Diversity of Culturable Chitinolytic Bacteria from Rhizospheres of Agronomic Plants in Japan

Microbes Environ. Vol. 26, No. 1, 7–14, 2011 http://wwwsoc.nii.ac.jp/jsme2/ doi:10.1264/jsme2.ME10149

NOBUTAKA SOMEYA1*, SEISHI IKEDA1, TOMOHIRO MOROHOSHI2, MASAKO NOGUCHI TSUJIMOTO3, TAKANOBU YOSHIDA4, HIROYUKI SAWADA5, TSUKASA IKEDA2, and KENICHI TSUCHIYA6 1National Agricultural Research Center for Hokkaido Region (NARCH), National Agriculture and Food Research Organization (NARO), 9–4 Shinsei-minami, Memuro-cho, Kasai-gun, Hokkaido 082–0081, Japan; 2Department of Applied Chemistry, Utsunomiya University, 7–1–2 Yoto, Utsunomiya 321–8585, Japan; 3National Agricultural Research Center (NARC), NARO, 3–1–1 Kannondai, Tsukuba, Ibaraki 305–8666, Japan; 4Bio-oriented Technology Research Advancement Institution, NARO, 1–40–2 Nisshin, Kita, Saitama, Saitama 331–8537, Japan; 5National Institute of Agrobiological Sciences, 2–1–2 Kannondai, Tsukuba, Ibaraki 305–8602, Japan; and 6Faculty of Agriculture, Kyushu University, Hakozaki, Higashi-ku, Fukuoka 812–8581, Japan (Received July 13, 2010—Accepted September 25, 2010—Published online November 6, 2010)

A total of 100 isolates of chitinolytic bacteria were obtained from the rhizospheres of various agronomic plants, and the 16S rRNA gene sequences of these isolates were determined. Phylogenetic analyses revealed that 81 isolates belonged to the classes Betaproteobacteria (39 isolates) and Gammaproteobacteria (42 isolates). Of the remaining 19 isolates, 16 belonged to the phylum Firmicutes. Clustering analysis identified 6 and 3 operational taxonomic units (OTUs) in Gammaproteobacteria and Betaproteobacteria, respectively, at the genus level. The majority of chitinolytic bacteria in Gammaproteobacteria belonged to the genera Serratia, Stenotrophomonas, and Lysobacter (14, 15, and 7 isolates, respectively) while those in Betaproteobacteria belonged to the genus Mitsuaria (37 isolates). The 16 isolates placed in Firmicutes belonged to 2 genera, Paenibacillus and Bacillus (8 isolates each). The isolates in the remaining OTUs belonged to the genera Erwinia, Aeromonas, Pseudomonas, Achromobacter, Flavobacterium, and Microbacterium, in less abundance. These results showed a wide distribution of culturable chitinolytic bacteria in the rhizospheres of various agronomic plants. Considering the potential antagonistic activity of chitinolytic enzymes against phytopathogenic fungi, which is exhibited by fungal cell wall degradation, the above-mentioned native chitinolytic bacteria in rhizospheres could potentially be utilized for the biological control of soil-borne phytopathogenic fungi. Key words: chitinase, rhizosphere, 16S rRNA gene, biological control, quorum sensing

Chitin, a homopolymer of N-acetyl-D-glucosamine, is the 34, 46). However, despite numerous attempts to utilize major structural component of various organisms including chitinolytic bacteria as biological control agents, the ecology fungi, insects, and crustaceans (11). Chitinase is a hydrolase of these bacteria, including phylogenetic diversity and host that degrades the chitin polymer, and like chitin, it is also specificity, in rhizospheres is not well understood. Most present in a wide range of organisms. In some cases, bacteria in nature are known to be unculturable (4). It has chitinases play a role in morphogenesis and/or autolysis in been shown that both culturable and unculturable bacteria chitin-containing organisms. Despite that bacteria do not harboring chitinase genes are also widely distributed in contain chitin as a native component, numerous bacteria are environments (7, 15, 39). However, it is difficult to utilize able to hydrolyze chitin. Thus, it is conceivable that chi- unculturable bacteria as biocontrol agents with current tinolytic bacteria utilize chitin as a carbon source via the microbial technologies. activity of chitinolytic enzymes. The present study aimed to determine the phylogenetic Most plant diseases are caused by phytopathogenic fungi diversity and spatial heterogeneity of culturable chitinolytic (13, 20, 22). Similar to other filamentous fungi, phyto- bacteria in the rhizospheres of various agronomic plants and pathogenic fungi contain chitin as the main component of of different geographical locations. In addition, since recent their cell walls. Therefore, chitinolytic bacteria show poten- studies have revealed that chitinolytic activity in some tial antagonistic activity against phytopathogenic fungi by Gram-negative bacteria is regulated by quorum sensing degrading the cell walls of these fungi. In fact, it has been re- via signal molecules, N-acylhomoserine lactones (AHLs) ported that various chitinolytic bacteria such as those belong- (6, 38), AHL production by chitinolytic bacteria was also ing to the genera Aeromonas, Alcaligenes, Arthrobacter, investigated. Bacillus, Cytophaga, Lysobacter, Pantoea, Pseudomonas, Serratia, and Stenotrophomonas have potential for the Materials and Methods biocontrol of phytopathogenic fungi (14, 17, 18, 24, 25, 30, Plant sampling and isolation of culturable chitinolytic bacteria A total of 36 root samples were collected from 16 species of * Corresponding author. E-mail: [email protected]; agronomic plants growing in 6 fields located in Ibaraki Prefecture, Tel: +81–155–62–9280; Fax: +81–155–61–2127. Japan (Fig. 1). The 16 species were beafsteak [Perilla frutescens 8 SOMEYA et al.

