1001

Arch. Biol. Sci., Belgrade, 67(3), 1001-1008, 2015 DOI:10.2298/ABS141212063Y

INTRODUCTION

Endophytic bacteria are bacteria that live in various tissues and organs of healthy plants at certain stages or all stages of their life cycle. The bacteria have estab-lished a mutualistic relationship with the plants (Ryan et al., 2008; Xu, 2011). Because they may confer an ecological advantage, endophytic bacteria can estab-lish long-term colonies in plants and be transmitted through generations of offspring with little influence from environmental conditions. Endophytic bacteria are natural biological resources and they have a wide range of biological functions during plant growth and resistance to disease and adverse environmental conditions (Guo et al., 2011; Lu et al., 2006; He et al., 2004); therefore, there is a great potential to research and develop endophytic bacteria for use in agricul-tural production. Various endophytic bacteria have been isolated from tomato, pepper, citrus, lemon, cot-ton, rice, poplar, tobacco and Dendrobium candidum (Berg et al., 2005; Mano et al., 2006; Gaulner et al.,

1982). The endophytic bacteria with functions such as disease resistance, growth promotion and nitro-gen fixation have been identified. Endophytic bacteria have been isolated that can degrade organic pollutants or promote plant growth in soils containing heavy metals (Sheng, 2008 a, b). Because of the potential advantages they may offer to crop plants, it is impor-tant to study endophytic bacteria and to establish a resource database of species with various functions.

Moso bamboo (Phyllostachys edulis) is a genus of bamboo found throughout the paleotropics. It is an important forest resource in South China because it is fast growing, matures early and has many uses and economic benefits. According to statistics in China’s Sixth Forest Resource Inventory, Moso bamboo forests in China cover an area of 3 372 million hectares, or approximately 47% of the world’s total Moso bamboo forest area. Research on Moso bamboo has focused on improving yields, optimizing the physical and chemi-cal properties of the soil, and studying the species’

ISOLATION OF CULTURABLE ENDOPHYTIC BACTERIA FROM MOSO BAMBOO (PHYLLOSTACHYS EDULIS) AND 16S RDNA DIVERSITY ANALYSIS

Zong-Sheng Yuan1, Fang Liu2 and Guo-Fang Zhang1,*

1 College of Forestry, Fujian Agriculture and Forestry University, Fuzhou, China2 Mycological Research Center of Fujian Agriculture and Forestry University, Fuzhou, China

*Corresponding author: fjzgfzgf@126.com

Abstract: We analyzed culturable endophytic bacteria from Moso bamboo (Phyllostachys edulis) using traditional bacterial isolation and culture methods and then studied the colony characteristics and diversity with a 16S rDNA sequence analysis. We isolated 82 endophytic bacteria strains belonging to 47 species in 26 genera from the root, rhizome, stem and leaves of Moso bamboo species from populations on Wuyi Mountain, and in the Jiangle and Changting regions. There were signifi-cant differences in the composition of the culturable endophytic bacteria isolated from the different areas and from different tissues. The dominant bacteria strains from the Wuyi Mountain samples were Arthrobacter, Staphylococcus, Bacillus and Enterobacter, while the dominant bacteria from the Jiangle samples were Bacillus, Staphylococcus and Curtobacterium, and the dominant bacteria in the Changting samples were Alcaligenes, Pseudomonas, Staphylococcus and Bacillus. Our results demonstrate the abundant diversity of endophytic bacteria in Moso bamboo.

Key words: Phyllostachys edulis; culturable endophytic bacteria; 16S rDNA; diversity

Received December 12, 2014; Revised February 23, 2015; Accepted February 24, 2015

1002 Yuan et al.

diversity (Huang et al., 2006; Gao et al., 2006; Zhang et al., 2007). In recent years, Li et al. (2008), Qi et al. (2006) and others have conducted research on the mi-croorganisms in the Phyllostachys edulis and Fargesia rhizosphere and on the soil bacteria. Xia et al. (2009) has explored the potential for selection of the plant’s growth medium on root endophytic bacteria isola-tion and culture. Han et al. (2009) has also studied the diversity of culturable bacteria isolated from root domains of Moso bamboo. However, there has been no research on the endophytic bacteria present in dif-ferent tissues of Moso bamboo or a comparison of the bacterial diversity in different Moso bamboo popula-tions. In this study, we selected three Moso bamboo populations in Fujian Province, China, and we isolated the culturable endophytic bacterial from the roots, rhi-zome, stems and leaves of Moso bamboo species in these regions to study the composition and diversity of these bacteria. This work establishes the founda-tion for screening bacteria for functions related to disease prevention and growth promotion, as well as the manufacture and application of microbial agents.

