Seasonal Variation Imparts The Endophytic Bacterial Community Dynamics in Mango Plants and Its Hemiparasites

Assessment of bacterial community dynamics helps to estimate the endophytic community structure and ecological behaviour imposed by them. Such community composition is essential to understand the molecular interplay that lies between them and the host plants. The present study aims to explore the endophytic bacterial communities and their dynamics in the pre-owering and post-owering seasons in the horticulturally important Mango (Mangifera indica L.) and its hemiparasites Loranthus sp., and Macrosolen sp. through a metagenomic approach using the sequence of V3 region of 16S rRNA gene. Bacillus was found to be the most abundant genera, followed by Acinetobacter, and Corynebacterium, which belong to the phyla Firmicutes, Proteobacteria, and Actinobacteria. It has been found that during the post-owering season, twigs and leaves of mango have lower endophytic bacterial loads. Furthermore, the alpha-diversity indices of the representative genera were highest in Loranthus sp. during the post-owering seasons of mango. The ecological, taxonomic, and complex correlation studies unravelled that the hemiparasites act as the potent reservoirs of endophytic community throughout the year, and during favourable conditions, these bacterial communities disseminate to the mango plant. (a), and (d) represents ecological ecological evenness, alpha-diversities, and Bray-Curtis beta-diversities of families, respectively; whereas (e), (f), (g), and (h) represents ecological dominance, ecological evenness, alpha-diversities, and Bray-Curtis beta-diversities of genera, respectively. Here, in (c) and (g), the black bars represent Simpson indices and hollow bars represent Shannon indices. In (d) and (h) represents the diversity of endophytes in mango (cross), Loranthus (square) and Macrosolen (triangle) in post-owering season, and in mango (circle), Loranthus (rhombus) and Macrosolen (star) in pre-owering season. Here all the data were represented as considering the abundance ≥ 0.75% OTUs.


Introduction
Endophytic bacteria reside in the plant system with a wide community structure without any disease syndrome and bene ts the host system by different plant growth-promoting properties, such as the production of phytohormones (indole acetic acid (IAA), cytokines, gibberellins), HCN, enzymes (ACC deaminase), synthesis of nitrogen, nitrates or solubilizes metals (phosphorus, iron, etc.) and also protect from different phytopathogens like fungi, bacteria, nematodes, etc. ( The culture-dependent endophytic communities were assessed by conventional media-dependent methods. But in this method, only a few community structures were observed due to alteration of growth conditions and nutrient variations (Mashiane et al. 2017). Thus, the total microbial community assessment can be possible in culture-independent metagenomic analysis. However, the signi cant disadvantages of this method are there are mitochondrial, chloroplast, and ribosomal DNAs also ampli ed during the PCR ampli cation process (Lucaciu et al. 2019). To overcome this constrain, the locked nucleic acid (LNA) oligonucleotide-PCR clamping technique has been adopted to selective ampli cation of the endophytic bacterial genes (Ikenaga and Sakai 2014).
The endophytic bacterial community in plants varies according to the host plant's environmental changes and physiological changes (Ding and Melcher 2016). These community variations are also observed when a plant hosts one or more hemiparasitic plants in its system. In this system, the endophytic bacterial community may shift from one plant to another in different seasons due to its favourable environment. In the earlier study, the culture-independent bacterial community structures were observed in several medicinal, horticultural, and crop plants in seasonal and temporal variations (Ou et al. 2019).
However, the endophytic bacterial community variations in the horticultural plant mango (Mangifera indica L., family Anacardiaceae) is not reported yet. Mango is an important economic horticultural plant that originated in Sri Lanka and distributed in India, China, Thailand, Pakistan, Bangladesh, Maldives, Myanmar, and the south Asian countries. India is the highest producer of mango (40.5% of the World's total production) and earn 67 million during the 2016-17 season (https://www.arjunfoods.com/Mango). Additionally, the mango plant also hosts two hemiparasite plants such as Scurrula parasitica L. (accepted name; synonym: Loranthus parasiticus (L.) Merr.) (Family: Loranthaceae) and Macrosolen colchinchinensis (Lour.) Tiegh. (Family: Loranthaceae). The endophytic bacterial communities in these plants were also not assessed to date. In this study, the culture-independent endophytic bacterial communities of mango and its two hemiparasites were observed in the pre-owering and post-owering seasons of mango. The possible endophytic bacterial community shift between three plants was also studied in this communication.

