ISOLATION of CULTURABLE ENDOPHYTIC BACTERIA from MOSO BAMBOO (PHYLLOSTACHYS EDULIS) and 16S Rdna DIVERSITY ANALYSIS

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

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, China 2 Mycological Research Center of Fujian Agriculture and Forestry University, Fuzhou, China

*Corresponding author: [email protected]

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

INTRODUCTION 1982). The endophytic bacteria with functions such as disease resistance, growth promotion and nitro- Endophytic bacteria are bacteria that live in various gen fixation have been identified. Endophytic bacteria tissues and organs of healthy plants at certain stages have been isolated that can degrade organic pollutants or all stages of their life cycle. The bacteria have estab- or promote plant growth in soils containing heavy lished a mutualistic relationship with the plants (Ryan metals (Sheng, 2008 a, b). Because of the potential et al., 2008; Xu, 2011). Because they may confer an advantages they may offer to crop plants, it is impor- ecological advantage, endophytic bacteria can estab- tant to study endophytic bacteria and to establish a lish long-term colonies in plants and be transmitted resource database of species with various functions. through generations of offspring with little influence from environmental conditions. Endophytic bacteria Moso bamboo (Phyllostachys edulis) is a genus of are natural biological resources and they have a wide bamboo found throughout the paleotropics. It is an range of biological functions during plant growth important forest resource in South China because it and resistance to disease and adverse environmental is fast growing, matures early and has many uses and conditions (Guo et al., 2011; Lu et al., 2006; He et al., economic benefits. According to statistics in China’s 2004); therefore, there is a great potential to research Sixth Forest Resource Inventory, Moso bamboo forests and develop endophytic bacteria for use in agricul- in China cover an area of 3 372 million hectares, or tural production. Various endophytic bacteria have approximately 47% of the world’s total Moso bamboo been isolated from tomato, pepper, citrus, lemon, cot- forest area. Research on Moso bamboo has focused on ton, rice, poplar, tobacco and Dendrobium candidum improving yields, optimizing the physical and chemi- (Berg et al., 2005; Mano et al., 2006; Gaulner et al., cal properties of the soil, and studying the species’

1001 1002 Yuan et al. diversity (Huang et al., 2006; Gao et al., 2006; Zhang Medium and main reagents et al., 2007). In recent years, Li et al. (2008), Qi et al. (2006) and others have conducted research on the mi- Endophytic bacteria were isolated and cultured in NA croorganisms in the Phyllostachys edulis and Fargesia culture media containing 3 g beef extract, 5 g pep- rhizosphere and on the soil bacteria. Xia et al. (2009) tone, 5 g NaCl, 18 g agar, and 1000 ml water with has explored the potential for selection of the plant’s pH 7.0-7.2. An endophytic bacteria DNA extraction growth medium on root endophytic bacteria isola- kit, primers, markers, dNTPs, buffers and lysozyme tion and culture. Han et al. (2009) has also studied were purchased from Shenggong Biotechnology Co. the diversity of culturable bacteria isolated from root Ltd (Shanghai, China). All other reagents were ana- domains of Moso bamboo. However, there has been lytically pure and made in China. no research on the endophytic bacteria present in different tissues of Moso bamboo or a comparison of the Isolation of endophytic bacteria bacterial diversity in different Moso bamboo populations. In this study, we selected three Moso bamboo The collected samples were rinsed with sterile water populations in Fujian Province, China, and we isolated and dried with sterile filter paper on a sterile bench. the culturable endophytic bacterial from the roots, rhi- One gram of tissue from each of the different plant zome, stems and leaves of Moso bamboo species in tissues was weighed, immersed in 75% ethanol for these regions to study the composition and diversity 5 min, and then immersed in 5% Clorox bleach for of these bacteria. This work establishes the founda- 3 min. After rinsing with sterile water 3-5 times and tion for screening bacteria for functions related to drying with sterile filter paper, the sample surface was disease prevention and growth promotion, as well as placed in contact with the surface of the NA culture the manufacture and application of microbial agents. plate for 3-5 min. The water from the last rinse was also placed on the NA plate. The plates were incubated at 30oC. If there was no colony growing on the MATERIALS AND METHODS plate surface, disinfection of the plant surface was considered successful; otherwise, the isolation of Sample collection 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 Three samples of Moso bamboo were selected in each sterile water were added and ground until fully ho- of the following regions in March 2014: Wuyi Moun- mogenized. The homogenized sample was diluted to tains (Wuyishan Xingcun), Jiangle (Longxi Moun- 10-2, 10-3, 10-4, and 10-5 gradient dilutions. 100 μl of tain nature reserve, Jiangle County), and Changting the diluted sample was spread on the NA plates. Each (Sidu, Changting County). The Moso bamboo tissues sample was plated three times. were chosen based on the following criteria: selected roots were 30 cm beneath the soil surface; selected Survey of the endophytic bacteria colonies rhizomes were at least 50 cm from their junction with the stem and with obvious rhizome buds; se- Samples were incubated at 28oC for 2-3 days. The total lected stems were 130-150 cm above the ground, and number of colonies was counted after the colonies ap- leaves were randomly selected. Tissues from the same peared and the average colony number per gram fresh structures of the three Moso bamboo samples from weight tissue was calculated and expressed as cfu/g FW. each region were mixed, placed in a sterile sample bag Colonies were chosen according to their characteris- immediately after collection, and then cryogenically tics, such as color, morphology, glossiness, mobility, preserved. The isolation of the endophytic bacteria edge roughness. A single colony typical for each com- was conducted within 48 h of the fieldwork. bination of characteristics was picked after the count E nDOPHYTIC bacteria from Moso bamboo and diversity ANALYSIS 1003 and grown on NA plates as a back-up. The isolated contained the least number of bacteria with only colonies were named with a combination of letters and 1.00 × 102 cfu/g FW (Table 1). numbers, following this template: (1) source of sam- Table 1. Total number of culturable endophytic bacteria popula- ples: WYS − samples from Wuyi Mountain; JL samples tions in different tissu from Jiangle; CT samples from Changting; (2) tissue: Moso bamboo culturable endophytic bacteria A − roots; B − rhizomes; C − stems, and D − leaves. Sampling sites Root Rhizome Stem Leaf 16S rDNA identification of endophytic bacteria Wuyishan 4.50×104 1.73×104 2.58×103 1.00×102