MATERIALS AND METHODS

Sample collection

Three samples of Moso bamboo were selected in each of the following regions in March 2014: Wuyi Moun-tains (Wuyishan Xingcun), Jiangle (Longxi Moun-tain nature reserve, Jiangle County), and Changting (Sidu, Changting County). The Moso bamboo tissues were chosen based on the following criteria: selected roots were 30 cm beneath the soil surface; selected rhizomes were at least 50 cm from their junction with the stem and with obvious rhizome buds; se-lected stems were 130-150 cm above the ground, and leaves were randomly selected. Tissues from the same structures of the three Moso bamboo samples from each region were mixed, placed in a sterile sample bag immediately after collection, and then cryogenically preserved. The isolation of the endophytic bacteria was conducted within 48 h of the fieldwork.

Medium and main reagents

Endophytic bacteria were isolated and cultured in NA culture media containing 3 g beef extract, 5 g pep-tone, 5 g NaCl, 18 g agar, and 1000 ml water with pH 7.0-7.2. An endophytic bacteria DNA extraction kit, primers, markers, dNTPs, buffers and lysozyme were purchased from Shenggong Biotechnology Co. Ltd (Shanghai, China). All other reagents were ana-lytically pure and made in China.

Isolation of endophytic bacteria

The collected samples were rinsed with sterile water and dried with sterile filter paper on a sterile bench. One gram of tissue from each of the different plant tissues was weighed, immersed in 75% ethanol for 5 min, and then immersed in 5% Clorox bleach for 3 min. After rinsing with sterile water 3-5 times and drying with sterile filter paper, the sample surface was placed in contact with the surface of the NA culture plate for 3-5 min. The water from the last rinse was also placed on the NA plate. The plates were incu-bated at 30oC. If there was no colony growing on the plate surface, disinfection of the plant surface was considered successful; otherwise, the isolation of endophytic fungi might be contaminated. Samples were cut with sterile scissors and placed in a sterile mortar. Sterilized quartz sand and a small amount of sterile water were added and ground until fully ho-mogenized. The homogenized sample was diluted to 10-2, 10-3, 10-4, and 10-5 gradient dilutions. 100 μl of the diluted sample was spread on the NA plates. Each sample was plated three times.

Survey of the endophytic bacteria colonies

Samples were incubated at 28oC for 2-3 days. The total number of colonies was counted after the colonies ap-peared and the average colony number per gram fresh weight tissue was calculated and expressed as cfu/g FW. Colonies were chosen according to their characteris-tics, such as color, morphology, glossiness, mobility, edge roughness. A single colony typical for each com-bination of characteristics was picked after the count

ENDOPHYTIC BACTERIA FROM MOSO BAMBOO AND DIVERSITY ANALYSIS 1003

and grown on NA plates as a back-up. The isolated colonies were named with a combination of letters and numbers, following this template: (1) source of sam-ples: WYS − samples from Wuyi Mountain; JL samples from Jiangle; CT samples from Changting; (2) tissue: A − roots; B − rhizomes; C − stems, and D − leaves.

16S rDNA identification of endophytic bacteria

After 24-h incubation in liquid NA medium, DNA from the endophytic bacteria was extracted using the Bacterial Genome Extraction Kit (Shenggong Biotechnology Co. Ltd, Shanghai). 16S rDNA gene sequences were ampli- fied using universal primers 27F (5’-AGAGTTT-GATCCTGGCTCAG-3’) and 1492R (5’-GGTTACCTT-GTTACGACTT-3’), and PCR products (1500 bp).