each OTU (2). Phylogenetic trees based on the sequences of each OTU were also constructed by the NJ method. Assay of chitinolytic activity of isolates and evaluation of quorum-sensing signal molecule production The isolates were grown in Luria-Bertani (LB; Sigma-Aldrich, St. Louis, USA) medium at 25°C for 24 h. The bacterial cells were centrifuged and suspended in sterile water at 108 colony forming units (CFU) mL−1. A cell suspension (1 μL) was inoculated on a dNBYC plate (3 replicates) and incubated at 25°C in the dark for 7 days. Chitinolytic activity was measured by observing the size of the halo formed around the colonies as follows: ±, halo < 1 mm; +, 1 mm ≤ halo < 5 mm; ++, halo ≥ 5 mm. Production of the quorum-sensing signal molecule, AHL, was evaluated using 2 AHL-reporter strains, Chromobacterium violaceum CV026 and VIR24. The response to AHL differs between CV026 and VIR24 and use of both strains enables the detection of a wide range of AHLs. The method for the detection of AHLs has been described previously (37). Fig. 1. Sampling locations of this study. Root samples were collected from 16 species of agronomic plants growing at 6 different sites: Ami, Accession numbers of nucleotide sequences Bando, Hitachi, Kasama, Mito, and Tsukuba. The sites included both Nucleotide sequences of the partial 16S rRNA genes of the commercial and experimental fields located in Ibaraki Prefecture, chitinolytic bacteria have been deposited in the DNA Data Bank of Japan. Japan (DDBJ) under the accession numbers AB560531–AB560630.

(L.) Britton; Pf], welsh onion (Allium fistulosum L.; Af), green Results foxtail [Setaria viridis; (L.) P. Beauv.: Sev] kiwano [Cucumis metuliferus E. Mey. ex Naud.; Cmt], Aztec marigold (Tagetes Culturable chitinolytic bacteria isolated from rhizospheres erecta L.; Te), edible burdock (Arctium lappa L.; Al), Chinese cabbage (Brassica campestris L. subsp. pekinensis; Bc), taro Numerous colonies were observed on a dNBYC plate [Colocasia esculenta (L.) Schott; Ce], soybean [Glycine max (L.) after incubation for 3 days. The numbers of colonies ranged 5 8 −1 Merr.; Gm], sunflower (Helianthus annuus L.; Ha), sweet potato from approximately 10 to 10 g fresh root weight. The [Ipomoea batatas (L.) Lam; Ib], garden petunia (Petunia hybrida chitinolytic bacteria identified in the present study were Vilm; Ph), Japanese radish (Raphanus sativus L. var. longipinnatus <0.1% of the total culturable bacteria (data not shown). Thus, L. H. Bailey; Rs), scarlet runner bean (Phaseolus coccineus L.; Pc), 100 isolates of chitinolytic bacteria were obtained from the Asiatic dayflower (Commelina communis L.; Coc), and lettuce root samples. The number of isolates obtained, host plants, (Lactuca sativa L.; Ls). Root tissue (1 g) was washed with 9 mL of sterile 15 mM and collection sites of the isolates are summarized in Fig. 2. phosphate buffer (pH 7.0), and sonicated to release the microbial cells attached to the roots. Serial dilutions of the cells were Phylogenetic diversity of culturable chitinolytic bacteria in cultivated on diluted nutrient broth medium (0.08% nutrient broth rhizospheres plus 0.05% yeast extract; Difco, Detroit, MI, USA) agar containing A total of 100 isolates of chitinolytic bacteria were 0.2% colloidal chitin (hereafter dNBYC) by incubating at 25°C in obtained from the rhizospheres of agronomic plant species, the dark for 3 days. After cultivation, 5 colonies were isolated at and their phylogenetic diversity was determined by sequenc- random from a sample by observing the halo formed around the colonies. ing the 16S rRNA gene. Using the RDP Classifier, the chitinolytic isolates were classified into the following 4 bacte- Sequence analysis of 16S rRNA genes rial phyla: Proteobacteria, Bacteroidetes, Firmicutes, and Nucleotide sequences of the 16S rRNA genes were determined Actinobacteria. The most dominant phylum among all the using the direct polymerase chain reaction (PCR) method (36) and isolates was Proteobacteria (81%), followed by Firmicutes placed in a taxonomic hierarchy by using the Ribosomal Database (16%). Project (RDP) Classifier (42). The relative abundance of each of the main phyla was determined at an 80% confidence level. The Clustering analysis of the 16S rRNA gene sequences sequences were aligned using CLUSTAL_X, and the alignments identified 6, 3, 4, and 1 OTUs in Gammaproteobacteria, were written in a PHYLogeny Inference Package (PHYLIP) format Betaproteobacteria, Firmicutes, Bacteroidetes, and Actino- file. By using the information from this file, distance matrices were bacteria, respectively, at the genus level (Fig. 2). The domi- constructed using the DNADIST program with the default param- nant OTUs in Gammaproteobacteria were classified into 2 eters (10, 41). The resulting matrices were used as the input for a families—Enterobacteriaceae and Xanthomonadaceae (Fig. software, DOTUR [Distance-based Operational Taxonomic Units (OTUs) and Richness] to generate diversity indices and richness 3). These families mostly comprised the genera Serratia, indicators of the different species (27). The default setting was used Stenotrophomonas, and Lysobacter (GP1, GP5, and GP6, in for DOTUR to generate a list file of OTUs for clustering analysis Table 1). The 2 dominant OTUs in Betaproteobacteria with Shared OTUs and Similarity (SONS) (28). In the analysis, the belonged to the genus Mitsuaria (BP1 and BP2 in Table 1), OTUs were defined by 95% sequence identity. The representative and the 2 dominant OTUs in Firmicutes belonged to the sequences of OTUs were aligned using CLUSTAL_X (41), and a genera Paenibacillus and Bacillus (F1 and F4, respectively, tree was constructed by the neighbor-joining (NJ) method (26). A in Table 1). It should be noted that the isolates in GP1, GP5 search of the GenBank database with the Nucleotide-nucleotide Basic Local Alignment Search Tool (BLASTn) was used to identify and F4 were obtained from 5, 4 and 3 different collection the sequence most closely related to the representative sequence of sites, respectively. In terms of the number of isolates, BP1 Chitinolytic Bacteria in Rhizosphere 9

