Composition and Diversity of Endophytic Bacterial Communities in Noni (Morinda Citrifolia L.) Seeds

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 The objective of this study is to investigate the endophytic bacterial Pengpeng , 1Cao Yanhua , communities in Noni (Morinda citrifolia L.) seeds. By using high-throughput 1 1 sequencing (HTS) technique, a research on the diversity of endophytic Li Jinxia , Wang bacterial communities in Noni seed samples was conducted by this paper, 3 Jiemiao , Tan Wangqiao, which were collected from four planting locations in the introduction and 1Cheng Chi cultivation base of Hainan Noni Biological Engineering Development Co., Ltd. in Sanya. A total of 268159 sequences and 2870 Operational Taxonomic Units 1 China National Research Institute (OTUs) were obtained from the seed samples, which were classified into N1, of Food and Fermentation N2, N3 and N4, through HTS by bioinformatics analysis. The results showed Industries, Beijing 100015, People’s that there was a good consistency between bacterial community structure and Republic of China distribution of dominant genus in all samples. A mount of 10 OTUs of the most 2 Beijing Geneway Technology Co., Ltd., Beijing 100010, People’s abundance associated with the four sample libraries were found to be related Republic of China. to Pelomonas sp., Ochrobactrum intermedium, Kinneretia sp., Serratia sp., 3 Hainan Noni Biological Paucibacter sp., Leptothrix sp., Inhella sp., Ewingella sp., Thiomonas sp. and Engineering Development Co., Ltd., Massilia sp..This study has laid a solid foundation for further study in Haikou 570125, People’s Republic beneficial microbes, which would help to improve the growth and efficacy of of China. component production in Noni plants.

*Corresponding Author Email: [email protected] Key words Noni Seed, endophytic bacteria, bacterial community, HTS Tel.:+ 86-10-53218288

INTRODUCTION

Seeds, which are reproductive organs in gymnosperm and medical field for thousands of years. Noni is native to angiosperm plants, play an important role in agriculture Southeast Asia and Australia. Some wild Noni is distributed production (Guan 2009). Due to the economic importance of in Paracel Islands in South China Sea, and there are also seeds, scientists have been working on seed science for some introductions in Hainan province of China. Noni is said years. According to some studies conducted by these to have so great a nutritional value that it is even regarded to scientists, various microbial communities inside different be the “super fruit”. A lot of researches have been carried seeds are identified, and there are also some microbes out to explore the nutritional characteristics of Noni, and the carried on the surface (Nelson 2004). Microbes which can results proved that the broad biological activities, anti- live in plant without causing any disease are defined as microbial properties, anti-cancer effects, anti-oxidant endophytic microorganisms (Kloepper and Beauchamp activities, anti-inﬂammatory, analgesic, and cardiovascular 1992). According to recent researches, endophytic bacteria of Noni are all involved (Wang et al. 2002). are found to be able to affect plants through many different As of now, focus has been put on the nutritional ways. Through endophytic nitrogen-fixation, growth- characteristics of Noni by basically all the international or promoting effects and biological control function, healthy domestic researches; very limited information about the plant growth and high quality agricultural products are endophytes is available, which may play an important role in expected to be obtained (Glick et al. 1999). the nutritional characteristics of Noni. This situation makes Noni (Morinda citrifolia L.) is a tropical plant that belongs it necessary to fill the gaps in Noni study through scientific to the Rubiaceae, and it has been applied to food and researches in the future. Furthermore, it is of great Int. J. Agric. Pol. Res. 099

significance to study the endophytic microbes, in view of the minutes, the second denaturation at 94°C for 30 seconds, fact that natural fermentation is the most important annealing at 52°C for 30 seconds, elongation at 72°C for 45 processing technology to produce Noni products. seconds, and extension at 72°C for 10 minutes after 30 In this research, Miseq Illumina sequencing platform is circulations. The band at approximately 400bp was excised used to identify the composition and diversity of endophytes and purified using the Wizard SV Gel and PCR Clean-up in Noni, and the Xisha Noni seed is used as the study object. System (Promega) as described by the manufacturer. The goal of this research is to offer background knowledge The purified PCR products were mixed in equal for further study, which may focus on relationships between concentration, and sequenced by Miseq (Illumina, USA.) endophytic microbes and Noni plants, and accordingly following the instructions at Chinese National Human improve the fermentation process of Noni products. Genome Center (SinoGenomax).