After 24-h incubation in liquid NA medium, DNA from Jiangle 1.75×104 8.53×103 2.50×103 1.00×102 the endophytic bacteria was extracted using the Bacterial Genome Extraction Kit (Shenggong Biotechnology Co. Changting 2.25×104 3.43×104 1.00×103 1.00×102 Ltd, Shanghai). 16S rDNA gene sequences were ampli- fied using universal primers 27F (5’-AGAGTTT- Composition and distribution GATCCTGGCTCAG-3’) and 1492R (5’-GGTTACCTT- of culturable endophytic bacteria GTTACGACTT-3’), and PCR products (1500 bp).

PCR products were separated by 1% (W/V) agarose Based on features such as color, character, edge smooth- gel electrophoresis and sent to Poshang Biotechnology ness, glossiness and liquidity, 82 different bacterial Co., Ltd. (Shanghai) for sequencing. After determi- strains were isolated from the roots, rhizomes, stems nation of the DNA sequences, homologous sequence and leaves of Moso bamboo on Wuyi Mountain and retrieval and analysis were conducted using BLAST in the Jiangle and Changting regions. Twenty-seven software from http:/www.ncbi.nlm.nih.gov/Blast/. The strains were isolated from the Wuyi Mountain sam- phylogenetic analysis was performed using the software ples: nine from the root, 8 from the rhizome, 8 from package MEGA (version 5.0) (Kumar et al. 2001), and the stem and 2 from the leaf. Twenty-four strains were the taxonomic status of the strains were determined. 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 RESULTS samples: 13 from the root, 16 from the rhizome, 1 from the stem, and one from the leaf (Fig. 1). Among the Endophytic bacteria isolation isolated endophytic bacteria strains, most were white, glossy, round or irregular in shape, with no liquidity. Using the dilution plate method, endophytic bac- This combination of characters describes 43 strains and teria was cultured from the roots, rhizomes, stems accounted for 52.44% of the total isolated strains. and leaves of Moso bamboo from three geographical regions: Wuyi Mountain, Jiangle, and Changting. No 16S rDNA sequence analysis bacteria grew on the NA plates in contact with the disinfected samples or on the NA plates with the After cloning and sequencing, the 16SrDNA se- last rinse of sterile water, indicating that the samples quences of the 82 isolated endophytic bacteria strains were disinfected thoroughly and the bacteria isolat- were analyzed using BLAST (http:/www.ncbi.nlm. ed were growing within the tissue. In a comparison nih.gov/Blast/) to analyze the similarity between of the endogenous bacteria isolated from root, rhi- our sequences and the NCBI database in order to zome, stem and leaf samples of Moso bamboo, the identify the bacterial strains in our samples. Each most abundant endophytic bacteria were isolated 16SrDNA query sequence recovered hits with known from the roots and rhizomes, with colony num- taxonomic status with more than 98% similarity. bers from 8.53 × 103 to 4.50 × 104 cfu/g FW. Leaves The similarities of the majority of the isolates with 1004 Yuan et al. the known strains in the nucleic acid database were culturable endophytic bacterial community in Moso 99-100% as shown in Table 2. bamboo (Fig. 2).