Fig. 2. Phylogenetic tree based on the 16S rRNA gene sequences of bacterial isolates, and the host plants and collection sites. The dendrogram (left) indicates the phylogenetic relationships among the 15 representative sequences defined by 95% sequence identity. Aquifex pyrophilus (M83548) was used as an out-group for the dendrogram. Operational taxonomic units (OTUs) were expressed as consecutively numbered prefixes, which were derived from phylum identity (GP, Gammaproteobacteria; BP, Betaproteobacteria; B, Bacteroidetes; F, Firmicutes; A, Actinobacteria). The table (right) indicates the number of isolates obtained from host plants and collection sites. The collection sites are indicated in Fig. 1. The host plants were as follows: beefsteak (Pf), welsh onion (Af), green foxtail (Sev), kiwano (Cmt), Aztec marigold (Te), edible burdock (Al), Chinese cabbage (Bc), taro (Ce), soybean (Gm), sunflower (Ha), sweet potato (Ib), petunia (Ph), Japanese radish (Rs), scarlet runner bean (Pc), Asiatic dayflower (Coc), and lettuce (Ls).

Table 1. Chitinolytic bacteria isolated from rhizospheres, chitinolytic activity and quorum sensing signal molecule production

d) a) b) Chitinolytic AHL production OTUs Closest species Accession no. Identity (%) c) activity CV026 VIR24 GP1 Serratia marcescens GU046545 100 ++ 11/14 11/14 GP2 Erwinia rhapontici GQ131583 98 ++ 3/3 3/3 GP3 Aeromonas hydrophila AB473026 99 + 0/1 1/1 GP4 Pseudomonas frederiksbergensis FJ796428 100 + ND ND GP5 Stenotrophomonas maltophilia AF417866 100 + 0/15 2/15 GP6 Lysobacter capsici FN357198 99 + ND ND BP1 Mitsuaria chitosanitabida FJ796447 99 + ND ND BP2 Mitsuaria chitosanitabida FJ796447 93 + ND ND BP3 Achromobacter xylosoxidans DQ659433 100 + 0/2 1/2 B1 Flavobacterium johnsoniae EU221404 97 + ND ND F1 Paenibacillus pabuli FJ189798 99 + ND ND F2 Paenibacillus chitinolyticus FJ174602 99 + ND ND F3 Paenibacillus humicus EU867348 99 + ND ND F4 Bacillus thuringiensis GQ479945 100 + ND ND A1 Microbacterium trichothecenolyticum EF204433 99 ± ND ND a) Representative isolates of each operational taxonomic unit (OTU) were as follows: CmtRB001 (GP1), LsRB018 (GP2), CeRB004 (GP3), CeRB009 (GP4), AfRB002 (GP5), RsRB001 (GP6), AlRB002 (BP1), AlRB002 (BP2), GmRB005 (BP3), CeRB005 (B1), HaRB006 (F1), IbRB007 (F2), PcRB018 (F3), HaRB001 (F4), and PcRB024 (A1). b) Closest species was determined by comparing the 16S rRNA gene sequence of the representative isolate with the sequences in the database. c) Chitinolytic activity was evaluated by measuring the size of the halo around the colonies as follows: ±, halo<1 mm; +, 1 mm≤halo<5 mm; ++, halo≥5 mm. d) N-acyl homoserine lactone (AHL)-positive isolates/Total isolates. AHL production was evaluated using 2 AHL-reporter strains, Chromo- bacterium violaceum CV026 (for the detection of short acyl-chain AHLs) and VIR24 (for the detection of long acyl-chain AHLs) (37). ND, not detected was the most dominant OTU containing 36 isolates, which Results of a phylogenetic analysis conducted with type were obtained from the roots of a wide range of plant strains related to each OTU and of detailed analyses of the species. However, the isolates in BP1 were all obtained isolates in OTUs GP1, GP5, and BP1 are shown in Fig. 4, from Mito. Fig. 5, and Fig. 6, respectively. GP1 consisted of 14 isolates, The results of the BLASTn search indicated that most of which were separately collected from 5 different plant the sequences showed 99% identity with those of known species growing of 5 different sites. All the isolates in GP1 species, with the exception of 5 sequences (93–98%). Phylo- showed a close relationship with Serratia marcescens (Fig. genetic analyses of the sequences of representative isolates 4). GP5 consisted of 15 isolates, which were separately of each OTU confirmed that most of the isolates obtained in collected from 8 species of agronomic plants growing at 4 the present study showed high identity with known species different sites. The isolates in GP5 were grouped into 2 clus- (Fig. 3). ters, STE1 and STE2. Most of the isolates in STE1 showed 10 SOMEYA et al.