Sequencing analysis MATERIALS AND METHODS

Sample collection and surface sterilization In order to obtain more accurate sequences, sequences which were shorter than 200bp or whose quality value was Seed samples of Noni (Morinda citrifolia L.) were randomly lower than 18 were eliminated. More than 80% of the total collected in June 2012 from four planting locations in the raw sequences were kept for the analysis. High-throughput introduction and cultivation base of Hainan Noni Biological sequencing data were processed using the Mothur software Engineering Development Co., Ltd. in Sanya, Hainan as described in the pipeline of “Costello stool analysis” (18.300160133600 N, 109.526580864400 E, southern (Costello et al. 2009). The observed richness, inverse China), which were labeled N1, N2, N3 and N4 respectively Simpson diversity and Shannon–Wiener index were and were stored at the temperature of 4℃. measured based on the frequency of OTUs and genera in the Noni seeds were first washed in sterile water and sequence collections. immersed in 70% ethanol for 3 minutes; and then they Miseq paired-end reads had to meet a mean quality score were washed with fresh sodium hypochlorite solution (2.5 of 21 and were limited to those with a minimum length of % available Cl-) for 5 minutes; after that, Noni seeds were 200 nucleotide base pairs (bp). Efforts to reduce sequencing rinsed with 70% alcohol for 30 seconds; and finally they errors that were an artifact of sequencing relied on the washed for 5-7 more times with sterile water. The aliquots “denoising” algorithm (Huse et al. 2010). The V6-V8 region of the final rinsing water were spread on Luria–Bertani was extracted from the sequences, and the subsequent solid medium plates and were cultured for 3 days at the reads were checked for chimeras using UCHIME (Edgar et al. temperature of 28℃, in order to confirm that the seeds were 2011) and were clustered into operational taxonomic units sterilized and no bacteria remained on the seed surface. (OTUs) at a similarity of 97% using the furthest neighbor Only the seed samples that were confirmed to be sterile algorithm by ClustalW. were used for subsequent analysis. 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 DNA extraction, amplification, and sequencing would become much larger. With the identity being at the level of 97%, the final OTU table consisted of 195722 About 5.0g of surface-sterilized Noni seeds were frozen sequences (average of 48930 sequences per sample) which with liquid nitrogen and quickly ground into a fine powder were distributed into 74769 OTUs, of those 2870 OTUs with a precooled sterile mortar. Then, the CTAB procedure were represented by more than 3 sequences summed up was used to extract the seed and bacterial DNA of all from the four samples. samples (Sun et al. 2008). The DNA was then resuspended in 30μL sterile Milli-Q water, and was stored at the Community analyses and taxonomy assignment temperature of -20℃. The primers used for amplification were 968F (5’- AACGCGAAGAACCTTAC-3’) and 1378R (5’- To explore the potential of microbial community CGGTGTGTACAAGGCCCGGGAACG-3’), which amplified about composition analysis using Miseq sequencing, and to learn 400bp DNA fragments flanking the V6 - V8 regions of the about how classification accuracy varies with reads of 16S rRNA gene (Yu and Morrison 2004). In order to different length and quality, a high-quality reference set was distinguish samples from each other in the mixed action, a compiled. The reference set was based on the SILVA SSURef 6-base long barcode was added to the 5’ of V6-V8 forward database Release 111 (Pruesse et al. 2007), comprising primers. The 50μL PCR reaction mixture contained 50ng about 739633 sequences which were near full-length 16S DNA extract, 1×Taq reaction buffer, 20pmol of each primer, rRNA. The optimized reads were taxonomically assigned 200μmol dNTP and 1.5 units Taq enzyme (Ferments). using the RDP-classifier with a bootstrap cut-off of 80% Reaction procedure: initial denaturation at 94°C for 5 (Wang et al. 2007). Liu et al. 100

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 Figure 1. Rarefaction analysis of the four samples richness, the inverse Simpson diversity, and the Shannon– (Rarefaction curves of OTUs clustered at 97% sequence Wiener index (Table 1). identity across the samples.)

