Isolation , characterization , and antimicrobial activity of endophytic bacteria from Polygonum cuspidatum

1 National Engineering Laboratory for Clean Technology of Leather Manufacture, Sichuan University, 24 South Section 1, Yihuan Road, Chengdu, Sichuan 610065, China. 2 Department of Environmental Science and Engineering, Sichuan University, 24 South Section 1, Yihuan Road, Chengdu, Sichuan 610065, China. 3 Department of Pharmaceutical and Biological Engineering, School of Chemical Engineering, Sichuan University, 24 South Section 1, Yihuan Road, Chengdu, Sichuan 610065, China.


INTRODUCTION
Plant-associated microorganisms fulfill important functions for plant growth and health.On the one hand, plants protect themselves by producing some compounds called secondary metabolites against pathogenic microbes.Some endophytic microorganisms produce antibiotics or growth-stimulating factor to benefit plant.Plant endophytes are microorganisms that live in the internal organs or cell gaps of healthy plants.Many factors, such as soil conditions and phytopathogen populations, influence the population structures of endophytic bacteria (Asraful Islam et al., 2010).Trivedi et al. (2010) found that infection of citrus plants by "Ca.
Liberibacter asiaticus" has a profound effect on the structure and composition of the bacterial community associated with citrus roots.
Endophytic bacteria have been isolated from the interior of the stems and roots of many plants, such as ginseng, cotton, sweet corn, canola, wheat, and others (Cho et al., 2007;Justin et al., 2003).
Many endophytes have antimicrobial activity.Seo et al. (2010) found that some endophytic bacteria isolated from young radishes can be used as biocontrol agents against human and plant pathogens.Castillo et al. (2002) found that the endophytic Streptomyces sp.NRRL 30562 obtained from snakevine produces novel peptide antibiotics that possess wide-spectrum activity against many pathogenic fungi and bacteria.Endophytic bacteria are considered potential resource of antimicrobial agents for biocontrol and pharmaceutical use.In recent years, endophytes have become a hot research topic.Polygonum cuspidatum Sieb.et Zucc., a traditional Chinese medicinal plant known in the UK as Japanese knotweed, is mainly distributed in the southern regions of China and is harvested in spring and autumn.In China, P. cuspidatum roots are dried and they have been used for centuries as a traditional herbal remedy for many diseases, including heart disease and stroke (Kimura et al., 1985).The active ingredients of this medicine are believed to be trans-resveratrol and glucoside (Soleas et al., 1997).The aims of the present study were as follows: (1) to examine the population structures of endophytic bacteria in P. cuspidatum from Shuangliu, Longquan, and Qingcheng in Sichuan Province, China; and (2) to investigate the inhibitory activity of the endophytic bacteria in P. cuspidatum against pathogenic microorganisms such as Gibberella fujikuroi, Aspergillus niger, Aspergillus fumigatus, Klebsiella pneumoniae, Staphylococcus aureus, Escherichia coli, and Bacillus subtilis for their utility as biological control agents or pharmaceutical use.Endophytic bacteria with antifungal and antibacterial activity are reported in this paper.

Sample collection
Samples of P. cuspidatum root were collected from pot-grown plants in Qingcheng, Shuangliu, and Longquan, around Chengdu, the capital of Sichuan Province (Figure 1), from April 2010 to May 2010.Immediately after collection, the plants were washed with tap water and processed for the isolation of endophytic bacteria.

Isolation of endophytic bacteria
Endophytic bacteria were isolated from P. cuspidatum roots from three different locations in Sichuan: Qingcheng, Shuangliu, and Longquan.The roots of P. cuspidatum were surface sterilized with 99% ethanol for 60 s followed by 3.125% sodium hypochlorite for 6 min, washed in 99% ethanol for 30 s, and finally rinsed in sterile water.The surface-sterilized roots were then aseptically sectioned into 1 cm fragments, distributed onto the isolation media, and incubated at 28°C for 2 to 15 days.The only bacterial colonies that developed in the media were separately transferred to fresh media to obtain a pure culture.