Fig. 3. Neighbor-joining tree based on the 16S rRNA gene sequences of the representative isolates of 15 OTUs (bold). Both GP1 and GP2 belonged to the family Entero- bacteriaceae. GP3 and GP4 belonged to the families Aeromonadaceae and Pseudomo- nadaceae, respectively. Both GP5 and GP6 belonged to the family Xanthomonadaceae. BP1, BP2, and BP3 belonged to the order Burkholderiales. B1 belonged to the family Flavobacteriaceae. F1, F2, and F3 belonged to the family Paenibacillaceae, and F4 belonged to the family Bacillaceae. A1 belonged to the family Microbacteriaceae. A. pyrophilus (M83548) was used as an out- group for the dendrogram. The scale bar represents 0.05 substitutions per nucleotide position. Bootstrap values of ≥50 (from 100 replicates) are indicated at the nodes.

Fig. 4. Neighbor-joining tree based on the 16S rRNA gene sequences of bacterial isolates in GP1 (genus Serratia). The bacterial isolates used in the present study are shown in bold. Host plants and collection sites of the isolates are indicated on the right side. Phylogenetic analysis of the 16S rRNA gene sequences of 10 type strains of the genus Serratia identified 14 chitinolytic isolates from rhizospheres. A. pyrophilus (M83548) was used as an out-group for the dendrogram. The scale bar represents 0.005 substitutions per nucleotide position. Bootstrap values of ≥50 (from 100 replicates) are indicated at the nodes. a close relationship with Stenotrophomonas maltophila, AHL production by chitinolytic bacteria Stenotrophomonas geniculata, and Stenotrophomonas After 7 days of incubation, all the 100 isolates degraded hibiscicola. In STE2, 2 isolates showed a close relationship colloidal chitin on the dNBYC plate. The chitinolytic activity with Stenotrophomonas rhizosphila. BP1 consisted of 36 was evaluated by measuring the halo formed around the isolates, which were collected from 6 different plant species colonies (Table 1). The representative isolates of GP1 and growing only at Mito. The isolates in BP1 showed a close GP2 showed strong chitinolytic activities (Table 1), while relationship with Mitsuaria chitosanitabida (formerly those of GP4 and A1 showed weak chitinolytic activities. Matsuebacter chitosanotabidus). The isolates in BP1 were AHL production by the isolates was evaluated using 2 largely grouped into 3 clusters, MIT1, MIT2, and MIT3 AHL-reporter strains, CV026 and VIR24, of the bacterium (Fig. 6), which distinctly separated from the type strain C. violaceum. A total of 18 isolates produced AHL under T M. chitosanitabida 3001 . MIT1 containing 15 isolates was the experimental conditions (Table 1). AHL-positive isolates the closest relative of M. chitosanitabida CAH4. MIT2 in GP1 and GP2 produced both short acyl- and long acyl- containing 15 isolates was the closest relative of M. chain homoserine lactone (HL), while those in GP3, GP5, chitosanitabida IMER-B4-4. MIT3 containing 6 isolates and BP3 produced only long acyl-chain HL (Table 1). The T was most distantly related to 3001 , CAH4, and IMER-B4-4. productivity of each isolate is shown in Table S1 (see supple- mentary material). Chitinolytic Bacteria in Rhizosphere 11

Fig. 5. Neighbor-joining tree based on the 16S rRNA gene sequences of bacterial isolates in GP5 (genus Stenotrophomonas). The bacterial isolates used in the present study are shown in bold. Host plants and collection sites of the isolates are indicated on the right side. Phylogenetic analysis of the 16S rRNA gene sequences of 9 type strains of the genus Stenotrophomonas identified 14 chitinolytic isolates from rhizospheres. Isolates in GP5 were classified into 2 distinct clusters, STE1 and STE2, by phylogenetic analysis. A. pyrophilus (M83548) was used as an out-group for the dendrogram. The scale bar represents 0.01 substitutions per nucleotide position. Bootstrap values of ≥50 (from 100 replicates) are indicated at the nodes.

Fig. 6. Neighbor-joining tree based on the 16S rRNA gene sequences of bacterial isolates in BP1 (genus Mit- suaria). The bacterial isolates used in the present study are shown in bold. Host plants and collection sites of the isolates are indicated on the right side. Phylogenetic analysis of the 16S rRNA gene sequences of one type strain of the genus Mitsuaria (M. chitosanitabida) and 6 strains belonging to the genus Mitsuaria identified 36 chitinolytic isolates from rhizospheres. Isolates in BP1 were classified into 3 distinct clusters, MIT1, MIT2 and MIT3, by phylogenetic analysis. A. pyrophilus (M83548) was used as an out-group for the dendrogram. The scale bar represents 0.002 substitutions per nucleotide position. Bootstrap values of ≥50 (from 100 replicates) are indicated at the nodes.

