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

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 significant 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.


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 established a mutualistic relationship with the plants (Ryan et al., 2008;Xu, 2011).Because they may confer an ecological advantage, endophytic bacteria can establish 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 agricultural production.Various endophytic bacteria have been isolated from tomato, pepper, citrus, lemon, cotton, 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 nitrogen 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 important 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 chemical 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 Yuan 1 , Fang Liu 2 and Guo-Fang Zhang  (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 microorganisms 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 isolation 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 different tissues of Moso bamboo or a comparison of the bacterial diversity in different Moso bamboo populations.In this study, we selected three Moso bamboo populations in Fujian Province, China, and we isolated the culturable endophytic bacterial from the roots, rhizome, stems and leaves of Moso bamboo species in these regions to study the composition and diversity of these bacteria.This work establishes the foundation for screening bacteria for functions related to disease prevention and growth promotion, as well as the manufacture and application of microbial agents.

Sample collection
Three samples of Moso bamboo were selected in each of the following regions in March 2014: Wuyi Mountains (Wuyishan Xingcun), Jiangle (Longxi Mountain 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; selected 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 peptone, 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 analytically 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 incubated at 30 o C. 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 homogenized.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 28 o C for 2-3 days.The total number of colonies was counted after the colonies appeared and the average colony number per gram fresh weight tissue was calculated and expressed as cfu/g FW.Colonies were chosen according to their characteristics, such as color, morphology, glossiness, mobility, edge roughness.A single colony typical for each combination of characteristics was picked after the count 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 samples: WYS − samples from Wuyi Mountain; JL samples from Jiangle; CT samples from Changting; (2) tissue: A − roots; B − rhizomes; C − stems, and D − leaves.
PCR products were separated by 1% (W/V) agarose gel electrophoresis and sent to Poshang Biotechnology Co., Ltd.(Shanghai) for sequencing.After determination 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.

Endophytic bacteria isolation
Using the dilution plate method, endophytic bacteria 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 isolated were growing within the tissue.In a comparison of the endogenous bacteria isolated from root, rhizome, stem and leaf samples of Moso bamboo, the most abundant endophytic bacteria were isolated from the roots and rhizomes, with colony numbers from 8.53 × 10 3 to 4.50 × 10 4 cfu/g FW.Leaves contained the least number of bacteria with only 1.00 × 10 2 cfu/g FW (Table 1).

Composition and distribution of culturable endophytic bacteria
Based on features such as color, character, edge smoothness, 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 samples: 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 sequences 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 the known strains in the nucleic acid database were 99-100% as shown in Table 2.The 82 endophytic bacteria strains isolated from the roots, rhizomes, stems and leaves of Moso bamboo 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, Bacillus, 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 Bacillus and Burkholderia.Strains of Alcaligenes and Staphylococcus were the dominant endophytic bacteria strains from Moso bamboo rhizomes.In addition, Staphylococcus and Ochrobactrum were the main endophytic bacterial strains in Moso bamboo stems.These findings provide a framework for understanding 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 genera of bacteria have been discovered in a variety of grains, fruit trees and other commercial crops.These endophytic bacteria are mainly of Bacillus, Pseudomonas, Xanthomonas, Erwinia and Curtobacterium (He et al., 2006).A majority of these species of endophytic bacteria are soil microbes.In this paper, the culturable endophytic bacterial community structure and its diversity in the roots, rhizomes, stems and leaves of Moso bamboo were studied.Through observations 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, soybeans, 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 ability 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.

Staphylococcus equorum(KM036089)
JL-C05,JL-C07,JL-A09,JL-B02,WYS- Twenty-nine endophytic bacteria strains were isolated from Moso bamboo root by using NA culture media.They belong to 22 species from 14 genera.The dominant bacteria were Bacillus (24.14%) and Burkholderia (13.8%).Similar research was conducted by Han et al. (2009), who isolated 40 endophytic bacteria strains from Moso bamboo root using LB and KB media that belong to 16 species from 9 genera, the dominant bacteria being Burkholderia (35%) and Pseudomonas (17.5) followed by Burkholderia, Pseudomonas, Bacillus and Arthrobacter.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, Stenotrophomonas, Lysinibacillus and Kocuria.As compared to Han et al. (2009), in our study we isolated a further 9 genera of endophytic bacteria, such as Alcaligenes, Staphylococcus, Brevibacterium, Curtobacterium, Enterobacter, Leclercia, Microbacterium, Planomicrobium and Moraxella.The differences the strains of endophytic bacteria from Moso bamboo growing in different areas, and between 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 culturable 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 endophytic bacteria of different ecological structure exists.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 isolate bacterial species from plants that have adopted the plant-endophytic bacteria system, and build an endophytic bacteria resource data base.Through the combination of traditional isolation methods and non-culture, molecular biology methods, we can select for advantageous bacteria strains with beneficial functions, and  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.
Fig. 1.Fig. 1.Comparison of the numbers of endophytic bacteria strains in different tissues of Moso bamboo.
1,* 1 College of Forestry, Fujian Agriculture and Forestry University, Fuzhou, China 2 Mycological Research Center of Fujian Agriculture and Forestry University, Fuzhou, China diversity

Table 1 .
Total number of culturable endophytic bacteria populations in different tissu

Table 2 .
Classification and distribution of the culturable endophytic bacteria