DNA extraction, PCR, Sequencing
After subculturing the bacterial strains on LB agar medium for 2 d, fresh mycelia were inoculated in a 100 mL Erlenmeyer flask containing 25 mL of liquid LB medium and cultured in a shaking incubator in darkness at 28°C and 220 rpm for 2 d.The bacterial cells were obtained by centrifugation, suspended in 500 μL of lysozyme solution, and incubated at 37°C for 60 min.Subsequently, 250 μL of 2% SDS was added and the mixture was agitated for 1 min.Then, 250 μL of neutral phenol chloroform was added and the mixture was agitated for 5 min, and then centrifugated at 1500 g for 5 min.Then, 80 μL of 3 M sodium acetate (pH 4.8) and 800 μL of isopropyl alcohol were added to the supernatant liquid, mixed gently, and then centrifugated at 1500 g for 2 min.Finally, the supernatant liquid was poured off and the DNA pellets were washed with ice-cold 70% ethanol.The DNA pellets were vacuum dried and dissolved in 50 μL of TE (pH 8.0).
Each bacterial DNA was amplified with the universal primers: 887F: 5′-CGGAGAGTTTGATCCTGG-3′; and 878R: 5′-TACGGCTACCTTGTTAGCGAC-3′.Amplification was performed in a 25 μL reaction system that contained 2 μL of template DNA, 2.5 μL of 10 mmol/L of each primer, 0.25 μL of 1 U/μL Taq polymerase, 0.75 μL/L of 2.5 mmol/L dNTP, 2.5 μL of 10× Buffer, 2.5 μL of MgCl 2 , and 14 μL of distilled water.The PCR was carried out according to the following protocol: initial denaturation 94°C for 3 min; denaturation 94°C for 45 s; annealing 50°C for 45 s, extension 72°C for 1 min.From each PCR reaction, 5 μL was obtained and the PCR products were examined through agarose gel electrophoresis (0.8% w/v) using ethidium bromide staining.The anticipated product, approximately 750 bp, was isolated from the amplified mixture after agarose gel electrophoresis using the SanPrep column DNA gel extraction kit, and was sequenced by Sangon Biotech (Shanghai) Co., Ltd.The partial sequences of the 16S rDNA of the isolated strains were submitted to GenBank and the accession numbers of the sequences are listed in Table 1.

Phylogenetic analysis
The sequence-based identification and phylogenetic analysis were based on data on sequences obtained from BLAST searches using the EzTaxon server 2.1 (Chun et al., 2007).Sequences were aligned using BioEdit (Hall 1999).The overhanging ends were removed from both ends to ensure that all sequences were of the same length.The tree was constructed with the MEGA 4.1 software package using the neighbor-joining method.Bootstrap tests were performed using 1000 replicates (Tamura et al., 2007).

Antimicrobial assay
The inhibitory activity of P. cuspidatum endophytic bacteria on the growth of G. fujikuroi, A. niger, A. fumigatus, K. pneumoniae, S. aureus, E. coli, and B. subtilis was determined through the filter paper method.Before final concentration of 2-5×10 7 cells mL -1 was regulated, microbe suspension (for bacteria) or spores suspension (for fungi) was mingled with LB or PDA medium respectively.Paper disc were immersed in bacterial fermentation liquor and placed on the surface of assay plates .After incubation at 37°C (for bacteria) or at 28°C (for fungi) for 24~48 h, the inhibition zones around each disc was measured in diameter Φ.The antimicrobial activity was qualitatively evaluated.according to the diameter of clear zone of growth inhibition.
For the minimal inhibitory concentration (MIC) determination, the indicator bacteria cultured on slants were washed with sterile water.Then, the bacterial or bacterial spore cultures were diluted to 10 7 CFU/mL and stored at 4°C.Before use, the cultures were mixed with appropriate amounts of medium (the final approximate bacterial concentration was 10 5 CFU/mL and the final approximate fungal concentration was 10 4 CFU/mL).The samples were separately dissolved and the resulting mixtures were diluted twofold, and were introduced into the 96-cell plates.The culture medium containing only the indicator bacteria, without the sample was used as the negative control, and the culture medium was used as the blank control.Both the bacterial and fungal cultures were cultured at 28°C for 24 h.The absence of bacterial growths and the MIC was determined by visual observation.To obtain statistically significant results, test of inhibitory activity to indicator strains were carried out three times, the data of the diameter were the average of three assays.

