseptically at three different stages of ing maturation by dividing the maturation process into three maturation, i.e., on the 10th (the early stage), 20th (the middle stages: early, middle and late. We found that a great change stage) and 30th (the late stage) day after flowering. A rice takes place in the culturable bacterial flora within the seed plant average in height and growing appearance was select- during maturation, in contrast to the slight change at the ed from the 2 m×2 m plot. The samples were brought back seed’s surface. to the laboratory in a sterilized box to prevent contamina- To build on and make comparisons with the findings ob- tion, and were used in the experiments within 1 hour. tained using pot-cultivated rice, our next task was to exam- ine the culturable bacterial flora of rice seeds cultivated in Plate count of surface and endophytic bacteria an environment similar to a paddy field. Thus, in the present Bacteria were isolated from the rice-seed surface as fol- study, we tried to clarify the characteristics of the culturable lows. The hulls were removed from the rice seeds with ster- bacterial flora of rice cultivated on an experimental plot ad- ilized forceps, and the seeds (0.5 g; about 30 seeds) were jacent to a paddy field. put into a test tube containing 10 mL of sterilized water. Af- Endophytes are considered to enter plants through dam- ter washing by pipetting, the supernatant was serially dilut- aged surface tissue and/or stoma17). This seems to be the ed with sterilized distilled water. Then, a portion of the su- case for the endophytes of rice seeds. In our previous study pernatant (1.0 mL) was mixed with ca. 20 mL of a nutrient concerning pot-cultivated rice25) and the study by broth (NB) agar (1.5 wt %) medium (containing 10 g of Elbeltagy5) concerning wild rice and cultivated rice, many Polypepton (Wako Pure Chemical Industries, Osaka, Ja- of the endophytes were motile. Motility may play an im- pan), 10 g of bonito extract (Wako Pure Chemical Indus- portant role in the entering of plants. During the maturation tries), 5 g of NaCl in 1000 mL of tap water; the pH was ad- process, the endophytes seem to be exposed to high osmotic justed to 7.2) or mixed with a 100-fold diluted nutrient broth pressure caused by two parallel events, the accumulation of (DNB) agar (1.5 wt %) medium. These plates were incubat- starch and the loss of water. Endophytes may also possess ed at 27°C. amylase activity to utilize starch and to resume growth after For the isolation of the rice-seed endophytes, the surface survival for long periods in seeds. Therefore we evaluated of the rice seeds (0.5 g) was washed with distilled water and the growth of isolates from rice in the presence of high con- then with a 75% ethanol solution for 1 minute. The seeds centrations of sucrose (0.6, 1.2 or 1.8 M), and determined were again washed with sterilized distilled water, and then whether the isolates have amylase activity. with a 1% sodium hypochlorite solution (Nacalai Tesque, The present study revealed the following regarding rice Kyoto, Japan) for 1 minute. After another wash with steril- seeds cultivated in an environment similar to a paddy field: ized water, the surface-sterilized rice seeds were crushed 1) the culturable bacterial flora inside and on the surface of with a sterilized mortar and pestle. The sub-samples were the rice seed differs; 2) it changes as the rice plant matures; ultrasonically washed (BRANSONIC 2510 J-MT, 100 W; and 3) in the later stages of the maturation process, the en- Yamato Scientific Co., Tokyo, Japan) for 15 seconds and dophytes develop tolerance to high osmotic pressure. then serially diluted with sterilized water. The confirmation of surface sterilization was conducted by culturing the ster- Materials and Methods ilized rice seeds on NB and DNB agar media. The diluted suspension was mixed with the NB and DNB agar media. Preparation of the rice seeds The plates were incubated at 27°C. Oryza sativa (cultivar Kinuhikari) was cultivated on a The colonies appearing on the plates were counted every small experimental plot (2 m×2 m) adjacent to a paddy field day for about 30 days. After 30 days, strains were isolated at in Otsu City in 2003. The rice seedlings were planted in random from the colonies that appeared on the plates. mid-May, herbicide was sprayed in late May, the soil was additionally fertilized in early June, the water was drained DNA extraction off between the middle and the end of June, the soil was fer- We isolated the surface and endophytic bacteria from the tilized for cropping in mid-July, and the rice was harvested colonies as follows. Fifteen strains were isolated at random in mid-September. Usually, the rice plant flowers about 100 from each sample, from either the surface or the inside of 88 MANO et al. the rice seed, at different stages of maturation and in differ- termined by searching the DDBJ (http://www.ddbj.nig. ent media. Fewer isolates from the NB agar medium could ac.jp/Welcome-j.html) database with the BLAST program, be used for further investigation, being non-culturable when and the sequences were then aligned using the CLUSTAL X re-inoculated to NB agar medium from the stab soft-agar program (version 1.83)32). A phylogenetic tree was con- medium used for preservation. The isolates were identified structed by the neighbor-joining method28) with 1000 boot- based on an analysis of the 16S rRNA gene sequence. The strap replicates in CLUSTAL X. cells in the NB liquid medium were harvested at the expo- nential-growth phase (1.5 mL) by centrifugation and resus- Accession numbers pended in 40 µL of TE buffer. Then, 10 µL of proteinase K All of the partial sequences of the 16S rRNA gene deter- (1 mg/mL) and 50 µL of BL buffer (containing 40 mM Tris mined in this study have been submitted to the DDBJ data- aminomethane, 1 mM of EDTA·2Na, 1% of Tween 20 and base under the accession numbers AB242926–AB242988. 0.5% of Nonidet P40) were added to the suspension. After incubation at 60°C for 30 minutes, the supernatant was cen- Morphological and physiological characterization of the trifuged and used for amplification by PCR. isolates All of the isolates were characterized by examining their PCR amplification and sequencing of the 16S rRNA morphological and physiological traits. We examined the genes motility, tolerance to high osmotic pressure and amylase ac- The PCR mixture consisted of 0.75 units of ExTaq (Taka- tivity of the cells cultured in NB or DNB liquid medium for ra Bio, Shiga, Japan), 1×Taq polymerase buffer, 200 µM 1–2 days at 100 rpm and 27°C. When a strain was isolated dNTPs, 50 pmol of each primer and the extracted DNA (50– from the NB (or DNB) agar medium, the isolate was cul- 100 ng) in a 20 µL reaction mixture. The primers used for tured in NB (or DNB) liquid medium. Endospore formation amplifying the 16S rRNA genes of the isolated bacteria was checked with an optical microscope after a few weeks were 25F (5'-AGTTTGATCCTGGCTC-3') as the forward of culture in a liquid medium. primer and 1510R (5'-GGCTACCTTGTTACGA-3') as the reverse primer, corresponding to positions 10–26 and 1495– The growth of isolates under high osmotic pressure 1510, respectively, of the 16S rRNA gene sequence of To determine whether the isolates could grow under high Escherichia coli. osmotic pressure, they were cultured in NB medium con- The thermal cycling program used was as follows: initial taining 0.6, 1.2 or 1.8 M sucrose. Escherichia coli strain denaturation at 95°C for 5 min, 30 cycles of 95°C for 1 min, IAM 12119, Bacillus subtilis strain 168, Micrococcus luteus 52°C for 2 min, and 72°C for 2 min, and a final extension at strain IFO 03763, and Pseudomonas syringae strain NIAES 72°C for 10 min. The amplified PCR products were ana- 1309 were also examined for comparison. The strains exam- lyzed by electrophoresis on 1% LO3 (Takara Bio) agarose ined were cultured in NB medium with or without sucrose gels in 1×TAE. A 200-bp DNA Marker (BEXEL Biotech- for 3 days, and the optical density at 540 nm was measured. nology, Union, USA) was used as the molecular weight standard, and stained with ethidium bromide (1 µg/mL with Amylase activity 1×TAE Buffer) for visualization. The PCR products were Each strain was cultured in LB liquid medium containing purified with a PCR-M CleanUp System (VIOGENE, 1% soluble starch for 2 days at 100 rpm and 27°C. After Sunnyvale, USA) in accordance with the manufacturer’s in- centrifugation, the supernatant (75 µL) was soaked on a structions. sterilized paper disc (diameter 8 mm) put on an 2% agar The sequences were determined using the 907R (5'- medium containing 1% starch and incubated for 24 hours at CCGTCAATTCCTTTGAGTTT-3') primer with the genetic 27°C. After removal of the paper disk, ca. 80 µL of a Lugol analyzer ABI PRISM AVANT 3100 (PE Biosystems, Fos- solution containing 0.067% iodine and 0.13% KI was ter, USA). The BigDye Terminator Cycle Sequencing poured on the agar plate. The extent of transparence of the Ready Reaction Kit ver. 3.1 (PE Biosystems) was used in clear zone around the position where the paper disk had accordance with the manufacturer’s directions. been placed, because there was no reaction between the iodine and decomposed starch, was compared with Bacillus Phylogenetic analysis subtilis strain 168 used as a control strain. Approximately 550 bp were used for the phylogenetic analysis. The phylogenetic position of the isolates were de- Culturable Bacterial Flora in a Paddy Field Rice-Seed 89

