International Journal of Agricultural Policy and Research Vol.2 (3), pp. 098-104, March 2014 Available online at http://www.journalissues.org/ijapr/ © 2014 Journal Issues

ISSN 2350-1561

Original Research Paper

Composition and diversity of endophytic bacterial communities in noni (Morinda citrifolia L.) seeds Accepted 14 February, 2014 *1Liu

Yang, 1Yao Su , 2Xu Pengpeng , 1Cao Yanhua , 1Li Jinxia , 1Wang Jiemiao , 3Tan Wangqiao, 1Cheng Chi 1 China

National Research Institute of Food and Fermentation Industries, Beijing 100015, People’s Republic of China 2 Beijing Geneway Technology Co., Ltd., Beijing 100010, People’s Republic of China. 3 Hainan Noni Biological Engineering Development Co., Ltd., Haikou 570125, People’s Republic of China. *Corresponding Author Email: [email protected] Tel.:+ 86-10-53218288

The objective of this study is to investigate the endophytic bacterial communities in Noni (Morinda citrifolia L.) seeds. By using high-throughput sequencing (HTS) technique, a research on the diversity of endophytic bacterial communities in Noni seed samples was conducted by this paper, which were collected from four planting locations in the introduction and cultivation base of Hainan Noni Biological Engineering Development Co., Ltd. in Sanya. A total of 268159 sequences and 2870 Operational Taxonomic Units (OTUs) were obtained from the seed samples, which were classified into N1, N2, N3 and N4, through HTS by bioinformatics analysis. The results showed that there was a good consistency between bacterial community structure and distribution of dominant genus in all samples. A mount of 10 OTUs of the most abundance associated with the four sample libraries were found to be related to Pelomonas sp., Ochrobactrum intermedium, Kinneretia sp., Serratia sp., Paucibacter sp., Leptothrix sp., Inhella sp., Ewingella sp., Thiomonas sp. and Massilia sp..This study has laid a solid foundation for further study in beneficial microbes, which would help to improve the growth and efficacy of component production in Noni plants. Key words Noni Seed, endophytic bacteria, bacterial community, HTS

INTRODUCTION Seeds, which are reproductive organs in gymnosperm and angiosperm plants, play an important role in agriculture production (Guan 2009). Due to the economic importance of seeds, scientists have been working on seed science for years. According to some studies conducted by these scientists, various microbial communities inside different seeds are identified, and there are also some microbes carried on the surface (Nelson 2004). Microbes which can live in plant without causing any disease are defined as endophytic microorganisms (Kloepper and Beauchamp 1992). According to recent researches, endophytic bacteria are found to be able to affect plants through many different ways. Through endophytic nitrogen-fixation, growthpromoting effects and biological control function, healthy plant growth and high quality agricultural products are expected to be obtained (Glick et al. 1999). Noni (Morinda citrifolia L.) is a tropical plant that belongs to the Rubiaceae, and it has been applied to food and

medical field for thousands of years. Noni is native to Southeast Asia and Australia. Some wild Noni is distributed in Paracel Islands in South China Sea, and there are also some introductions in Hainan province of China. Noni is said to have so great a nutritional value that it is even regarded to be the “super fruit”. A lot of researches have been carried out to explore the nutritional characteristics of Noni, and the results proved that the broad biological activities, antimicrobial properties, anti-cancer effects, anti-oxidant activities, anti-inflammatory, analgesic, and cardiovascular of Noni are all involved (Wang et al. 2002). As of now, focus has been put on the nutritional characteristics of Noni by basically all the international or domestic researches; very limited information about the endophytes is available, which may play an important role in the nutritional characteristics of Noni. This situation makes it necessary to fill the gaps in Noni study through scientific researches in the future. Furthermore, it is of great