Isolation and phylogenetic placement of endophytic bacteria from P. cuspidatum roots
The diversity of endophytic bacteria in P. cuspidatum was assessed using root samples collected from three different locations: Shuangliu, Qingcheng, and Longquan (Figure 1).In total, 244 culturable endophytes were recovered from the interior of P. cuspidatum roots (Table 1).Among them, 80 strains were obtained from Shuangliu, 82 strains from Longquan, and the remainder, from Qingcheng.
The results of the phylogenetic analysis of P. cuspidatum endophytic bacteria isolated from Shuangliu are shown in Figure 2A.Based on the 16S rDNA sequence analysis, three orders were identified, namely, Bacillales, Actinomycetales, and Pseudomonadales, and 80 endophytic bacteria were classified into 6 genera, including 11 distinct species (Table 1): Lysinibacillus  Figure 2B shows the phylogenetic analysis of P. cuspidatum endophytic bacteria from Longquan.Five orders were identified: Actinomycetales, Bacillales, Rhizobiales, Pseudomonadales, and Enterobacteriales.The 82 isolates from Longquan represented 10 genus and 14 species (Table 1); the species are as follows:  Streptomyces ederensis (CB25, 2 isolates).The 16S rDNA sequences of these 82 isolates were 98-100% similar to the sequences in databases, except CB20, which was 97.718% similar to its corresponding sequence.
The strains under the order Bacillales were broadly distributed in the roots of P. cuspidatum collected from the three sites.Of the 244 strains collected, 71% (173/244) belonged to this order.Bacteria most closely related to B. thuringiensis ATCC 10792 were found in all three sites.The strains B. cereus, L. sphaericus, P. pabuli, P. alvei, and B. atrophaeus were the predominant species; they were found in two sample sites and were isolated more than once in each site.Actinomycetes are an important group in the root of P. cuspidatum.Streptomycetes was the predominant group of endophytic actinobacteria found.Streptomyces strains were found in samples from all three sites.

Antimicrobial activity of endophytic bacteria from P. cuspidatum
Selected endophytic bacteria from the three sites were studied to determine their in vitro inhibitory activity against pathogenic fungi (G.fujikuroi, A. niger, and A. fumigatus) and pathogenic bacteria (K.pneumoniae, S. aureus, E. coli, and B. subtilis) (Table 2).Five isolates (CB22, CB23, CB25, CB35, and CB36) appeared to have a broad spectrum of antifungal and antibacterial activity in vitro.The isolates were all Streptomyces sp.CB36 (S. murinus) in particular, exhibited a notable antifungal activity against all pathogenic fungi tested.CB23, isolated from Longquan, not only had a strong activity against pathogenic fungi (A.niger and A. fumigatus), but also against pathogenic bacteria (K.pneumoniae and B. subtilis).CB10 (S. umbrinus) and CB22 (S. atroolivaceus) showed activity against pathogenic S. aureus.In this study, some Bacillus strains showed significant antifungal activities, such as B. thuringiensis (CB6, CB15, and CB29), which showed inhibitory activity against G. fujikuroi, and B. cereus (CB5 and CB19) and B. atrophaeus (CB16 and CB28), which showed inhibitory activity against A. niger.Rhizobium sp.(CB14 and CB33) against A. fumigatus, CB10 against Staphylococcus aureus, and CB23 against K. pneumoniae was high.Hence, we chose to determine the minimal inhibitory concentration (MIC) of these three strains as follows: CB10 was 542 μg/mL, CB 23 was 407 μg/mL, and CB36 was 272 μg/mL respectively.