appeared. It has been revealed that the isolates taking longer 18) Results and Discussion to form visible colonies, show smaller growth rates . The culturable bacterial flora seems to contain mainly fast- The formation of colonies on the NB and DNB agar growing strains, because in our experiment, almost all colo- media nies appeared within 10 days. The number of endophytic The number of colonies appearing on the NB and DNB bacterial colonies on the NB agar medium was greater than agar media during the 30 days of incubation increased, as or comparable with that on DNB medium. Surface bacteria shown in Fig. 1. Most surface and endophytic bacterial col- formed a slightly greater number of colonies on the DNB onies appeared within 10 days after the initiation of incuba- than NB agar medium in the early stages of the maturation tion, whereas few colonies appeared after 10 days. This ten- process, although colony numbers were almost the same in dency was observed on both the NB and the DNB medium, the later stages (Figs. 1–2). These results may indicate that regardless of the stage in the maturation of the rice seed the bacteria from the surface and the inside of the rice seed (early (10th day), middle (20th day) or late (30th day)). From adapt to high-nutrient conditions, in contrast to bacteria in the pattern of increase in the number of colonies with the in- soil, which form more colonies on DNB than on NB cubation time (colony forming curve; CFC), it is possible to medium11,18,24). divide the isolates into groups according to when colonies The total number of colonies which appeared during the

Fig. 1. Increase in the number of colonies on NB ( ) and DNB ( ) agar media with incubation for surface (1) and endophytic bacteria (2) of the rice seed. The plates were incubated at 27°C. The average of 2 plates was plotted as the colony number. 90 MANO et al.

Fig. 2. The total number of colonies which appeared during 30 days of incubation on NB ( ) and DNB ( ) agar medium for the surface bacteria (A) and endophytes (B) of rice seeds at each maturation stage.