20 18 16 Wuyishan DISCUSSION 14 Jiangle 12 Changting Currently, more than 129 species belonging to 54 gen- 10 era of bacteria have been discovered in a variety of 8 6 grains, fruit trees and other commercial crops. These 4 endophytic bacteria are mainly of Bacillus, Pseudomo- 2 Number of endophytic bacteria strains bacteria endophytic of Number nas, Xanthomonas, Erwinia and Curtobacterium (He 0 Root Rhizome Stem Leaf et al., 2006). A majority of these species of endophytic

bacteria are soil microbes. In this paper, the cultur- Fig. 1. Fig. 1. Comparison of the numbers of endophytic bacteria strains able endophytic bacterial community structure and in different tissues of Moso bamboo. its diversity in the roots, rhizomes, stems and leaves of Moso bamboo were studied. Through observa- The 82 endophytic bacteria strains isolated from tions of the bacterial colonies’ characteristics and a the roots, rhizomes, stems and leaves of Moso bam- 16S rDNA sequence analysis, 82 endophytic bacteria boo on Wuyi Mountain and the Jiangle and Changting strains, belonging to 47 species from 26 genera were regions belong to 47 species from 26 genera (Table 2). isolated. Alcaligenes, Bacillus and Staphylococcus were The majority of the bacteria were Alcaligenes, Bacillus, the dominant genera of bacteria, of which Bacillus is Staphylococcus, Pseudomonas and Curtobacterium, the endophytic bacterium that can be isolated from representing 13.41%, 12.20%, 12.20%, 7.32% and most plants (Sturz, 2000). Strains of Alcaligenes and 7.32% of the total diversity of the isolated colonies, Staphylococcus have been isolated from grapes, soy- respectively. Among these, there were 20 species from beans, and cantaloupe plants (Tripathi et al., 2006; Liu 14 genera isolated from the Wuyi Mountain samples; et al., 2011; Hung, 2004). 17 species from 14 genera isolated from the Jiangle samples; and 18 species from 11 genera isolated from Because endophytic bacteria and pathogenic the Changting samples. bacteria have the same ecological niche (Hvozdiak et al., 2008), they compete for space and nutrition. The dominant endophytic bacteria strains from The existence of endophytic bacteria can enhance Wuyi Mountain were Arthrobacter, Staphylococcus, plants’ ability to resist disease. Some endogenous Bacillus and Enterobacter, (Table 2). In addition, Ba- bacteria can promote induced systemic resistance cillus, Staphylococcus and Curtobacterium were the (ISR) in plants (Harish et al., 2008; Rajesh et al., most common endophytic bacterial strains from 2014). The results from our research demonstrate Jiangle. The dominant endophytic bacteria strains that the endophytic bacteria community in Moso from Changting were Alcaligenes, Pseudomonas, bamboo is very rich and diverse and can reflect the Staphylococcus and Bacillus. The main endophytic soil microbial abundance of the sampling site. This bacteria strains from Moso bamboo roots were Ba- is a rich resource for further isolation of endophytic cillus and Burkholderia. Strains of Alcaligenes and bacteria strains that have phosphate-dissolving abil- Staphylococcus were the dominant endophytic bacte- ity and nitrogen-fixing functions to promote growth ria strains from Moso bamboo rhizomes. In addition, of plants and resistance to diseases. This research Staphylococcus and Ochrobactrum were the main en- provides a scientific foundation for the screening of dophytic bacterial strains in Moso bamboo stems. bioactive endophytic bacteria from Moso bamboo, These findings provide a framework for understand- and for further exploration to develop and utilize ing the diversity, structure and characteristics of the these resources. E nDOPHYTIC bacteria from Moso bamboo and diversity ANALYSIS 1005