PCR products were separated by 1% (W/V) agarose gel electrophoresis and sent to Poshang Biotechnology Co., Ltd. (Shanghai) for sequencing. After determi-nation of the DNA sequences, homologous sequence retrieval and analysis were conducted using BLAST software from http:/www.ncbi.nlm.nih.gov/Blast/. The phylogenetic analysis was performed using the software package MEGA (version 5.0) (Kumar et al. 2001), and the taxonomic status of the strains were determined.

RESULTS

Endophytic bacteria isolation

Using the dilution plate method, endophytic bac-teria was cultured from the roots, rhizomes, stems and leaves of Moso bamboo from three geographical regions: Wuyi Mountain, Jiangle, and Changting. No bacteria grew on the NA plates in contact with the disinfected samples or on the NA plates with the last rinse of sterile water, indicating that the samples were disinfected thoroughly and the bacteria isolat-ed were growing within the tissue. In a comparison of the endogenous bacteria isolated from root, rhi-zome, stem and leaf samples of Moso bamboo, the most abundant endophytic bacteria were isolated from the roots and rhizomes, with colony num-bers from 8.53 × 103 to 4.50 × 104 cfu/g FW. Leaves

contained the least number of bacteria with only 1.00 × 102 cfu/g FW (Table 1). Table 1. Total number of culturable endophytic bacteria popula-tions in different tissu

Sampling sites

Moso bamboo culturable endophytic bacteria

Root Rhizome Stem Leaf

Wuyishan 4.50×104 1.73×104 2.58×103 1.00×102

Jiangle 1.75×104 8.53×103 2.50×103 1.00×102

Changting 2.25×104 3.43×104 1.00×103 1.00×102

Composition and distribution of culturable endophytic bacteria

Based on features such as color, character, edge smooth-ness, glossiness and liquidity, 82 different bacterial strains were isolated from the roots, rhizomes, stems and leaves of Moso bamboo on Wuyi Mountain and in the Jiangle and Changting regions. Twenty-seven strains were isolated from the Wuyi Mountain sam-ples: nine from the root, 8 from the rhizome, 8 from the stem and 2 from the leaf. Twenty-four strains were isolated from the Jiangle samples: 7 from the root, 11 from the rhizome, 4 from the stem and 2 from the leaf. Thirty-one strains were isolated from the Changting samples: 13 from the root, 16 from the rhizome, 1 from the stem, and one from the leaf (Fig. 1). Among the isolated endophytic bacteria strains, most were white, glossy, round or irregular in shape, with no liquidity. This combination of characters describes 43 strains and accounted for 52.44% of the total isolated strains.  

16S rDNA sequence analysis

After cloning and sequencing, the 16SrDNA se-quences of the 82 isolated endophytic bacteria strains were analyzed using BLAST (http:/www.ncbi.nlm.nih.gov/Blast/) to analyze the similarity between our sequences and the NCBI database in order to identify the bacterial strains in our samples. Each 16SrDNA query sequence recovered hits with known taxonomic status with more than 98% similarity. The similarities of the majority of the isolates with

1004 Yuan et al.

the known strains in the nucleic acid database were 99-100% as shown in Table 2.

0

2

4

6

8

10

12

14

16

18

20

Root Rhizome Stem Leaf

Num

ber o

f en

doph

ytic

bac

teria

stra

ins

WuyishanJiangle

Changting

Fig. 1. Fig. 1. Comparison of the numbers of endophytic bacteria strains in different tissues of Moso bamboo.

The 82 endophytic bacteria strains isolated from the roots, rhizomes, stems and leaves of Moso bam-boo on Wuyi Mountain and the Jiangle and Changting regions belong to 47 species from 26 genera (Table 2). The majority of the bacteria were Alcaligenes, Bacillus, Staphylococcus, Pseudomonas and Curtobacterium, representing 13.41%, 12.20%, 12.20%, 7.32% and 7.32% of the total diversity of the isolated colonies, respectively. Among these, there were 20 species from 14 genera isolated from the Wuyi Mountain samples; 17 species from 14 genera isolated from the Jiangle samples; and 18 species from 11 genera isolated from the Changting samples.