30 days of incubation on the NB and DNB agar media at 84% in the xylem of citrus roots10) and 94% in alfalfa each stage of maturation is shown in Fig. 2. The total num- roots9). ber of endophytes per gram of rice capable of forming colo- However, the ratio of Gram-negative strains among the nies ranged from 102 to 104 on the NB agar medium and 103 isolates changed greatly with maturation of the seeds. In the to 104 on the DNB agar medium. The number of surface seeds early on (10 days after flowering), all of the isolates bacteria capable of forming colonies was greater than that were Gram-negative, whereas Gram-positive isolates ap- of endophytic bacteria, ranging from 106 to 107 on both the peared with the maturation of rice seeds: 70% at the middle NB and the DNB agar medium. stage (20 days after flowering) and 45% at the late stage (30 days after flowering) (see Table 1). The culturable bacterial flora in rice seeds and the The environment inside the rice seed changes with the changes it undergoes during the maturing process maturation process and this change seems to affect the mi- The isolates from the inside of the rice seeds were ana- crobes inhabiting it. Starch begins to accumulate in the rice lyzed based on their 16S rRNA gene sequences. The closest plant at about 4 days and stops at 30 to 35 days after the relatives of the isolates are shown in Table 1, and phyloge- flowering. During this period, the water content of the rice netic trees of these isolates are shown with some reference seed decreases from ca. 80% to 20–25% and remains con- strains in Fig. 3. stant thereafter16). The endophytes seem to be exposed to the It is noteworthy that fewer of the strains grown on the NB drastic changes taking place inside the seed, which results in medium could be re-cultured after isolation compared with a change in ratio between the Gram-negative and Gram- the strains grown on DNB medium. These strains may re- positive isolates, as mentioned above, and in the successive quire a hitherto undetected rice-seed constituent as a nutri- predominance of different bacterial species, as discussed ent for growth or may easily enter an unculturable state. We below. are now investigating the growth conditions of these strains. The closest relatives of the isolates from inside the seed Among the endophytes, more Gram-negative strains belonged to 6 main genera, as shown in Fig. 3. These genera (63%) were isolated than Gram-positive strains (see Table seemed to be divided into 3 groups: 1) the genera whose 1); strains belonging to the Gammaproteobacteria were strains decreased in number with the maturation process, dominant (34%), and many strains belonged to the phyla such as Sphingomonas and Methylobacterium, 2) the genera Firmicutes (28%) and Alphaproteobacteria (25%) (see Fig. whose strains increased in number, such as Bacillus and 3). It has been reported that, among endophytic bacteria, Curtobacterium, and 3) the genera whose strains were Gram-negative strains are usually dominant14). Seventy one found at the early and late stages, such as Xanthomonas and percent of the endophytes from wild and cultivated rice Pantoea. plants have been reported to be Gram-negative5). Other At the early and middle stages, strains closely related to studies have also reported a dominance of Gram-negative Sphingomonas paucimobilis were isolated. Strains close to strains among endophytes: ca. 75% in corn and cotton21), S. paucimobilis, which have nitrogen-fixing ability, have al- Culturable Bacterial Flora in a Paddy Field Rice-Seed 91

Table 1. The physiological characteristics of the isolates from the surface and inside of rice seed