Materials And Methods
Sampling Plant samples such as leaves, stem, and clinging stems of mango (Mangifera indica L., Family: Anacardiaceae) and its two hemiparasites Scurrula parasitica L. Metagenomic DNA preparation, 16SrDNA library generation, and Illumina MiSeq sequencing were done by AgriGenome Labs, India. The metagenomic DNA was prepared separately using the DNeasy Power Soil Kit (GIAGEN GmbH, Germany), following the manufacturer's protocol. The concentration and purity of DNA were determined by Nanodrop 2000c spectrophotometer and 1% agarose gel electrophoresis. The V3 region of 16SrRNA was ampli ed using speci c V3 forward primer 5'-CCTACGGGAGGCAGCAG-3' and reverse primer 5'-ATTACCGCGGCTGCTGG-3'. The PCR amplicons were used to construct libraries and then subjected to pair-end sequencing using an Illumina MiSeq sequencing platform.

Data analysis
The quality of the raw metagenomic sequences was then checked according to the base quality, base composition, and GC content. The adapters were trimmed accordingly, and the chimaera of the raw sequences was removed by QIIME pipeline v1.9.1 (Caporaso et al. 2010). After that, the V3 regions were ltered and identi ed from the paired-end data, and the consensus sequences were constructed from these pair-end data through FLASH or clustelO programs. The trimmed consensus sequence of the V3 region was run through QIIME v1.9.1, and these sequences were simultaneously run through the MG-RAST server at http://metagenomics.anl.gov (Meyer et al. 2008). The operational taxonomic units (OTUs) were recorded, ltered (with <5 reads), and similarity of representative sequences searched and analyzed using Uclust (similarity cutoff = 0.97) and QIIME v1.9.1 programs. The taxonomic classi cations were performed using the SILVA database ( Figure S1). The sharing percentages of OTUs of the three plants and two seasons were observed through the Venn diagram using Venny 2.1.0 (Oliveros 2015). The species richness, evenness, Shannon, and Simpson diversity indices were estimated for each of the samples, and these indices were compared between habitats and seasons using Kruskal-Wallis test. The beta diversity was estimated using the Cody index and Bray-Curtis dissimilarity using Past4.1.0. Then, non-metric multidimensional scaling (NM-MDS) was used to compare dissimilarities of the bacterial community structure between the two seasons with the Bray-Curtis similarity coe cient using Past v4.1.0. The detailed methods are described in schematic approaches ( Figure S1). All the plant samples were collected from the same mango plant in a triplicate manner. First, the unweighted pair group method with arithmetic mean (UPGMA) tree was constructed based on endophytic bacterial genera present within mango and its hemiparasites plants during two seasons of consecutive years Past v4.1.0 software. Next, the analysis of variance (ANOVA) studies was performed from the OTUs and ecological data of the two years endophytic community, keeping p≤0.05 as a signi cant level through GraphPad Prism 8.0 software. The mean values of the two seasons' data were represented as mean±SD (standard deviation).

Characterization of potential pathogens in endophytic communities
According to standard protocol, the potential of pathogenicity in the plant and humans of the identi ed genera in the endophytic community was determined through a literature survey to determine the agricultural, industrial, and economic threat in the total endophytome of the three plants (Maropola et al. 2015).

Data deposition
Raw reads of six samples were deposited in fastq format to the National Center for Biotechnology Information. Sequences were deposited under Bioproject PRJNA737054. The accession numbers for the submitted sequence of the endophytic community of pre-owering season present in mango, Loranthus, and Macrosolen samples are SAMN19460308, SAMN19460304, and SAMN19460306, respectively. In contrast, the accession numbers for the submitted sequence of the endophytic community of postowering season present in mango, Loranthus, and Macrosolen samples are SAMN19460309, SAMN19460305, and SAMN19460307, respectively. (Following links https://www.ncbi.nlm.nih.gov/bioproject/PRJNA737054).