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

The bacterial species in the sample libraries N1, N2, N3 and Figure 2. Correlation among the microbial communities of the N4 were further researched for there were core endophytic four samples (The correlation values were calculated using the “cor. test” microbes in Noni seeds. The four sample libraries have 948 function in R.) 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 RESULTS 3). 937 of the shared OTUs (98.83% in proportion) and 100598 shared reads (99.7% in proportion) were contained The diversity and richness of endophytic communities in Proteobacteria and Firmicutes. Within the two phyla, Alpha proteobacteria (29.85% in proportion), Beta Employing Miseq sequencing, 268159 sequences in total proteobacteria (6.22% in proportion), Gamma Int. J. Agric. Pol. Res. 101

Table 1. Endophytic diversity and richness index of four seed samples

Sample name Shannon–Wienera Species richnessb InvSimpsonc N1 7.0193 22277 17.5725 N2 7.8777 23541 35.8782 N3 6.2635 12296 15.2615 N4 7.9057 24890 27.9090

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

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

proteobacteria (35.23% in proportion), Bacilli (12.13% in at random, which are located in the introduction and proportion) and SK259 (15.30% in proportion) represented cultivation base of Hainan Noni Biological Engineering the types of the most abundance that were common to the Development Co., Ltd. in Sanya. four libraries. 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 DISCUSSION indicates the distribution of the endophytics in this very area. By using the 16S rDNA clone library technique, 42 Nowadays，researches on the plant seeds are getting Operational Taxonomic Units (OTUs) were identified from integrated with the microbiology science more closely. 33 genuses of the Proteobacteria, Firmicutes, Bacteroidetes According to our previous research, about 50 genus and and Actinobacteria in previous research. The dominant 100 species endophytics have been identified in the plant endophytic species in wild Noni seeds are Enterobacter sp. seeds by now, most of which are found to be Gram negative and Bacillus sp., while Pelomonas sp. and Ochrobactrum sp. bacteria; barley, rice, bean, cole, cotton, sugar beet, tomato are the dominant endophytic species in the samples of this are the plants involved. However, compared to researches on research; Pelomonas sp. and Ochrobactrum sp. are also plant roots, limited information about the endophytic found in certain amount in wild Noni seeds. Since microorganisms in the seeds is available (Cankar et al. compositions of the endophytics are basically the same, it 2005); in addition, focus has been put on traditional crops can be concluded that the genetic-related Noni seeds usually and vegetables by most of these studies, and knowledge share the same endophytic community structure. about plant seeds used in functional food remains to be The soil condition where the plant grow has a significant explored. influence on the community structure and the diversity of For the sake of scientificalness, the study material N1, N2, the endophytic microorganisms (Jefferey et al. 1999), and N3 and N4 are selected from four different planting regions the fact that most identified bacteria of this project are from Liu et al. 102