Isolatea Motility The effect of osmotic pressureb Amylasec Closest related straind (accession no.) Similarity Early stage NB (NB+0.6 M) (NB+1.2 M) (NB+1.8 M) /(NB) /(NB) /(NB) Surface Pd-S-(s)-e-N-1(3) + 0.41 0.41 0.37 0.19 (+) Bacillus subtilis (AB177641)** 662/663 (99.8) Pd-S-(s)-e-D-1(4) + 1.13 0.51 0.08 0.00 − Stenotrophomonas maltophilia (AY445079) 707/709 (99.7) Pd-S-(s)-e-D-2(5) + 1.31 0.52 0.05 0.00 − Stenotrophomonas maltophilia (AY445079) 664/665 (99.8) Pd-S-(s)-e-D-3(5) + 1.23 0.38 0.09 0.01 − Curtobacterium flaccumfaciens pv. basellae 566/571 (99.1) (AY273210)** Pd-S-(s)-e-D-4(6) + 1.52 0.46 0.03 0.00 − Stenotrophomonas maltophilia (AY445079) 548/549 (99.8) Pd-S-(s)-e-D-5(20) + 1.13 0.29 0.00 0.00 +++ Stenotrophomonas maltophilia (AY445079) 567/567 (100) Pd-S-(s)-e-D-6(4) + 1.49 0.24 0.00 0.00 − Sphingomonas yabuuchiae (AB071955) 561/562 (99.8) Pd-S-(s)-e-D-7(4) + 1.60 0.05 0.00 0.00 − Sphingomonas yabuuchiae (AB071955) 689/691 (99.7) Pd-S-(s)-e-D-8(4) + 1.55 0.25 0.03 0.00 − Sphingomonas yabuuchiae (AB071955) 655/655 (100) Pd-S-(s)-e-D-9(16) + 1.64 0.43 0.00 0.00 − Sphingomonas yabuuchiae (AB071955) 736/737 (99.9) Endophyte Pd-E-(s)-e-N-1(3) + 1.05 0.55 0.48 0.18 − Xanthomonas translucens pv. poae 746/747 (99.9) (AY572961) Pd-E-(s)-e-N-2(19) − 1.15 1.32 0.53 0.12 − Pantoea ananatis (DQ133548) 610/611 (99.8) Pd-E-(s)-e-D-1(1) + 0.97 1.38 0.59 0.17 − Pantoea ananatis (DQ133548) 662/662 (100) Pd-E-(s)-e-D-2(3) + 1.14 0.63 0.22 0.07 − Methylobacterium aquaticum (AJ785572) 540/542 (99.6) Pd-E-(s)-e-D-3(3) + 0.86 0.43 0.11 0.03 + Methylobacterium aquaticum (AJ785572) 566/567 (99.8) Pd-E-(s)-e-D-4(3) + 0.42 0.23 0.00 0.00 − Sphingomonas melonis (AB055863) 578/581 (99.5) Pd-E-(s)-e-D-5(9) + 1.27 0.21 0.00 0.00 − Sphingomonas yabuuchiae (AB071955) 657/657 (100) Pd-E-(s)-e-D-6(3) + 1.25 0.16 0.00 0.00 − Sphingomonas yabuuchiae (AB071955) 610/612 (99.7) Pd-E-(s)-e-D-7(3)* +−Methylobacterium aquaticum (AJ785572) 593/594 (99.8) Pd-E-(s)-e-D-8(1) + 1.07 1.28 0.39 0.19 − Pantoea ananatis (DQ133548) 570/572 (99.7) Pd-E-(s)-e-D-9(15) + 0.87 1.75 0.46 0.20 − Pantoea ananatis (DQ133548) 597/600 (99.5) Middle stage Surface Pd-S-(s)-m-N-1(2) − 0.22 0.24 0.12 0.00 − Micrococcus luteus (AY167858)** 689/690 (99.9) Pd-S-(s)-m-D-1(2) + 1.22 0.41 0.06 0.01 − Sphingomonas yabuuchiae (AB071955) 713/715 (99.7) Pd-S-(s)-m-D-2(2) + 0.78 0.83 0.21 0.13 − Sphingomonas yabuuchiae (AB071955) 664/664 (100) Pd-S-(s)-m-D-3(3) + 0.21 0.00 0.00 0.00 − Methylobacterium aquaticum (AJ785572) 588/592 (99.3) Pd-S-(s)-m-D-4(3) + 0.88 0.73 0.35 0.15 − Methylobacterium aquaticum (AJ785572) 607/612 (99.2) Pd-S-(s)-m-D-5(13) + 0.08 0.00 0.06 0.00 − Methylobacterium aquaticum (AJ785572) 604/606 (99.7) Pd-S-(s)-m-D-6(3) + 0.23 0.00 0.00 0.00 − Methylobacterium aquaticum (AJ785572) 583/585 (99.7) Pd-S-(s)-m-D-7(4) + 0.55 0.00 0.00 0.00 − Methylobacterium aquaticum (AJ785572) 624/626 (99.7) Pd-S-(s)-m-D-8(4) + 1.46 0.41 0.10 0.04 − Sphingomonas yabuuchiae (AB071955) 681/685 (99.4) Pd-S-(s)-m-D-9(3) + 1.11 0.06 0.00 0.00 − Sphingomonas yabuuchiae (AB071955) 662/670 (98.8) Pd-S-(s)-m-D-10(11) + 1.13 1.41 0.42 0.12 − Sphingomonas yabuuchiae (AB071955) 618/618 (100) Endophyte Pd-E-(s)-m-N-1(2) + 0.23 1.82 0.89 0.70 + Bacillus subtilis (AB177641)** 569/571 (99.6) Pd-E-(s)-m-N-2(2)* +−Bacillus subtilis (AB177641)** 700/702 (99.7) Pd-E-(s)-m-N-3(2) − 0.34 1.18 0.70 0.35 + Bacillus subtilis (AB177641)** 743/747 (99.5) Pd-E-(s)-m-N-4(2) + 0.26 1.14 0.62 0.31 − Bacillus subtilis (AB177641)** 705/706 (99.9) Pd-E-(s)-m-N-5(11) + 1.04 1.01 0.37 0.14 − Bacillus pumilus (AM062682)** 675/678 (99.6) Pd-E-(s)-m-N-6(2) − 0.59 0.44 0.07 0.02 − Micrococcus luteus (AF501366)** 695/698 (99.6)

(Continued) 92 MANO et al.

(Continued)