The dominant endophytic bacteria strains from Wuyi Mountain were Arthrobacter, Staphylococcus, Bacillus and Enterobacter, (Table 2). In addition, Ba-cillus, Staphylococcus and Curtobacterium were the most common endophytic bacterial strains from Jiangle. The dominant endophytic bacteria strains from Changting were Alcaligenes, Pseudomonas, Staphylococcus and Bacillus. The main endophytic bacteria strains from Moso bamboo roots were Ba-cillus and Burkholderia. Strains of Alcaligenes and Staphylococcus were the dominant endophytic bacte-ria strains from Moso bamboo rhizomes. In addition, Staphylococcus and Ochrobactrum were the main en-dophytic bacterial strains in Moso bamboo stems. These findings provide a framework for understand-ing the diversity, structure and characteristics of the

culturable endophytic bacterial community in Moso bamboo (Fig. 2).

DISCUSSION

Currently, more than 129 species belonging to 54 gen-era of bacteria have been discovered in a variety of grains, fruit trees and other commercial crops. These endophytic bacteria are mainly of Bacillus, Pseudomo-nas, Xanthomonas, Erwinia and Curtobacterium (He et al., 2006). A majority of these species of endophytic bacteria are soil microbes. In this paper, the cultur-able endophytic bacterial community structure and its diversity in the roots, rhizomes, stems and leaves of Moso bamboo were studied. Through observa-tions of the bacterial colonies’ characteristics and a 16S rDNA sequence analysis, 82 endophytic bacteria strains, belonging to 47 species from 26 genera were isolated. Alcaligenes, Bacillus and Staphylococcus were the dominant genera of bacteria, of which Bacillus is the endophytic bacterium that can be isolated from most plants (Sturz, 2000). Strains of Alcaligenes and Staphylococcus have been isolated from grapes, soy-beans, and cantaloupe plants (Tripathi et al., 2006; Liu et al., 2011; Hung, 2004).

Because endophytic bacteria and pathogenic bacteria have the same ecological niche (Hvozdiak et al., 2008), they compete for space and nutrition. The existence of endophytic bacteria can enhance plants’ ability to resist disease. Some endogenous bacteria can promote induced systemic resistance (ISR) in plants (Harish et al., 2008; Rajesh et al., 2014). The results from our research demonstrate that the endophytic bacteria community in Moso bamboo is very rich and diverse and can reflect the soil microbial abundance of the sampling site. This is a rich resource for further isolation of endophytic bacteria strains that have phosphate-dissolving abil-ity and nitrogen-fixing functions to promote growth of plants and resistance to diseases. This research provides a scientific foundation for the screening of bioactive endophytic bacteria from Moso bamboo, and for further exploration to develop and utilize these resources.

ENDOPHYTIC BACTERIA FROM MOSO BAMBOO AND DIVERSITY ANALYSIS 1005

Table 2. Classification and distribution of the culturable endophytic bacteria

Genus Closest strains (accession no.) strains Sequence similarity

Number of isolates from different parts

Root Rhizome Stem Leaf

Bacillus Bacillus sp.(KJ733996) CT-A03 100% 1Bacillus amyloliquefaciens(KM117160) CT-A06, JL-A04,JL-B05,JL-B06 100% 2 2

Bacillus sp.(KM251856) JL-A05 100% 1

Bacillus thuringiensis(KJ542769) CT-A19,WYS-C09 99-100% 1 1

Bacillus pichinotyi(KF453786) WYS-A04,WYS-A07 98% 2

Burkholderia Burkholderia sp.(KF788188) CT-A12,CT-A13,WYS-A02-1 100% 3

Burkholderia sp.(KF479551) WYS-A03-1 100% 1

Alcaligenes Alcaligenes sp.(JN836756) CT-A17,CT-B04-1,CT-B07,CT-B09-1, CT-B17,CT-B20,CT-B20-1 99% 1 6

Alcaligenes sp.(JF710958) CT-B01 100% 1

Alcaligenes faecalis(LK391652) CT-B19,JL-B08,JL-C02-1 100% 2 1

Pseudomonas Pseudomonas sp.(JF740045) CT-A14 99% 1

Pseudomonas sp.(GU120660) CT-B11,CT-B21,CT-D06,WYS-A02 99% 1 2 1Pseudomonas fluorescens(AB680523) JL-A02-2 99% 1

Staphylococcus Staphylococcus sciuri(KM243920) CT-B01-1,CT-B03 100% 2

Staphylococcus sciuri(NR074999) CT-B10 99% 1

Staphylococcus equorum(KM036089)