Table 2. Classification and distribution of the culturable endophytic bacteria

Number of isolates Sequence Genus Closest strains (accession no.) strains from different parts similarity Root Rhizome Stem Leaf Bacillus Bacillus sp.(KJ733996) CT-A03 100% 1 Bacillus CT-A06, JL-A04,JL-B05,JL-B06 100% 2 2 amyloliquefaciens(KM117160) 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

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

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 1 Pseudomonas JL-A02-2 99% 1 fluorescens(AB680523) Staphylococcus Staphylococcus sciuri(KM243920) CT-B01-1,CT-B03 100% 2 Staphylococcus sciuri(NR074999) CT-B10 99% 1

Staphylococcus JL-C05,JL-C07,JL-A09,JL-B02,WYS- 100% 1 1 4 equorum(KM036089) C08, WYS-C10

Staphylococcus warneri(KF876869) WYS-B04-1 100% 1 Brevibacterium Brevibacterium aureum(KF002253) CT-A16,CT-A18 99% 2 Curtobacterium Curtobacterium CT-A04,CT-A05,CT-A15,JL-B16 99% 3 1 flaccumfaciens(JX113236) 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% 1 Arthrobacter JL-A07 100% 1 dextranolyticus(AB117515) 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.

Table 2 continued:

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

Twenty-nine endophytic bacteria strains were erating conditions, climate or culturing conditions. isolated from Moso bamboo root by using NA cul- Further study is needed for clarification. According ture media. They belong to 22 species from 14 gen- to the study of the community structure of cultur- era. The dominant bacteria were Bacillus (24.14%) able endophytic bacteria in different regions and and Burkholderia (13.8%). Similar research was different parts of bamboo, it was found that Bacillus conducted by Han et al. (2009), who isolated 40 en- and Burkholderia mainly existed in bamboo root, dophytic bacteria strains from Moso bamboo root with less in the rhizomes. Strains of Alcaligenes and using LB and KB media that belong to 16 species Staphylococcus mainly existed in rhizomes and in from 9 genera, the dominant bacteria being Burk- smaller number in bamboo root. This showed that holderia (35%) and Pseudomonas (17.5) followed by the different organizational structure of the endo- Burkholderia, Pseudomonas, Bacillus and Arthrobac- phytic bacteria of different ecological structure ex- ter. Han et al. (2009) isolated Pantoea, and we also ists. These results are consistent with results of Liu isolated this strain in Moso bamboo leaf. Our study et al. (2011) in citrus research. did not isolate the bacteria of Xanthomonas, Steno- trophomonas, Lysinibacillus and Kocuria. As com- Since endophytic bacteria can enter the plant pared to Han et al. (2009), in our study we isolated through the epidermis, colonize the inside of the plant a further 9 genera of endophytic bacteria, such as and establish a mutualistic relationship with the plant Alcaligenes, Staphylococcus, Brevibacterium, Curto- over evolutionary time, it is possible to screen and iso- bacterium, Enterobacter, Leclercia, Microbacterium, late bacterial species from plants that have adopted the Planomicrobium and Moraxella. The differences plant-endophytic bacteria system, and build an endo- between the strains of endophytic bacteria from phytic bacteria resource data base. Through the combi- Moso bamboo growing in different areas, and be- nation of traditional isolation methods and non-culture, tween bacteria in different tissues of Moso bamboo molecular biology methods, we can select for advan- might be related to the influence of the habitat, op- tageous bacteria strains with beneficial functions, and E nDOPHYTIC bacteria from Moso bamboo and diversity ANALYSIS 1007

Fig. 2. Phylogenetic analysis of 16S rDNA sequences of Moso bamboo culturable endophytic bacteria. the foundation for further utilization can be established. Conflict of interest disclosure: All authors have read and Further understanding of the process of colonization, approved this version of the article, and due care has been breeding and propagation of endophytic bacteria are taken to ensure the integrity of the work. Neither the entire needed before we can selectively use them. Appropriate paper nor any part of its content has been published or has adjustments to the microbial community structure are been accepted elsewhere. It is not being submitted to any necessary to avoid disadvantages to the host plant and other journal. self-regulating, stable bacterial communities, so that a more advanced microbial community structure can be developed for agricultural production systems. REFERENCES

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