Pd-E-(s)-m-D-1(4) + 1.58 0.02 0.00 0.00 − Sphingomonas yabuuchiae (AB071955) 592/597 (99.2) Pd-E-(s)-m-D-2(4) + 1.48 0.18 0.04 0.00 − Sphingomonas yabuuchiae (AB071955) 610/620 (98.4) Pd-E-(s)-m-D-3(4) + 1.50 0.21 0.00 0.00 − Acidovorax sp. (AY566579) 681/691 (98.4) Pd-E-(s)-m-D-4(12) + 0.74 1.24 0.39 0.15 ++ Curtobacterium flaccumfaciens pv. basellae 587/588 (99.8) (AY273210)** Late stage Surface Pd-S-(s)-l-D-1(2) + 0.40 0.50 0.01 0.00 − Agrobacterium larrymoorei (Z30542) 680/680 (100) Pd-S-(s)-l-D-2(2) + 0.97 0.32 0.00 0.00 + Xanthomonas translucens pv. poae 730/731 (99.9) (AY572961) Pd-S-(s)-l-D-3(2) + 0.87 0.30 0.00 0.00 − Xanthomonas translucens pv. poae 677/678 (99.9) (AY572961) Pd-S-(s)-l-D-4(3) + 0.21 0.00 0.00 0.00 − Methylobacterium aquaticum (AJ785572) 632/636 (99.4) Pd-S-(s)-l-D-5(2) + 1.42 0.16 0.00 0.00 − Sphingomonas yabuuchiae (AB071955) 686/687 (99.9) Pd-S-(s)-l-D-6(2) − 0.21 0.59 0.00 0.00 − Agrobacterium larrymoorei (Z30542) 725/726 (99.9) Pd-S-(s)-l-D-7(4) + 1.38 0.00 0.00 0.00 − Methylobacterium aquaticum (AJ785572) 565/567 (99.6) Pd-S-(s)-l-D-8(4) + 1.07 0.27 0.00 0.00 ++ Xanthomonas translucens pv. poae 572/574 (99.7) (AY572961) Pd-S-(s)-l-D-9(6) + 1.00 1.33 0.52 0.18 − Xanthomonas translucens pv. poae 732/737 (99.3) (AY572961) Pd-S-(s)-l-D-10(3) + 1.25 0.38 0.00 0.00 + Xanthomonas translucens pv. poae 662/663 (99.8) (AY572961) Endophyte Pd-E-(s)-l-N-1(1) + 0.20 0.71 0.22 0.00 + Paenibacillus amylolyticus (AB115960)** 675/675 (100) Pd-E-(s)-l-N-2(5) + 1.18 1.12 0.33 0.19 − Pantoea ananatis (DQ133548) 600/601 (99.9) Pd-E-(s)-l-D-1(2) + 0.84 1.28 0.64 0.18 − Pantoea ananatis (DQ133548) 576/589 (97.8) Pd-E-(s)-l-D-2(2) + 1.27 1.02 0.36 0.10 − Bacillus pumilus (AM062682)** 689/691 (99.7) Pd-E-(s)-l-D-3(2) + 0.81 1.65 0.57 0.25 − Pantoea ananatis (DQ133548) 739/742 (99.6) Pd-E-(s)-l-D-4(3) + 1.28 0.30 0.00 0.00 ++ Xanthomonas translucens pv. poae 605/605 (100) (AY572961) Pd-E-(s)-l-D-5(3) + 0.88 1.75 0.54 0.14 (+) Bacillus pumilus (AM062682)** 702/703 (99.9) Pd-E-(s)-l-D-6(4) + 0.15 3.84 0.99 0.01 − Curtobacterium flaccumfaciens pv. basellae 777/778 (99.9) (AY273210)** Pd-E-(s)-l-D-7(6) + 1.11 0.20 0.00 0.00 − Xanthomonas translucens pv. poae 553/556 (99.5) (AY572961) Pd-E-(s)-l-D-8(3) + 0.97 1.49 0.62 0.26 − Bacillus pumilus (AM062682)** 643/643 (100) Pd-E-(s)-l-D-9(20) + 0.96 0.28 0.00 0.00 +++ Xanthomonas translucens pv. poae 573/574 (99.8) (AY572961) a The first abbreviation ‘Pd’ means that the seed was cultivated in an experimental plot near a paddy field. The second indicates Surface (S) or Endophytic (E) bacteria. The third in brackets means ‘Seed’. The fourth, e, m or l means early, middle and late stage of maturation, respectively. The fifth means NB (N) or DNB (D) medium. The digit following the letters is the serial number of the strain. The number in brackets is the day when the colony appeared on the agar plate. b The first value indicates the optical density (O.D. 540 nm) of the liquid NB medium cultured for 3 days for each strain. The second, third and fourth values indicate the ratio of O.D. 540 nm for 3 days of culture in NB medium containing 0.6 M, 1.2 M and 1.8 M sucrose to that of NB medium, respectively. c When the extent of transparence of the clear zone due to no reaction of iodine with decomposed starch was similar to that of Bacillus subtilis 168, the symbol ‘+’ was used. The symbol ‘++’ means that the extent of clearness was higher and the symbol ‘+++’ means the instantaneous appearance of the clear zone after addition of the iodine solution. The symbol ‘(+)’ and ‘−’ indicates a weaker clearness and no appearance of a clear zone, respectively. d The closest relative by sequence comparison. * No or little growth. ** Gram-positive bacterium. Culturable Bacterial Flora in a Paddy Field Rice-Seed 93