JL-C05,JL-C07,JL-A09,JL-B02,WYS-C08, WYS-C10 100% 1 1 4

Staphylococcus warneri(KF876869) WYS-B04-1 100% 1

Brevibacterium Brevibacterium aureum(KF002253) CT-A16,CT-A18 99% 2

Curtobacterium Curtobacterium flaccumfaciens(JX113236) CT-A04,CT-A05,CT-A15,JL-B16 99% 3 1

Curtobacterium sp.(JN084144) JL-B10,JL-B17 99% 2

Enterobacter Enterobacter sp.(JQ660204) CT-B09-2 99% 1

Enterobacter sp.(KC736654) WYS-A02-2 99% 1

Enterobacter sp.(JX566614) WYS-C01-1 99% 1

Enterobacter sp.(KC355280) WYS-B12 99% 1

Arthrobacter Arthrobacter sp.(KF055023) WYS-C05,WYS-B03,WYS-B04 99-100% 2 1

Arthrobacter sp.(KJ878607) WYS-B08 99% 1Arthrobacter dextranolyticus(AB117515) JL-A07 100% 1

Leclercia Leclercia sp.(KJ000855) JL-A03 100% 1

Leclercia sp.(HM159983) JL-B11 99% 1

Rhizobium Rhizobium tropici(FN178365) CT-B06 1

Rhizobium sp.(FJ784124) WYS-B02 100% 1

Micrococcus Micrococcus sp.(KJ782615) WYS-C03,WYS-D01-1 99% 1 1

1006 Yuan et al.

Twenty-nine endophytic bacteria strains were isolated from Moso bamboo root by using NA cul-ture media. They belong to 22 species from 14 gen-era. The dominant bacteria were Bacillus (24.14%) and Burkholderia (13.8%). Similar research was conducted by Han et al. (2009), who isolated 40 en-dophytic bacteria strains from Moso bamboo root using LB and KB media that belong to 16 species from 9 genera, the dominant bacteria being Burk-holderia (35%) and Pseudomonas (17.5) followed by Burkholderia, Pseudomonas, Bacillus and Arthrobac-ter. Han et al. (2009) isolated Pantoea, and we also isolated this strain in Moso bamboo leaf. Our study did not isolate the bacteria of Xanthomonas, Steno-trophomonas, Lysinibacillus and Kocuria. As com-pared to Han et al. (2009), in our study we isolated a further 9 genera of endophytic bacteria, such as Alcaligenes, Staphylococcus, Brevibacterium, Curto-bacterium, Enterobacter, Leclercia, Microbacterium, Planomicrobium and Moraxella. The differences between the strains of endophytic bacteria from Moso bamboo growing in different areas, and be-tween bacteria in different tissues of Moso bamboo might be related to the influence of the habitat, op-

erating conditions, climate or culturing conditions. Further study is needed for clarification. According to the study of the community structure of cultur-able endophytic bacteria in different regions and different parts of bamboo, it was found that Bacillus and Burkholderia mainly existed in bamboo root, with less in the rhizomes. Strains of Alcaligenes and Staphylococcus mainly existed in rhizomes and in smaller number in bamboo root. This showed that the different organizational structure of the endo-phytic bacteria of different ecological structure ex-ists. These results are consistent with results of Liu et al. (2011) in citrus research.

Since endophytic bacteria can enter the plant through the epidermis, colonize the inside of the plant and establish a mutualistic relationship with the plant over evolutionary time, it is possible to screen and iso-late bacterial species from plants that have adopted the plant-endophytic bacteria system, and build an endo-phytic bacteria resource data base. Through the combi-nation of traditional isolation methods and non-culture, molecular biology methods, we can select for advan-tageous bacteria strains with beneficial functions, and