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significance to study the endophytic microbes, in view of the fact that natural fermentation is the most important processing technology to produce Noni products. In this research, Miseq Illumina sequencing platform is used to identify the composition and diversity of endophytes in Noni, and the Xisha Noni seed is used as the study object. The goal of this research is to offer background knowledge for further study, which may focus on relationships between endophytic microbes and Noni plants, and accordingly improve the fermentation process of Noni products. MATERIALS AND METHODS Sample collection and surface sterilization Seed samples of Noni (Morinda citrifolia L.) were randomly collected in June 2012 from four planting locations in the introduction and cultivation base of Hainan Noni Biological Engineering Development Co., Ltd. in Sanya, Hainan (18.300160133600 N, 109.526580864400 E, southern China), which were labeled N1, N2, N3 and N4 respectively and were stored at the temperature of 4℃. Noni seeds were first washed in sterile water and immersed in 70% ethanol for 3 minutes; and then they were washed with fresh sodium hypochlorite solution (2.5 % available Cl-) for 5 minutes; after that, Noni seeds were rinsed with 70% alcohol for 30 seconds; and finally they washed for 5-7 more times with sterile water. The aliquots of the final rinsing water were spread on Luria–Bertani solid medium plates and were cultured for 3 days at the temperature of 28℃, in order to confirm that the seeds were sterilized and no bacteria remained on the seed surface. Only the seed samples that were confirmed to be sterile were used for subsequent analysis. DNA extraction, amplification, and sequencing About 5.0g of surface-sterilized Noni seeds were frozen with liquid nitrogen and quickly ground into a fine powder with a precooled sterile mortar. Then, the CTAB procedure was used to extract the seed and bacterial DNA of all samples (Sun et al. 2008). The DNA was then resuspended in 30μL sterile Milli-Q water, and was stored at the temperature of -20℃. The primers used for amplification were 968F (5’AACGCGAAGAACCTTAC-3’) and 1378R (5’CGGTGTGTACAAGGCCCGGGAACG-3’), which amplified about 400bp DNA fragments flanking the V6 - V8 regions of the 16S rRNA gene (Yu and Morrison 2004). In order to distinguish samples from each other in the mixed action, a 6-base long barcode was added to the 5’ of V6-V8 forward primers. The 50μL PCR reaction mixture contained 50ng DNA extract, 1×Taq reaction buffer, 20pmol of each primer, 200μmol dNTP and 1.5 units Taq enzyme (Ferments). Reaction procedure: initial denaturation at 94°C for 5

minutes, the second denaturation at 94°C for 30 seconds, annealing at 52°C for 30 seconds, elongation at 72°C for 45 seconds, and extension at 72°C for 10 minutes after 30 circulations. The band at approximately 400bp was excised and purified using the Wizard SV Gel and PCR Clean-up System (Promega) as described by the manufacturer. The purified PCR products were mixed in equal concentration, and sequenced by Miseq (Illumina, USA.) following the instructions at Chinese National Human Genome Center (SinoGenomax). Sequencing analysis In order to obtain more accurate sequences, sequences which were shorter than 200bp or whose quality value was lower than 18 were eliminated. More than 80% of the total raw sequences were kept for the analysis. High-throughput sequencing data were processed using the Mothur software as described in the pipeline of “Costello stool analysis” (Costello et al. 2009). The observed richness, inverse Simpson diversity and Shannon–Wiener index were measured based on the frequency of OTUs and genera in the sequence collections. Miseq paired-end reads had to meet a mean quality score of 21 and were limited to those with a minimum length of 200 nucleotide base pairs (bp). Efforts to reduce sequencing errors that were an artifact of sequencing relied on the “denoising” algorithm (Huse et al. 2010). The V6-V8 region was extracted from the sequences, and the subsequent reads were checked for chimeras using UCHIME (Edgar et al. 2011) and were clustered into operational taxonomic units (OTUs) at a similarity of 97% using the furthest neighbor algorithm by ClustalW. When using the more standard ‘species-level’ OTU cutoff (97% sequence identity), it is more difficult to make data visualizations and analysis, because the resulting OTU table would become much larger. With the identity being at the level of 97%, the final OTU table consisted of 195722 sequences (average of 48930 sequences per sample) which were distributed into 74769 OTUs, of those 2870 OTUs were represented by more than 3 sequences summed up from the four samples. Community analyses and taxonomy assignment To explore the potential of microbial community composition analysis using Miseq sequencing, and to learn about how classification accuracy varies with reads of different length and quality, a high-quality reference set was compiled. The reference set was based on the SILVA SSURef database Release 111 (Pruesse et al. 2007), comprising about 739633 sequences which were near full-length 16S rRNA. The optimized reads were taxonomically assigned using the RDP-classifier with a bootstrap cut-off of 80% (Wang et al. 2007).

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Figure 1. Rarefaction analysis of the four samples (Rarefaction curves of OTUs clustered at 97% sequence identity across the samples.)