Fig. 3. Phylogenetic tree for the isolates from the inside of rice seed based on 16S rRNA gene sequences. The phylogenetic tree was calculated based on approximately 450 nucleotides by the neighbor-joining method. The results of 1000 bootstrap trials are shown at the nodes. Sym- bols: , isolates from the early stage; , isolates from the middle stage; , isolates from the late stage of maturation. 94 MANO et al. ready been isolated from the rice rhizosphere1). The nitro- In the present study, we isolated strains closely related to gen-fixing ability of the isolates examined in the present Pantoea ananatis as well as strains close to Xanthomonas study should be investigated further. All strains close to Me- translucens at the early and late stages in the maturation of thylobacterium aquaticum were isolated at an early stage. the rice seed. In a previous study, we also isolated many en- Methylobacterium spp. have also been isolated as endo- dophytes close to Pantoea ananatis (22%) (see Table 1). phytes from the plant body of several kinds of wild rice and Strains closely related to Pantoea ananatis have been isolat- cultivated rice5). The function fulfilled by the isolates close- ed from wild rice (Oryza alta)5), and pot-cultivated rice ly related to M. aquaticum in the rice plant is another sub- (Oryza sativa) as well25). Pantoea ananatis may be a com- ject for further study. mon endophytic bacterium in rice seeds regardless of the It is noteworthy that close relatives of Bacillus subtilis, B. species of rice and the cultivating conditions. The isolates pumilus, and Paenibacillus amylolyticus, which can form close to Pantoea ananatis examined in the present study endospores, were dominant at the later stages: 50% and were tolerant of high osmotic pressure. This may make Pan- 36% at the middle and late stage, respectively (see Table 1). toea ananatis a common endophytic bacterium. X. trans- In our previous study25), strains closely related to Bacillus lucens (X. campestris pv. translucens) is known as a patho- sp. were isolated at a much higher ratio (73%) from pot-cul- gen in barley and wheat (Graminieae), and has been shown tivated rice. The formation of endospores was confirmed to propagate via the seeds22). Although X. translucens has microscopically for some isolates examined in the present not been reported to cause disease in rice, it may propagate study. via rice seeds, as in barley and wheat. The tolerance of the isolates to high osmotic pressure was The endophytes of rice seeds found in other studies as examined, as shown in Table 1. It is worth noting that more well as those found in the present study are listed in Table 2. isolates tolerant of high osmotic pressure were found among In the present study, strains belonging to the following 9 the endophytes than the surface bacteria, especially at the genera were isolated; Acidovorax, Bacillus, Curtobacteri- later stages in the maturation of the rice seed. um, Methylobacterium, Micrococcus, Paenibacillus, Pan- By developing tolerance to high osmotic pressure and/or toea, Sphingomonas and Xanthomonas. Among them, Cur- through endospore formation, these isolates may be able to tobacterium and Methylobacterium were found in the stem survive the great environmental changes taking place inside of rice plants and Sphingomonas, in the stem and root of the rice seed during the seed’s maturation. Some strains of rice plants. As mentioned earlier Bacillus, Pantoea and B. subtilis and B. pumilus have been shown to exhibit am- Sphingomonas have been found in rice seeds in other stud- ylase activity29). Some endophytic isolates closely related to ies as well as the present study. By contrast, Acidovorax, Xanthomonas, Methylobacterium and Bacillus also exhibit- Micrococcus, Paenibacillus and Xanthomonas were found ed amylase activity in the present study (Table 1). These in rice plants for the first time in the present study. The gen- isolates may cause rice to soften and rot. Strains close to era Bacillus, Acidovorax, Micrococcus, Paenibacillus, Pan- Curtobacterium flaccumfaciens of the Actinobacteria were toea and Xanthomonas seem to be specific endophytes of isolated from inside the rice seeds at the middle and late rice seeds, although assertion must await the study of non- maturation stages, although these strains were small in num- diazotrophic endophytes in rice root. ber (6.3%) (see Table 1). C. flaccumfaciens can cause disease in beans, tulips and poinsettias19). Species of the genus The culturable bacterial flora on the surface of the rice Curtobacterium have been isolated from various plants; C. seed and the changes it undergoes during the maturation citreum, C. albidum and C. luteum have been isolated from process the rice plant19). Elbeltagy, et al. have also isolated a Curto- The isolates from the surface of rice seeds were analyzed bacterium sp. from inside the stem of wild and cultivated based on their 16S rRNA gene sequences. The closest rela- rice5). It has been reported that a Curtobacterium sp. pre- tives of the isolates are shown in Table 1, and the phyloge- vents disease caused by Erwinia30), and induces plant netic trees of these isolates are shown with some reference growth31). Dunleavy4) has reported that the bacteria of Cur- strains in Fig. 4. tobacterium spp. are transmitted via seeds. The isolates ex- The ratio of Gram-negative strains among the surface amined in the present study may also be transmitted via rice bacteria (90%) was greater than that among the endophytes seeds as endophytes. The characteristics of the isolates of the rice seeds (63%) (see Table 1). Most strains belonged closely related to Curtobacterium sp. examined in the to the Alphaproteobacteria (61%) or the Gammaproteobac- present study should be investigated further from this angle. teria (29%) (see Fig. 4). Strains closely related to the genus Culturable Bacterial Flora in a Paddy Field Rice-Seed 95

Table 2. Endophytes from various parts of rice plants

Part Bacterial species Plant species Reference

Herbaspirillum seropedicae O. meridionalis 5)

Klebsiella oxytoca O. sativa 5)

O. alta 5)

Pantoea ananatis O. sativa 25)

O. sativa this study

Bacillus cereus O. sativa 25)

Sphingomonas echinoides O. sativa 25)

Sphingomonas parapaucimobilis O. sativa 25)

Acidovorax sp. O. sativa this study seed Bacillus pumilus O. sativa this study

Bacillus subtilis O. sativa this study

Curtobacterium flaccumfaciens O. sativa this study

Methylobacterium aquaticum O. sativa this study

Micrococcus luteus O. sativa this study

Sphingomonas melonis O. sativa this study

Sphingomonas yabuuchiae O. sativa this study

Paenibacillus amylolyticus O. sativa this study

Xanthomonas translucens O. sativa this study

O. officinalis 7)

Azoarcus sp. O. minuta 7)

O. sativa 7)

Azoarcus indigens O. sativa 7)

Azorhizobium caulinodans O. sativa 7)

Azospirillum brasilense O.sativa 7)

Azospirillum lipoferum O. sativa 7)

root Burkholderia sp. O. sativa 7)

Gallionella sp. Oryza cf. nivara 7)

Herbaspirillum sp. O. sativa 7)

Klebsiella sp. O. granulata 7)

Klebsiella pneumoniae O. sativa 7)

Ochrobactrum sp. O. sativa 7)

O. officinalis 7) Sphingomonas paucimobilis O. sativa 7)

(Continued) 96 MANO et al.