Ochrobactrum Ochrobactrum sp.(KJ944018) WYS-C01,WYS-C14 100% 2

Acinetobacter Acinetobacter guillouiae(KJ147068) CT-A02 99% 1

Acinetobacter sp.(HM063913) WYS-A01-1 99% 1

Janibacter Janibacter sp.(HG423361) WYS-B05 99% 1

Janibacter sp.(HG423361) WYS-B09 99% 1

Microbacterium Microbacterium phyllosphaerae(FJ006871) JL-B15,WYS-A05 99% 1 1

Leucobacter Leucobacter aridicollis(KC764981) CT-B13-1 99% 1

Leifsonia Leifsonia xyli(CP006734) CT-C02 99% 1

Brevibacillus Brevibacillus sp.(HM453885) JL-B09 99% 1

Planomicrobium Planomicrobium sp.(HM224493) JL-A14 99% 1

Dermacoccus Dermacoccus sp.(EU330343) JL-D03 99% 1

Labedella Labedella sp.(KJ000855) JL-B03 99% 1

Pantoea Pantoea sp.(JX266331) JL-D02 99% 1

Brachybacterium Brachybacterium sp.(NR113401) JL-C04 99% 1

Moraxella Moraxella sp.(AY162144) WYS-A06 99% 1

Streptomyces Streptomyces flavofuscus(JQ924410) WYS-D01 100% 1

Table 2 continued:

ENDOPHYTIC BACTERIA FROM MOSO BAMBOO AND DIVERSITY ANALYSIS 1007

the foundation for further utilization can be established. Further understanding of the process of colonization, breeding and propagation of endophytic bacteria are needed before we can selectively use them. Appropriate adjustments to the microbial community structure are necessary to avoid disadvantages to the host plant and self-regulating, stable bacterial communities, so that a more advanced microbial community structure can be developed for agricultural production systems.

Authors’ contributions: The work presented here was car-ried out in collaboration between all authors. Zong-sheng Yuan, Fang Liu and Guo-Fang Zhang defined the research theme. Zong-sheng Yuan and Guo-Fang Zhang designed methods and experiments, carried out the laboratory ex-periments, analyzed the data, interpreted the results. Fang Liu co-designed experiments, and co-worked on associated data collection and their interpretation.

Conflict of interest disclosure: All authors have read and approved this version of the article, and due care has been taken to ensure the integrity of the work. Neither the entire paper nor any part of its content has been published or has been accepted elsewhere. It is not being submitted to any other journal.

REFERENCES

Berg, G., Krechel, A., Ditz, M., Sikora, R.A., Ulrich, A. and J. Hall-mann (2005). Endophytic and ectophytic potato-associated bacterial communities differ in structure and antagonistic function against plant pathogenic fungi. FEMS. Microbiol. Ecol. 51, 215-229.

Gao, Z.Q. and M.Y. Fu (2006). Characteristics of seasonal changes in soil carbon and nitrogen nutrients of different Phyllo-stachys pubescens stands. J. Zhejiang Coll. Forest. 23, 248-254.

Fig. 2. Phylogenetic analysis of 16S rDNA sequences of Moso bamboo culturable endophytic bacteria.

1008 Yuan et al.

Gaulner, J.M. Feldman, A.W. and R.M. Zablotowiez (1982). Iden-tity and behavior of xylem-residing bacteria in rough lemon of Florida citrus trees. Appl. Environ. Microb. 43, 1335-1342.

Guo H., Mao Z.Q. and X.L. Liu (2011). Research progress of inter-action between plant and microorganism. Chinese Agri. Sci. Bulle. 27, 28-33.

Han, J.G., Xia, D.L., Li, L.B., Sun, L., Yang, K. and L.P. Zhang (2009). Diversity of culturable bacteria isolated from root domains of Moso bamboo (Phyllostachys edulis). Microb. Ecol. 58, 363-373.

Harish, S., Kavino, M., Kumar, N., Saravanakumar, D., Soori-anathasundaram, K. and R. Samiyappan (2008). Biohard-ening with plant growth promoting rhizosphere and endo-phytic bacteria induces systemic resistance against banana bunchy top virus. Appl. Soil. Ecol. 39, 187-200.

He, H., Qiu, S.X., Hu, F.P. and X. Guang (2004). Advance in bio-logical effects of endophytic bacteria. J. Microbiol. 24, 40-45.

He, J., liu, Y.Z. and J.B. Kang (2006). Recent research advances in endophytic Mycelium and its utilization in medicine. Guizhou Agri. Sci. 34, 113-115.