Discussion chitinolytic bacteria identified was less than that reported. It is possible that the culture medium used created a bias Chitinolytic bacteria were isolated from the roots of which led to an underestimation of the population size of various agronomic plants grown in different agricultural chitinolytic bacteria. The 16S rRNA gene sequences of the fields in Ibaraki Prefecture, Japan. It is known that 2%–10% majority of isolates showed high identity with those of of bacteria isolated from soil and natural water can utilize known chitinolytic bacteria. However, a few isolates could chitin as a sole carbon source through the actions of chi- be considered as novel chitinolytic species (Table S1). tinases (11). Further, it has been reported that only 1% of It is generally considered that the diversity of chitinolytic rhizobacteria from maize, wheat, and rice show potential bacteria differs considerably among environments (5, 9, 17, chitinolytic activity (17). In the present study, at least one 39). In the present study, the diversity of culturable chi- isolate was obtained from each collection site, suggesting the tinolytic bacteria in the rhizospheres of agronomic plants was ubiquity of chitinolytic bacteria in rhizospheres. However, as determined. Most of the bacteria isolated from rhizospheres a percentage of all culturable bacteria, the proportion of or soil have been reported as chitinolytic bacteria. However, 12 SOMEYA et al. the isolates in GP3, BP3, and F1 have not been reported as revealed that chitinolytic populations of Mitsuaria spp. are rhizospheric or soil-associated bacteria. Furthermore, in this present in the rhizospheres of agronomic plants. The isolates study, the bacterial isolates in GP2, BP1, BP2, and A1 in BP3, belonging to the genus Achromobacter, were only showed the ability to produce chitinases. a minor population in the present study, but seemed to be All the isolates in GP1 and GP5 showed high identity with able to survive in rhizospheres, as reported previously (9). Serratia marcescens and Stenotrophomonas maltophilia, Paenibacillus (F1, F2, and F3) and Bacillus (F4) are the respectively, well-known chitinolytic bacterial species with most dominant genera of chitinolytic bacteria in diverse the potential to control phytopathogenic fungi (18, 30, 34, environments, including soils (1, 14, 25, 29). Further, our 46). Interestingly, all the isolates in GP1, which were col- observations indicated that these genera were widely distri- lected from 5 different plant species growing at 5 different buted in the rhizospheres of various plants. The isolate in A1 sites, showed a close relationship with Serratia marcescens showed high identity with the genus Microbacterium, which in the phylogenetic analysis of the 16S rRNA gene sequences has not been reported as a chitinolytic bacterium previously. (Fig. 4), suggesting high affinity of Serratia marcescens All isolates belonging to the genera Serratia (GP1) and for the rhizospheres of various agronomic plants. In fact, Erwinia (GP2) showed noticeably strong chitinolytic activity most of the biocontrol strains of the genus Serratia have (Table 1). It has been reported that a strain of Serratia been reported to belong to Serratia marcescens (18, 30, 34). marcescens and a chitinase gene-transfected Erwinia ananas In contrast, the isolates in GP5 (genus Stenotrophomonas) showed biocontrol activity against various phytopathogens, were classified into 2 distinct clusters, STE1 and STE2, the biocontrol being related to their chitinolytic activities by the phylogenetic analysis (Fig. 5). STE1 contained 12 (30–32). Therefore, highly chitinolytic isolates including GP1 isolates, which showed a close relationship with Steno- and GP2 might have the potential to control phytopathogens. trophomonas maltophilia, Stenotrophomonas geniculata, Production of chitinolytic enzymes in bacteria is regulated and Stenotrophomonas hibiscicola. STE2 contained 2 iso- by various mechanisms. Most of the chitinolytic organisms lates, which were related to Stenotrophomonas rhizophila. produce multiple chitinases (6, 31). Therefore, chitinolytic In a previous study, predominance of Stenotrophomonas activity depends on various factors such as the number of maltophilia over other pseudomonads has been observed isozymes, their activity, and amounts of the enzymes. Syner- in the rhizospheres of several plants (23). Meanwhile, gistic effects of multiple chitinases have been reported for Stenotrophomonas rhizophilia closely related to STE2 has both chitin degradation and antifungal activity (8, 31, 35). also been isolated from the rhizospheres of several plants Another regulatory mechanism of chitinolytic activity has (43). These results may imply that a subpopulation of recently been revealed in Gram-negative bacteria such as C. Stenotrophomonas spp. prefers to associate with rhizo- violaceum and Pseudomonas aeruginosa; in these bacteria, spheres. The isolates in GP1 and GP5 were collected from chitinolytic activity is regulated by quorum sensing via the different sites (Fig. 2); therefore, these bacterial groups could signal molecule AHL (6, 38). In the present study, 18 chi- be considered cosmopolitan with an ubiquitous distribution tinolytic isolates produced AHLs (Table 1 and Table S1). Of in agricultural fields. Aeromonas and Flavobacterium were these, 17 isolates belonged to Gammaproteobacteria (genera reported to be the dominant genera of chitinolytic bacteria Serratia, Erwinia, Aeromonas, and Stenotrophomonas) and in lake water, but only minor populations in rhizospheres (5, only one isolate belonged to Betaproteobacteria (genus 9). The genus Pseudomonas contains abundant culturable Achromobacter). Both AHL-producing and nonproducing rhizobacteria, some with the potential to hydrolyze chitin isolates were included in GP1, GP5 and BP3 (Table 1), and (12, 21). However, chitinolytic isolates belonging to this no relationship was observed between their chitinolytic genus and obtained from rhizospheres were not abundant in activity and AHL-production. In GP3, GP5 and BP3, AHL- the present study. The representative isolate of GP6 showed producing isolates were detected by VIR24, a new AHL high identity with Lysobacter capsici, which has recently reporter detecting isolates producing long-acyl-chain AHLs, been reported as a novel chitinolytic rhizobacterium (24). developed in a previous study (37). These isolates might not Interestingly, the isolates in BP1 and BP2 showed high have been detected using only the CV026 reporter. Chernin homology with M. chitosanitabida and were specifically et al. (6) reported that chitinase production is regulated by obtained from Mito. M. chitosanitabida is a novel bacterium N-hexanoyl-L-homoserine lactone (C6HSL), and an AHL- isolated from soil. It produces chitosan-degrading enzymes; deficient mutant could not produce chitinases. Further however, its chitinase productivity has not been investigated studies should focus on the role of AHLs in chitinase pro- (3). The isolates in BP1 were grouped into 3 clusters, MIT1, duction in AHL-producing chitinolytic isolates. Comparative MIT2, and MIT3, all of which were clearly separated from analyses of these isolates should clarify the ecological the type strain M. chitosanitabida 3001T. Since Mitsuaria role of quorum sensing via AHLs in chitinase production comprises only one species, M. chitosanitabida, to date, the or other functions in rhizospheres. isolates in MIT1, MIT2, and MIT3 might represent novel Does chitinase production in rhizospheres confer a species in this genus. Although chitinase activity has not selective advantage on the producer bacteria? It is a fact that been examined in the type strain, it has been reported that a rhizobacteria compete with other microorganisms, including betaproteobacterium, KNU3 (closely related to MIT2), fungi, in rhizospheres. If chitinolytic enzymes function as which showed high identity with M. chitosanitabida 3001T, antifungal agents, the presence of chitinolytic bacteria in can produce both a chitinase and a chitosanase (44). Most rhizospheres may be beneficial for plants. However, several of the isolates belonging to the genus Mitsuaria have been attempts to utilize only chitinolytic bacteria as biological isolated from soil or rhizospheres. Indeed, the present study control agents have failed (35, 40). One reason for these Chitinolytic Bacteria in Rhizosphere 13 failures is that both colonization and chitinase production by 7. Cottrell, M.T., D.N. Wood, L. Yu, and D.L. Kirchman. 2000. the bacteria in rhizospheres are affected by various factors Selected chitinase genes in cultured and uncultured marine bacteria in the α- and γ-subclasses of the proteobacteria. Appl. Environ. including plant species, cultivars, the physiological condition Microbiol. 66:1195–1201. of plants, soil type and indigenous microorganisms (16, 19, 8. Dahiya, N., R. Tewari, and G.S. Hoondal. 2006. Biotechnological 33, 45). aspects of chitinolytic enzymes: a review. Appl. Microbiol. Meanwhile, most bacteria in nature are known to be Biotechnol. 71:773–782. 9. Donderski, W., and M.S. Brzezińska. 2001. Occurrence of chi- unculturable (4). Therefore, the phylogenetic and functional tinolytic bacteria in water and bottom sediment of eutrophic lakes in diversity of bacterial communities in different environments Iławskie Lake district. Pol. J. Environ. Stud. 10:331–336. has been examined without using cultivation methods (16). 10. Felsenstein, J. 1997. An alternating least squares approach to infer- Further, by using culture-independent methods, it has been ring phylogenies from pairwise distances. Syst. Biol. 46:101–111. 11. Gooday, G.W. 1997. The many uses of chitinases in nature. Chitin shown that bacteria with chitinase genes are widely distri- Chitosan Res. 3:233–243. buted in rhizospheres and arable soils (15, 39). In those 12. Haas, D., and G. Défago. 2005. Biological control of soil-borne studies, the chitinase gene-containing bacteria in rhizo- pathogens by fluorescent pseudomonads. Nat. Rev. Microbiol. 3:307– spheres differed from the culturable chitinolytic bacteria in 319. 13. Hirano, Y., and T. Arie. 2009. Variation and phylogeny of Fusarium the present study; however, some chitinolytic bacteria were oxysporum isolates based on nucleotide sequences of poly- detected by both culture-dependent and culture-independent galacturonase genes. Microbes Environ. 24:113–120. methods. In order to understand the diversity and ecological 14. Huang, C.J., T.K. Wang, S.C. Chung, and C.Y. Chen. 2005. characteristics of chitinolytic bacteria, investigations should Identification of an antifungal chitinase from a potential biocontrol be conducted using both culture-dependent and culture- agent, Bacillus cereus 28-9. J. Biochem. Mol. Biol. 38:82–88. 