(Continued)

leaf sheath Methylobacterium sp. O. sativa 5)

Aureobacterium testaceum O. rufipogon 5)

Azospirillum amazonense O. alta 5)

Azospirillum brasilense O. rufipogon 6)

O. grandiglumis 6) Azospirillum lipoferum O. sativa 6)

Corynebacterium aquaticum O. punctata 5)

O. eichingeri 5)

O. longiglumis 5)

Curtobacterium citreum O. punctata 5)

O. rufipogon 5)

O. sativa 5)

Cytophagales str. MBIC4147 O. sativa 5)

Enterobacter cancerogenus O. rufipogon 6)

Flavobacterium gleum O. alta 5) stem Herbaspirillum rubrisubalbicans O. barthii 6)

O. officinalis 6) Herbaspirillum seropedicae O. rufipogon 6)

Ideonella dechloratans O. sativa 6)

Sphingomonas adhaesiva O. rufipogon 5)

O. sativa 5)

O. longiglumis 5)

O. brachyantha 5)

Methylobacterium sp. O. latifolia 5)

O. rufipogon 5)

O. minuta 5)

O. meridionalis 5)

Microbacterium sp. O. officinalis 5)

Rhodopseudomonas palustris O. ridleyi 5) * Elbeltagy et al. (reference 6) and Engelhard et al. (reference 7) used N-free medium to isolate diazotrophic endophytes.

Sphingomonas or Methylobacterium were dominant, ac- In the early stages, many strains close to Stenotrophomo- counting for 55% of all isolates (see Table 1). nas maltophilia, which were specific to the surface and not With the maturation of the rice seeds, the culturable bac- isolated as endophytes, were found. S. maltophilia, was terial flora isolated from the seed surface changed in a dif- once transferred to the genus Xanthomonas, but it has since ferent manner from the culturable endophytic flora. been given a new generic name, Stenotrophomonas, after Culturable Bacterial Flora in a Paddy Field Rice-Seed 97