DISCUSSION
Endophytic bacteria are found in virtually every plant on earth (Ryan et al., 2008).Knowledge on the diversity of endophytic bacteria is important for both ecological and biotechnological studies.Cho et al. (2007) have identified 13 different bacterial genera from 63 isolates from the interior of ginseng root.Similarly, Dias et al. (2009) have isolated 20 different endophytic bacterial genera from the meristematic tissues of three varieties of strawberry.Justin et al. (2003) isolated 49 actinobacteria from surface-sterilized wheat roots.Thus, endophytic communities are clearly distinct in different plant species and the diversity of the communities may vary significantly.Also, endophytic communities from different locations are different.In this study, 244 culturable endophytic bacteria associated with P. cuspidatum were isolated and identified.Interestingly, the predominance of Bacillales strains over the other bacterial endoflora of P. cuspidatum was observed in the present study.The most prevalent endophytic bacterial groups isolated from P. cuspidatum roots from Shuangliu and Qingchen belong to Bacillales.On the other hand, Actinomycetales was the dominant group in the plants from Longquan, because of the great number of CB17, which was most related to Leucobacter aridicollis (Table 1).The strains related to B. thuringiensis (similarity 99.8%) were the most frequently found in all locations.Furthermore, the other strains, which were related to L. sphaericus, P. pabuli, B. cereus, P. alvei, P. rettgeri, B. atrophaeus, and P. nitroreducens, were isolated from at least two sites.This suggests that the population structures of endophytic bacteria in the root tissues of P. cuspidatum are similar despite the large distance between collection sites.The results in this study indicate different ecological characteristics from those presented in previous reports, which imply that the distribution of endophytic bacteria mainly depend on environmental conditions such as temperature, humidity, UV irradiation, and nutrients in the apoplast, but not on the hosts (Durgude et al., 2009;Baker et al., 2010).The higher frequency of Bacillales in plants compared with the other groups observed may indicate that they have formed a beneficial association with plants.
The organisms that reside in the living tissues of host plants form a variety of relationships ranging from symbiotic to pathogenic (Chen et al., 2011).Endophytes may contribute to their host plants by producing a plethora of substances that provide protection, and ultimately have survival value, to the plants ( Soca-chafre Sun et al. 1503et al., 2011).Ultimately, these compounds, once isolated and characterized, may also have potential uses in modern medicine, agriculture, and in various industries.Miller et al. (1998) found that Pseudomonas viridiflava, a plant-associated bacterium, produces ecomycins, which have significant bioactivity against a wide range of human and plant pathogenic fungi.We have demonstrated that some endophytic bacteria from P. cuspidatum inhibit other microorganisms (Figure 3).Most of the crude extracts from 36 endophytes showed different degrees of inhibitory activity against the test organisms (Table 2).Up to 10 strains showed high antifungal activity; they belonged to Bacillus (CB5, CB6, CB15, CB16, CB19, CB28, and CB29) and Streptomyces (CB23, CB35, CB36).The antifungal activity of Bacillus has been reported in previous studies.Bacillus has been found to produce antifungal factors such as antifungal hydrolytic enzymes (Chang et al., 2007), spore-specific lipopeptides (Yao et al., 2003), and fengysin (Lin et al., 1999).Furthermore, Bacillus strains are stable in soil as spores, and this property is advantageous for their use as biocontrol agents.However, the antifungal activity of Streptomyces mainly depends on their secondary metabolites (Shimizu et al., 2000;Taechowisan et al., 2005).The endophytic bacteria from P. cuspidatum from Longquan showed apparently high inhibitory activity against G. fujikuroi (90%).Less than half of all the strains from the three sample sites were found to exude growth inhibitory substances towards A. niger when tested in vitro (Figure 3).In our study, Streptomyces sp.CB36 showed broad-spectrum antifungal activity and the strongest antifungal activity against Aspergillus.Streptomyces strains CB10, CB22, and CB23 showed strong antibacterial activity, whereas the other endophytes showed weak or no antibacterial activity.Many antibacterial agents have been reported and identified from Streptomyces by previous research (Raja et al., 2011).The results of these studies indicate that strains of endophytic Streptomyces play important roles in the antimicrobial mechanism of plants and that they are significant resources for novel antimicrobial agents.Microbe-plant interactions are far from being fully understood.Nevertheless, more evidence shows plantassociated microorganisms provide substantial benefits to agriculture, industry, and the environment.In brief, this study determined that there are regional differences between microbial communities inside the roots of P. cuspidatum.Most of the bacteria we examined had antimicrobial activity.These results show the potential use of endophytic bacteria for biocontrol to protect plants from fungal or bacterial diseases.Further studies are needed to separate and extract the active substances from endophytic bacteria.