15. Ikeda, S., N. Ytow, H. Ezura, K. Minamisawa, K. Miyashita, and T. independent methods. However, it is actually difficult to Fujimura. 2007. Analysis of molecular diversity of bacterial chitinase utilize unculturable bacteria as biocontrol agents. Therefore, genes in the maize rhizosphere using culture-independent methods. culturable chitinolytic bacteria collected in the present study Microbes Environ. 22:71–77. would be a valuable source for screening of biocontrol agents 16. Ikeda, S., T. Okubo, T. Kaneko, S. Inaba, T. Maekawa, S. Eda, S. Sato, S. Tabata, H. Mitsui, and K. Minamisawa. 2010. Community against phytopathogens. In the present study, the diversity of shifts of soybean stem-associated bacteria responding to different culturable chitinolytic bacteria in rhizospheres was partially nodulation phenotypes and N levels. ISME J. 4:315–326. evaluated by collecting isolates from various plant species 17. Kamil, Z., M. Rizk, M. Saleh, and S. Moustafa. 2007. Isolation growing at different sampling sites. This ecological infor- and identification of rhizosphere soil chitinolytic bacteria and their potential in antifungal biocontrol. Global J. Mol. Sci. 2:57–66. mation would be useful for the selection and utilization of 18. Kobayashi, D.Y., M. Guglielmoni, and B.B. Clarke. 1995. Isolation indigenous chitinolytic bacteria as candidates for the effec- of the chitinolytic bacteria Xanthomonas maltophilia and Serratia tive control of phytopathogenic fungi in an ecological way. marcescens as biological control agents for summer patch disease of turfgrass. Soil Biol. Biochem. 27:1479–1487. 19. Latour, X., T. Corberand, G. Laguerre, F. Allard, and P. Lemanceau. Acknowledgements 1996. The composition of fluorescent pseudomonad populations associated with roots is influenced by plant and soil type. Appl. We would like to thank the following people for their coop- Environ. Microbiol. 62:2449–2456. eration in collecting plant samples; K. Watanabe, Agricultural 20. Matsumoto, N. 2009. Snow molds: A group of fungi that prevail Research Institute, Ibaraki Agricultural Center, Japan; Y. Takatsu, under snow. Microbes Environ. 24:14–20. Plant Biotechnology Institute, Ibaraki Agricultural Center, Japan. 21. Nielsen, M.N., and J. Sørensen. 1999. Chitinolytic activity of We thank P. Williams, University of Nottingham, UK for providing Pseudomonas fluorescens isolates from barley and sugar beet C. violaceum strain CV026. We also thank H. Tokuji at the National rhizosphere. FEMS Microbiol. Ecol. 30:217–227. Agricultural Research Center for Hokkaido Region, Japan for 22. Nion, Y.A., and K. Toyota. 2008. Suppression of bacterial wilt technical assistance. and Fusarium wilt by a Burkholderia nodosa strain isolated from Kalimantan soils, Indonesia. Microbes Environ. 23:134–141. 23. Palleroni, N.J. 2005. Genus IX. Stenotrophomonas Palleroni and References Bradbury 1993, 608VP, pp. 107–115. In D.J. Brenner, N.R. Krieg, J.T. Staley, and G.M. Garrity (eds.), Bergey’s Manual of Systematic 1. Aktuganov, G.E., A.I. Melent’ev, N.F. Galimzyanova, and A.V. Bacteriology, 2nd ed., Part B The Gammaproteobacteria. Springer, Shirokov. 2008. The study of mycolytic properties of aerobic spore- New York, USA. forming bacteria producing extracellular chitinases. Microbiology 24. Park, J.H., R. Kim, Z. Aslam, C.O. Jeon, and Y.R. Chung. 2008. (Moscow) 77:700–709. Lysobacter capsici sp. nov., with antimicrobial activity, isolated from 2. Altschul, S.F., W. Gish, W. Miller, E.W. Myers, and D.J. Lipman. the rhizosphere of pepper, and emended description of the genus 1990. Basic local alignment search tool. J. Mol. Biol. 215:403–410. Lysobacter. Int. J. Syst. Evol. Microbiol. 58:387–392. 3. Amakata, D., Y. Matsuo, K. Shimono, J.K. Park, C.S. Yun, H. 25. Reyes-Ramírez, A., B.I. Escudero-Abarca, G. Aguilar-Uscanga, P.M. Matsuda, A. Yokota, and M. Kawamukai. 2005. Mitsuaria Hayward-Jones, and J.E. Barboza-Corona. 2004. Antifungal activity chitosanitabida gen. nov., sp. nov., an aerobic, chitosanase-producing of Bacillus thuringiensis chitinase and its potential for the biocontrol member of the ‘Betaproteobacteria’. Int. J. Syst. Evol. Microbiol. of phytopathogenic fungi in soybean seeds. J. Food Sci. 69:M131– 55:1927–1932. M134. 4. Amann, R.I., W. Ludwig, and K.H. Schleifer. 1995. Phylogenetic 26. Saitou, N., and M. Nei. 1987. The neighbor-joining method: A identification and in situ detection of individual microbial cells new method for reconstructing phylogenetic trees. Mol. Biol. Evol. without cultivation. Microbiol. Rev. 59:143–169. 4:406–425. 5. Brzezinska, M.S., and W. Donderski. 2006. Chitinolytic bacteria 27. Schloss, P.D., and J. Handelsman. 2005. Introducing DOTUR, a in two lakes of different trophic status. Pol. J. Ecol. 54:295–301. computer program for defining operational taxonomic units and 6. Chernin, L.S., M.K. Winson, J.M. Thompson, S. Haran, B.W. estimating species richness. Appl. Environ. Microbiol. 71:1501– Bycroft, I. Chet, P. Williams, and G.S.A.B. Stewart. 1998. 1506. Chitinolytic activity in Chromobacterium violaceum: Substrate 28. Schloss, P.D., and J. Handelsman. 2006. Introducing SONS, a tool analysis and regulation by quorum sensing. J. Bacteriol. 180:4435– for operational taxonomic unit-based comparisons of microbial 4441. community memberships and structures. Appl. Environ. Microbiol. 72:6773–6779. 14 SOMEYA et al.