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were generated by 4 samples. After filtering, 116092 effective sequences remained, accounting for nearly 43.2% of the total sequences. And 2870 OTUs were obtained from the four samples through sequencing analysis. Reads ranging from 25696 to 34700 were contained in each of the four bacterial communities, and the OTU ranged from 1807 to 2169. The rarefaction curves of all samples tended to approach the saturation plateau (Figure 1). As it is shown in Figure 1, the endophytic bacterial community structures of these four samples were quite similar to each other based on the correlation analysis, which was calculated by using the “cor. test” function in R, and the correlation ranged from 56% to 93% between each sample (Figure 2). The similarities of bacterial community structures of the four samples, which were collected from four different planting locations in Noni cultivation base, could also be concluded through three indices: the observed richness, the inverse Simpson diversity, and the Shannon– Wiener index (Table 1). Taxonomic composition All the sequences were classified from phylum to genus, according to the OTUs clustered through the furthest neighboring algorithm by ClustalW. 23 different phyla or groups were identified from these samples. The four libraries showed a high similarity of the 16S rDNA profiles in their distributions of phylum level (Figure 3). Proteobacteria and Firmicutes had absolute advantage in the bacterial communities of all the four samples, which accounted for about 97% in each sample. Ten OTUs of the most abundance within these samples could be determined to further understand the importance of the bacteria. The OTUs of the most abundance associated with the four sample libraries were related to Pelomonas (45.93%-59.68%), Ochrobactrum intermedium (7.18%9.09%), Kinneretia (5.16%-5.90%), Serratia (1.81%8.72%), Paucibacter (1.97%-2.61%), Leptothrix (1.60%2.63%), Inhella (1.52%-1.90%), Ewingella (1.75%-3.57%), Thiomonas (1.36%-2.67%) and Massilia (0.77%-2.35%) (Table 2 and Figure 4). Core endophytic bacteria

Figure 2. Correlation among the microbial communities of the four samples (The correlation values were calculated using the “cor. test” function in R.)

RESULTS The diversity and richness of endophytic communities Employing Miseq sequencing, 268159 sequences in total

The bacterial species in the sample libraries N1, N2, N3 and N4 were further researched for there were core endophytic microbes in Noni seeds. The four sample libraries have 948 OTUs in all (Figure 5). According to the statistical analysis, it was indicated that the OTUs common to the four libraries accounted for 80.07%, 84.21%, 90.04% and 84.90% of the reads in the N1, N2, N3 and N4 libraries respectively (Table 3). 937 of the shared OTUs (98.83% in proportion) and 100598 shared reads (99.7% in proportion) were contained in Proteobacteria and Firmicutes. Within the two phyla, Alpha proteobacteria (29.85% in proportion), Beta proteobacteria (6.22% in proportion), Gamma

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Table 1. Endophytic diversity and richness index of four seed samples Sample name N1 N2 N3 N4

Shannon–Wienera 7.0193 7.8777 6.2635 7.9057

Species richnessb 22277 23541 12296 24890

InvSimpsonc 17.5725 35.8782 15.2615 27.9090

Shannon-Wiener diversity index. The higher the number is, the more the diversity is. Species richness refers to the number of OTU classified by ClustalW, cutoff 97%. c Inverse Simpson stands for the diversity index. a

b

Figure 3:Bacterial composition of the communities in phylum level distributions

proteobacteria (35.23% in proportion), Bacilli (12.13% in proportion) and SK259 (15.30% in proportion) represented the types of the most abundance that were common to the four libraries. DISCUSSION Nowadays，researches on the plant seeds are getting integrated with the microbiology science more closely. According to our previous research, about 50 genus and 100 species endophytics have been identified in the plant seeds by now, most of which are found to be Gram negative bacteria; barley, rice, bean, cole, cotton, sugar beet, tomato are the plants involved. However, compared to researches on plant roots, limited information about the endophytic microorganisms in the seeds is available (Cankar et al. 2005); in addition, focus has been put on traditional crops and vegetables by most of these studies, and knowledge about plant seeds used in functional food remains to be explored. For the sake of scientificalness, the study material N1, N2, N3 and N4 are selected from four different planting regions

at random, which are located in the introduction and cultivation base of Hainan Noni Biological Engineering Development Co., Ltd. in Sanya. By comparing the results, it can be concluded that similar pattern is shared by the endophytic community and the structure of the four samples，which in some degree indicates the distribution of the endophytics in this very area. By using the 16S rDNA clone library technique, 42 Operational Taxonomic Units (OTUs) were identified from 33 genuses of the Proteobacteria, Firmicutes, Bacteroidetes and Actinobacteria in previous research. The dominant endophytic species in wild Noni seeds are Enterobacter sp. and Bacillus sp., while Pelomonas sp. and Ochrobactrum sp. are the dominant endophytic species in the samples of this research; Pelomonas sp. and Ochrobactrum sp. are also found in certain amount in wild Noni seeds. Since compositions of the endophytics are basically the same, it can be concluded that the genetic-related Noni seeds usually share the same endophytic community structure. The soil condition where the plant grow has a significant influence on the community structure and the diversity of the endophytic microorganisms (Jefferey et al. 1999), and the fact that most identified bacteria of this project are from