Fig. 4. Phylogenetic tree for isolates from the surface of rice seed based on 16S rRNA gene sequences. The phylogenetic tree was calculated based on approximately 450 nucleotides by the neighbor-joining method. The results of 1000 bootstrap trials are shown at the nodes. Sym- bols: , isolates from the early stage; , isolates from the middle stage; , isolates from the late stage of maturation. 98 MANO et al. the criticism of this transfer was recognized as valid26). strains may form a close bond with the rice plant, as in the Strains close to S. maltophilia may form a close bond with case of Agrobacterium larrymoorei, which was isolated the rice plant, as in the case of pathogenic Xanthomonas from the aerial tumors of Ficus benjamina L.2). spp., although they belong to a different cluster from the Strains close to Xanthomonas translucens were isolated strains close to Xanthomonas translucens, as shown in the at the late stage from the surface as well as from inside the phylogenetic tree (Fig. 4), and no pathogenicity has been re- seed, which indicates that they are common inhabitants of ported concerning S. maltophilia. S. maltophilia was iso- rice seeds. lated from the inside of sweet corn root (Zea mays L.)21). Stenotrophomonas sp. was also isolated from the rhizosphere The culturable bacterial flora and physiological traits of and inside of cucumber root20). In the present study, S. mal- the surface and endophytic bacteria tophila was isolated from the surface but not from the inside The phylogenetic trees based on the 16S rDNA sequenc- of rice seed. This organism may not exist in as great a num- es of the endophytic and surface bacteria are shown in Figs. ber as in endophytes or may not be able to adapt to the envi- 3 and 4, respectively. Gram-negative strains were dominant ronment inside the rice seed. Almost all of the strains close- both inside and on the surface of the seeds, although at dif- ly related to Sphingomonas sp. were isolated at the early and ferent ratios (63% and 90%, respectively) (see Table 1). The middle stages of the maturation process in the case of the culturable bacterial flora also differed at the two sites: in- seed surface. This was also the case for the endophytic side the seeds, isolates close to the Gammaproteobacteria strains close to Sphingomonas sp., as mentioned before. (34%), the Firmicutes (28%) and the Alphaproteobacteria These strains may move from the surface to the interior of (25%) were dominant, whereas at the surface, isolates close the seed and vice versa. The motility of these isolates (Table to the Alphaproteobacteria (61%) and the Gammaproteo- 1) supports this hypothesis. A process whereby the inside of bacteria (29%) were dominant (see Fig. 4). In the present the plant is invaded using cellulase or pectinase has been study, fewer of the strains grown on the NB medium could suggested for Azoarcus sp., Azospirillum irakense, and be re-cultured after isolation compared with the strains Pseudomonas fluorescens14). Elbeltagy et al.5) clarified that grown on DNB medium. Thus, the figures for each phylum many of the endophytes from rice seeds were motile as well as mentioned above are close to those of isolates from DNB as pectinase-positive and that about a half were cellulose- medium but not NB medium. An analysis using non-cultur- positive. The enzyme activity of cellulase and pectinase ing methods such as PCR-DGGE seems to be necessary for should be analyzed to clarify the invasion process of the iso- further investigation. lates in the present study. The isolates from the inside and from the surface of seeds Two strains, one close to Bacillus sp. and the other close belonged to 9 and 8 genera, respectively. Among these, 3 to Curtobacterium sp., were isolated from the surface early genera (Paenibacillus, Acidovorax and Pantoea) and 2 gen- on; they differed from the endophytes in number and the era (Stenotrophomonas and Rhizobium) were specific to the stage of appearance; 8 and 2 strains close to Bacillus sp. and inside and to the surface of the seeds, respectively. Six gen- Curtobacterium sp., respectively, were isolated from the in- era (Bacillus, Curtobacterium, Methylobacterium, Sphin- side of the rice seed at the middle and late stage. Strains gomonas, Xanthomonas and Micrococcus) were common to close to these genera may adapt to the environmental condi- both the inside and the surface. The isolates close to these tions inside the seed at these later stages. genera may be able to migrate from the surface to the interi- Strains closely related to Methylobacterium sp. were iso- or of the seeds and vice versa. lated from the surface as well as from inside the seed, al- Elbeltagy et al.6) revealed that gfp-labeled cells of though at different stages; at the middle and the late stages Herbaspirillum sp. formed colonies in inter-cellular spaces they were isolated from the surface and at the early stage in shoots of a wild rice plant (O. officinalis) using fluores- from inside. This genus is composed of a variety of pink- cence microscopy. In the present study, attempts to confirm pigmented, facultatively methylotrophic bacteria, which ex- the existence of the endophytes in rice seeds did not suc- hibit resistance to the stressful conditions possibly prevail- ceed. The use of a genetic tool may be needed. ing at the surface, such as dehydration and the absence of The culturable endophytic bacterial flora changed with nutrients12,13). the seed maturation process and could be divided into 3 Strains close to Agrobacterium larrymoorei (Rhizobium groups: 1) isolates closely related to Sphingomonas and Me- larrymoorei is preferable, according to Young33)) were iso- thylobacterium, 2) isolates closely related to Bacillus and lated at the late stage specifically from the surface. These Curtobacterium, and 3) isolates closely related to Xanth- Culturable Bacterial Flora in a Paddy Field Rice-Seed 99 omonas and Pantoea. The number of bacteria decreased in References group 1 and increased in group 2 as the maturation process 1) Bally, R., A. Givaudan, J. Bernillon, T. Heulin, J. Balandreau and progressed, whereas the bacteria in group 3 were found in R. Bardin. 1990. Numerical taxonomic study of three N2-fixing abundance at the early and the late stages of maturation. As yellow-pigmented bacteria related to Pseudomonas paucimobilis. the rice plants matured, the bacterial flora isolated from the Can. J. Microbiol. 36: 850–855. seed surface changed in a different manner from the endo- 2) Bouzar, H., W.S. Chilton, X. Nesme, Y. Dessaux, V. Vaudequin, A. Petit, J.B. Jones and N.C. Hodge. 1995. A new Agrobacterium phytes. Early on, many strains close to Stenotrophomonas strain isolated from aerial tumors on Ficus benjamina L. Appl. maltophilia, which were specific to the surface and not iso- Environ. Microbiol. 61: 65–73. lated as endophytes, were isolated along with strains close 3) Coombs, J.T. and C.M.M. Franco. 2003. Isolation and identifica- to Bacillus, Curtobacterium and Sphingomonas, which tion of Actinobacteria from surface-sterilized wheat roots. Appl. Environ. Microbiol. 69: 5603–5608. were found as endophytes. At the middle stage, isolates 4) Dunleavy, J.M. 1989. Curtobacterium plantarum sp. nov. is ubiq- close to the genera Sphingomonas and Methylobacterium uitous in plant leaves and is seed transmitted in soybean and corn. were dominant, whereas at the late stage, isolates close to Int. J. Syst. Bacteriol. 39: 240–249. the genera Rhizobium, Methylobacterium and Xanthomonas 5) Elbeltagy, A., K. Nishioka, H. Suzuki, T. Sato, Y. Sato, H. Morisaki, H. Mitsui and K. Minamisawa. 2000. Isolation and were dominant. Although a representative rice plant average characterization of endophytic bacteria from wild and traditional- in height and appearance was selected as a sample from the ly cultivated rice varieties. Soil Sci. Plant Nutr. 46: 617–629. experimental plot in the present study, there may be a differ- 6) Elbeltagy, A., K. Nishioka, T. Sato, H. Suzuki, B. Ye, T. Hama- ence in bacterial flora between plants of different heights da, T. Isawa, H. Mitsui and K. Minamisawa. 2001. Endophytic colonization and in planta nitrogen fixation by a Herbaspirillum and appearances. These potential differences must be taken sp. isolated from wild rice species. Appl. Environ. Microbiol. 67: into account and will be investigated further. 5285–5293. Not only the culturable bacterial flora, but also the physi- 7) Engelhard, M., T. Hurek and B. Reinhold-Hurek. 2000. Preferen- ological traits of the isolates changed with the maturation tial occurrence of diazotrophic endophytes, Azoarcus spp., in wild rice species and land races of Oryza sativa in comparison process both inside and on the surface of the seed. As shown with modern races. Environ. Microbiol. 2: 131–141. in Table 1, almost all of the isolates showed motility, as has 8) Freeman, S. and R.J. Rodriguez. 1993. Genetic conversion of a been revealed for isolates from pot-cultivated rice25). More fungal plant pathogen to a nonpathogenic, endophytic mutualist. isolates tolerant of high osmotic pressure were found among Science 260: 75–78. the endophytes than among the surface bacteria, especially 9) Gagné, S., C. Richard, H. Rousseau and H. Antoun. 1987. Xy- lem-residing bacteria in alfalfa roots. Can. J. Microbiol. 33: 996– at the later stages of the maturation of the rice seed. The 1000. number of endophytes having amylase activity increased at 10) Gardner, J.M., A.W. Feldman and R.M. Zablotowicz. 1982. Iden- the late stage. Interestingly, these isolates showed lower tol- tity and behavior of xylem-residing bacteria in rough lemon roots erance to high osmotic pressure and did not carry en- of Florida citrus trees. Appl. Environ. Microbiol. 43: 1335–1342. 11) Gorlach, K., R. Shingaki, H. Morisaki and T. Hattori. 1994. Con- dospores. 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