29. Singh, A.K., I. Ghodke, and H.S. Chhatpar. 2009. Pesticide tolerance 38. Swift, S., J.P. Throup, P. Williams, G.P.C. Salmond, and G.S.A.B. of Paenibacillus sp. D1 and its chitinase. J. Environ. Manage. Stewart. 1996. Quorum sensing: A population density component 91:358–362. in the determination of bacterial phenotype. Trends Biochem. Sci. 30. Someya, N., N. Kataoka, T. Komagata, K. Hirayae, T. Hibi, and K. 21:214–219. Akutsu. 2000. Biological control of cyclamen soilborne diseases by 39. Terahara, T., S. Ikeda, C. Noritake, K. Minamisawa, K. Ando, S. Serratia marcescens strain B2. Plant Dis. 84:334–340. Tsuneda, and S. Harayama. 2009. Molecular diversity of bacterial 31. Someya, N., M. Nakajima, K. Hirayae, T. Hibi, and K. Akutsu. 2001. chitinases in arable soils and the effects of environmental factors on Synergistic antifungal activity of chitinolytic enzymes and pro- the chitinolytic bacterial community. Soil Biol. Biochem. 41:473– digiosin produced by biocontrol bacterium, Serratia marcescens 480. strain B2 against gray mold pathogen, Botrytis cinerea. J. Gen. Plant 40. Thara, K.V., and S.S. Gnanamanickam. 1994. Biological control of Pathol. 67:312–317. rice sheath blight in India: Lack of correlation between chitinase 32. Someya, N., S. Numata, M. Nakajima, A. Hasebe, T. Hibi, and production by bacterial antagonists and sheath blight suppression. K. Akutsu. 2003. Biological control of rice blast by the epiphytic Plant Soil 160:277–280. bacterium Erwinia ananas transformed with a chitinolytic enzyme 41. Thompson, J.D., T.J. Gibson, F. Plewniak, F. Jeanmougin, and gene from an antagonistic bacterium, Serratia marcescens strain B2. D.G. Higgins. 1997. The CLUSTAL_X windows interface: Flexible J. Gen. Plant Pathol. 69:276–282. strategies for multiple sequence alignment aided by quality analysis 33. Someya, N., S. Numata, M. Nakajima, A. Hasebe, and K. Akutsu. tools. Nucleic Acids Res. 25:4876–4882. 2004. Influence of rice-isolated bacteria on chitinase production 42. Wang, Q., G.M. Garrity, J.M. Tiedje, and J.R. Cole. 2007. Naïve by the biocontrol bacterium Serratia marcescens strain B2 and Bayesian classifier for rapid assignment of rRNA sequences into the the genetically modified rice epiphytic bacterium. J. Gen. Plant new bacterial taxonomy. Appl. Environ. Microbiol. 73:5261–5267. Pathol. 70:371–375. 43. Wolf, A., A. Fritze, M. Hagemann, and G. Berg. 2002. Stenotro- 34. Someya, N., M. Nakajima, K. Watanabe, T. Hibi, and K. Akutsu. phomonas rhizophila sp. nov., a novel plant-associated bacterium 2005. Potential of Serratia marcescens strain B2 for biological with antifungal properties. Int. J. Syst. Evol. Microbiol. 52:1937– control of rice sheath blight. Biocontrol Sci. Technol. 15:105–109. 1944. 35. Someya, N., K. Tsuchiya, T. Yoshida, M.T. Noguchi, K. Akutsu, 44. Yi, J.H., H.K. Jang, S.J. Lee, K.E. Lee, and S.G. Choi. 2004. and H. Sawada. 2007. Fungal cell wall degrading enzyme-producing Purification and properties of chitosanase from chitinolytic β- bacterium enhances the biocontrol efficacy of antibiotic-producing Proteobacterium KNU3. J. Microbiol. Biotechnol. 14:337–343. bacterium against cabbage yellows. J. Plant Dis. Prot. 114:108–112. 45. Zeller, S.L., H. Brandl, and B. Schmid. 2007. Host-plant selectivity 36. Someya, N., and K. Akutsu. 2009. Indigenous bacteria may interfere of rhizobacteria in a crop/weed model system. PLoS ONE 2:e846. with the biocontrol of plant diseases. Naturwissenschaften 96:743– 46. Zhang, Z., G.Y. Yuen, G. Sarath, and A.R. Penheiter. 2001. 747. Chitinases from the plant disease biocontrol agent, Stenotrophomonas 37. Someya, N., T. Morohoshi, N. Okano, E. Otsu, K. Usuki, M. maltophilia C3. Phytopathology 91:204–211. Sayama, H. Sekiguchi, T. Ikeda, and S. Ishida. 2009. Distribution of N-acylhomoserine lactone-producing fluorescent pseudomonads in the phyllosphere and rhizosphere of potato (Solanum tuberosum L.). Microbes Environ. 24:305–314.