Table 2. Identities of the 10 most abundant OTUs in the bacterial communities

N1 N2 N3 N4 Abundance Taxa order (Abundance) 1 Pelomonas sp. Pelomonas sp. Pelomonas sp. Pelomonas sp. (59.68%) (45.93%) (45.99%) (54.16%) 2 Ochrobactrum intermedium Ochrobactrum intermedium Ochrobactrum intermedium Ochrobactrum intermedium (7.43%) (7.18%) (9.09%) (8.09%) 3 Kinneretia sp. Kinneretia sp. Serratia ficaria Kinneretia sp. (5.16%) (5.87%) (8.72%) (5.90%) 4 Serratia ficaria Serratia ficaria Kinneretia sp. Thiomonas cuprina (3.17%) (2.91%) (5.32%) (2.67%) 5 Paucibacter sp. Leptothrix sp. Ewingella Americana Paucibacter sp. (2.49%) (2.63%) (3.57%) (2.13%) 6 Leptothrix sp. Paucibacter sp. Paucibacter sp. Serratia ficaria (2.01%) (2.61%) (1.97%) (1.81%) 7 Inhella sp. Thiomonas cuprina Leptothrix sp. Ewingella Americana (1.90%) (2.37%) (1.80%) (1.76%) 8 Ewingella Americana Massilia sp. Inhella sp. Massilia sp. (1.75%) (2.35%) (1.52%) (1.76%) 9 Thiomonas cuprina Inhella sp. Thiomonas cuprina Inhella sp. (1.73%) (1.90%) (1.36%) (1.64%) 10 Massilia sp. Ewingella Americana Massilia sp. Leptothrix sp. (0.77%) (1.83%) (1.35%) (1.60%)

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

soil also confirms this theory. There are three reasons as to (van Overbeek and van Elsas 2008). why the soil bacteria become the dominate ones: one is that According to the result of this research, under certain the soil is the closest and the most common substrate fixed growth conditions, Pelomonas sp., Ochrobactrum sp. available during the growth process of plants, which offers a and Serratia sp. turn out to be the dominant bacteria in all good opportunity for the bacteria to enter the plant; one lies the four Noni seed samples, which indicate that all these in the fact that certain genes in the bacteria decided whether genus might exist in the growth environment. The ratio they this species can survive in the plant (or on the plant surface) account for may vary, depending on how well they adapt or not (Hardoim et al. 2008); and the grow stages of the themselves to the samples. plant also affect the community structure of the endophytics A positive influence on the plant growth is shown by some Int. J. Agric. Pol. Res. 103

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 Shared OTUs Shared reads N1 N2 N3 N4 Actinobacteria 8 57 90 36 104 Bacteroidetes 1 3 4 3 4 Firmicutes 115 1203 2082 756 1428 Proteobacteria 882 29287 20981 24585 20276 Verrucomicrobia 2 12 7 2 4 Total shared reads --- 30562 23164 25382 21816 Total reads --- 34700 27507 28189 25696 Shared reads/Total reads (%) --- 88.07 84.21 90.04 84.90

of the identified endophytics and these endophtics are found growth of plants to some extent, better bio-control during to be beneficial to the fermentation process. For example, the fermentation process or agricultural products of higher Ochrobactrum intermedium can produce nicotine-degrading quality can be obtained according to this project. enzymes, which can alleviate the ecological damages caused by the neonicotinoid insecticide (Li et al. 2007); Serratia ACKNOWLEDGMENTS marcescens can resist some of the plant pathogen by producing antibiotics or catalases (Wei et al. 1996; Li et al. This work was supported by the National Natural Science 2011); chitinase and 2-KDG can be obtained by a high Foundation of China (No. 31300008) and the Scientific and efficient fermentation of Serratia sp. PS-2 and Serratia sp. Technological Development Project of China National BK-98 (Pan et al. 2008; Zhang et al. 2011). Research Institute of Food and Fermentation Industries This is the first time that high-throughput sequencing (2012KJFZ-BS-01). We would also like to thank Demi van technology has been applied to the endophytic Wanrooij at the Wageningen University for his assistance microorganism study of the Xisha Noni seeds. Being far with English language and grammatical editing of the more than a microecology research of the plant seeds, this manuscript. 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 REFERENCES study among various gene-types or growth stages in the future. The commercial value of this project is significant as Cankar K, Kraigher H, Ravnikar M, Rupnik M (2005). well. Since endophytic microbes have influences on the Bacterial endophytes from seeds of Norway spruce (Picea Liu et al. 104

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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.