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Table 2. Identities of the 10 most abundant OTUs in the bacterial communities N1 Abundance order 1 2 3 4 5 6 7 8 9 10

N2

N3

N4

Pelomonas sp. (45.99%) Ochrobactrum intermedium (9.09%) Serratia ficaria (8.72%) Kinneretia sp. (5.32%) Ewingella Americana (3.57%) Paucibacter sp. (1.97%) Leptothrix sp. (1.80%) Inhella sp. (1.52%) Thiomonas cuprina (1.36%) Massilia sp. (1.35%)

Pelomonas sp. (54.16%) Ochrobactrum intermedium (8.09%) Kinneretia sp. (5.90%) Thiomonas cuprina (2.67%) Paucibacter sp. (2.13%) Serratia ficaria (1.81%) Ewingella Americana (1.76%) Massilia sp. (1.76%) Inhella sp. (1.64%) Leptothrix sp. (1.60%)

Taxa (Abundance) Pelomonas sp. (59.68%) Ochrobactrum intermedium (7.43%) Kinneretia sp. (5.16%) Serratia ficaria (3.17%) Paucibacter sp. (2.49%) Leptothrix sp. (2.01%) Inhella sp. (1.90%) Ewingella Americana (1.75%) Thiomonas cuprina (1.73%) Massilia sp. (0.77%)

Pelomonas sp. (45.93%) Ochrobactrum intermedium (7.18%) Kinneretia sp. (5.87%) Serratia ficaria (2.91%) Leptothrix sp. (2.63%) Paucibacter sp. (2.61%) Thiomonas cuprina (2.37%) Massilia sp. (2.35%) Inhella sp. (1.90%) Ewingella Americana (1.83%)

Figure 4: Bacterial composition of the communities in genus level distributions

soil also confirms this theory. There are three reasons as to why the soil bacteria become the dominate ones: one is that the soil is the closest and the most common substrate available during the growth process of plants, which offers a good opportunity for the bacteria to enter the plant; one lies in the fact that certain genes in the bacteria decided whether this species can survive in the plant (or on the plant surface) or not (Hardoim et al. 2008); and the grow stages of the plant also affect the community structure of the endophytics

(van Overbeek and van Elsas 2008). According to the result of this research, under certain fixed growth conditions, Pelomonas sp., Ochrobactrum sp. and Serratia sp. turn out to be the dominant bacteria in all the four Noni seed samples, which indicate that all these genus might exist in the growth environment. The ratio they account for may vary, depending on how well they adapt themselves to the samples. A positive influence on the plant growth is shown by some

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Figure 5: Shared OTUs analysis of the four libraries (Venn diagram showing the unique and shared OTUs (3% distance level) in the sample libraries of N1, N2, N3 and N4.)

Table 3 Shared phyla among the libraries of sample N1, N2, N3 and N4 Phylum Actinobacteria Bacteroidetes Firmicutes Proteobacteria Verrucomicrobia Total shared reads Total reads Shared reads/Total reads (%)

Shared OTUs 8 1 115 882 2 -------

of the identified endophytics and these endophtics are found to be beneficial to the fermentation process. For example, Ochrobactrum intermedium can produce nicotine-degrading enzymes, which can alleviate the ecological damages caused by the neonicotinoid insecticide (Li et al. 2007); Serratia marcescens can resist some of the plant pathogen by producing antibiotics or catalases (Wei et al. 1996; Li et al. 2011); chitinase and 2-KDG can be obtained by a high efficient fermentation of Serratia sp. PS-2 and Serratia sp. BK-98 (Pan et al. 2008; Zhang et al. 2011). This is the first time that high-throughput sequencing technology has been applied to the endophytic microorganism study of the Xisha Noni seeds. Being far more than a microecology research of the plant seeds, this project could make a contribution to the efficient selecting of the functional endophytics. The report of this research can also be used as a reference for the endophytic community study among various gene-types or growth stages in the future. The commercial value of this project is significant as well. Since endophytic microbes have influences on the

N1 57 3 1203 29287 12 30562 34700 88.07

Shared reads N2 N3 90 36 4 3 2082 756 20981 24585 7 2 23164 25382 27507 28189 84.21 90.04

N4 104 4 1428 20276 4 21816 25696 84.90

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Cite this article as : Liu Y, Cheng C, Yao S, Xu P, Cao Y, Li J, Wang J, Tan W (2014).Composition and diversity of endophytic bacterial communities in noni (Morinda citrifolia L.) seeds. Int. J. Agric. Pol. Res.2(3):098-104.