Huang, Q.T., Chen, A.L. and J. He (2006). Comparison of soil physical and chemical properties among various Phyllo-stachys pubescens plantation. J. Fujian CHung, P.G. and P.K. Anna (2004). Isolation and characterization of endophytic bacteria in soybean (Glycine sp.). Omonrice. 12, 92-101.

Hvozdiak, R.I., Pasichnyk, L.A. and V.P. Patyka (2008). Depart-ment of Plant Pathogenic Bacteria: the past and the present. Human Perfor. Extre. Environ. 70, 48-54.

Kumar, S., Tamura, K., Jakobsen, I. and M. Nei (2001). MEGA2: molecular evolutionary genetics analysis software. Bioin-formatics. 17, 1244-1245.

Li, L.L., Liu, M., Yang, S., Liu, L., Miao, K., Yang, K. and J. Han (2008). Cultivable microbial diversity at the rhizosphere of Phyllostachys pubescens. Acta. Microbiol. Sin. 48, 772-779.

Liu, J., Wang, E.T. and W.X. Chen (2011). Discovery and research progress of endophytic bacteria in the root nodules of legumes − A review. Acta. Microbiol. Sin. 51, 1001-1006.

Lu, Y. and M. Luo (2004). Isolation of endophytic bacteria from Hami melon and screening of antagonistic bacteria. J. Shi-hezi Univ. (Nat. Sci). 22, 104-109.

Lu, Z.Y., Yang, X.F. and Y.J. Feng (2006). Endophytic bacteria: sepa-ration, classification, colonization and application. Chinese Bull. Life. Sci. 18, 90-94.

Mano, H.F., Tananka, A., Watanabe, H., Kaga, S., Okunishi, S. and H. Morisaki (2006). Culturable surface and endophytic bac-terial flora of the maturing seeds of rice plants (Oryza sativa) cultivated in a paddy field. Microb. Environ. 21, 86-100.

Qi, Z.M. and W.Q. Yang (2006). Microbial quantity and enzyme activity in Fargesia denudata rhizosphere soil of western Sichuan. Chinese J. Ecolo. 25, 1370-1374.

Rajesh, P.S. and R.V. Ravishankar (2014). Quorum quenching activ-ity in cell-free lysate of endophytic bacteria isolated from Pterocarpus santalinus Linn., and its effect on quorum sens-ing regulated biofilm in Pseudomonas aeruginosa PAO1. Microbiol. Res. 169, 561-569.

Ryan, R.P., Germaine, K., Franks, A., Ryan, D.J. and D.N. Dowl-ing (2008). Bacterial endophytes: recent developments and application. FEMS. Microbiol. Lett. 278, l-9.

Sheng, X.F., Chen, X.B. and L.Y. He (2008). Characteristics of an endophytic bacteria pyrene-degrading Bacterium Enterobac-ter sp. 12JI from Allium macrostemon Bunge. Int. Biodeter. Biodegr. 62, 88-95.

Sheng, X.F., Xia, J.J., Jiang, C.Y., He, L.Y. and M. Qian (2008). Char-acterization of heavy metal-resistant endophytic bacteria from rape (Brassica napus) roots and their potential in pro-moting the growth and lead accumulation of rape. Environ. Pollut. 156, 1164-1170.

Sturz, A.V., Christie, B.R. and J. Norwak (2000). Bacterial endo-phytes: Potential role in developing sustainable systems of crop production. Crit. Rev. Plant Sci. 19, 1-30.

Tripathi, A.K., Verma, S.C., Chowdhury, S.P., Lebuhn, M., Gattinger, A. and M. Schloter (2006). Ochrobactrum oryzae sp.nov., an endophytic bacterial species isolated from deep-water rice in India. Int. J. Syst. Evol. Micr. 56, 1677-1680.

Xia, D.L., Ren, Y., Li, L.B., Sun, L. and J.G. Han (2009). Screening of isolating medium for Phyllostachys edulis Endophytic bacte-ria. J. Beijing Univ. Agr. 24, 15-19.

Xu, Y.J. (2011). Research progress on resources diversity of plant endophytes. Guangdong Agr. Sci. 4, 149-152.

Zhang, G.H., Xiao, J.H., Nie, J.Z., Chen, S.L. and Y.W. Guo (2007). Study on the species diversity at Moso bamboo stands of different type. Forest Res. 20, 615-621.