Results
Sample description, behaviour, and collection of plant samples The endophytic bacterial populations and their dynamic nature were studied on mango and its two hemiparasitic plants, Loranthus parasiticus (L.) Merr. and Macrosolen colchinchinensis (Lour.) Tiegh (Family: Loranthaceae). The primary host plant mango (Mangifera indica L., Anacardiaceae) is the national fruit of India, Haiti, Fillipines, and the national tree of Bangladesh, a tropical tree with juicy drupe fruit. The plant has owering seasons starts from the end of the winter in the tropical region, i.e., the end of January to March. The hemiparasite plants Loranthus sp., Macrosolen sp. are commonly grown on the branches of mature-aged mango plants. In this study, the sampling was done where both the hemiparasites infested the host mango plant, and the sampling time was done during the pre-owering seasons and post-owering seasons of mango plants. The leaf and stem samples of mango and its hemiparasites were collected from a selected mango plant with hemiparasites from the mango orchard of the Malda district, West Bengal, India (24.9624 ºN, 88.1823 ºE; Figure 1) and were carried out DNA extraction and sequence analysis.

Taxonomic composition analysis
The taxonomic diversity of all the sequences of the two seasons and the three hosts were classi ed from phylum to genus according to the default QIIME v1.9.1 program. More than 340,200 reads with GC% <50% were considered in each host plant during each host and season. There were 11 different phyla, 14 classes, 17 orders, 21 families, and 22 genera ( Figure S2). There were a large percentage of OTUs were from unidenti ed and uncultured populations. The population variations of the endophytic bacterial community were evident with the seasonal variations. On the overall account of the two seasons and three hosts, Actinobacteria, Proteobacteria, Firmicutes, Cyanobacteria, and Bacteroidetes were the ve most dominant phyla carrying 99.48% of the total reads while the rest phylum is containing 0.52% reads only. Among the top ve phyla, Proteobacteria (38.04%) are the most dominant, followed by Actinobacteria (25.03%), Firmicutes (17.97%), Cyanobacteria (15.56%), and Bacteroidetes (2.88%) ( Figure S2a, b). The total unculturable communities possessed total 21 families, and among them, 60.83% belong to the major 5 families, and the other 16 families contain the rest 39.17% reads. These major 5 families were Bacillaceae (27.56%), Moraxellaceae (15.76%), Corynebacteriaceae (6.76%), Propionibacteriaceae (6.08%), Acetobacteraceae (4.65%) (Figure 2a, Figure S2c, d). Among the unculturable genera, the top 5 reads share 69.2% reads which contain Bacillus (32.46%), Acinetobacter (20.12%), Corynebacterium (6.36%), Actinomycetospora (5.99%), Methylobacterium (4.27%) (Figure 2b, Figure S2e, f). These observations showed that although many reads were left unidenti ed, diverse numbers of reads were present in each taxon from phylum to genera.

Ecological diversity of the microbial populations in hosts and seasonal variations
The ecological diversity from the represented percentages of bacterial OTUs was assessed by measuring ecological dominance, evenness, alpha, and beta diversity indices (Figure 3). The maximum dominance of families (0.319) and genus (0.4329) were showed in the Macrosolen sp. during the pre-owering season of mango and lowest in Loranthus sp. in both families (0.1528) and genus (0.1879) during the post-owering season of mango (Figure 3a, e). The ecological evenness was reciprocal to the ecological dominance because ecological dominance and evenness are always inversely proportional (Figure 3b (Figure 3h). The Bray-Curtis dissimilarities indicate a wide range of dissimilarity indices because of the restricted relative abundance of the representative taxa (both families and genera). Additionally, most taxa have positive dissimilarity indices due to their absence or minimum relative abundance in their respective hosts and seasons. The ecological dominance (df=17, p>0.05) was statistically insigni cant, whereas the ecological evenness was signi cant (df=17, p<0.05) differences in different hosts and seasons. The ANOVA studies of alpha diversity showed statistically insigni cant differences in both Simpson (df=17, p>0.05) and Shannon diversity indices (df=17, p>0.05). This result indicates that depending on variable diversity indices, the signi cance may vary. The beta diversity of both family (df=125, p>0.05) and genus (df=89, p>0.05) rank showed insigni cant variations. The non-metric multidimensional scaling (NM-MDS) of the bacterial community analysis showed that the family and genera of the two seasons were intermingled between each other and distributed almost equally throughout the axis (Figure 4a, b). This observation indicates that the bacterial community was dispersed from the entire plot to the mid axis, i.e., the bacterial community is dispersed during the different seasons of mango.

Microbial community distribution
The majority of the phylum were present in both pre-owering and post-owering seasons. The microbial community dynamics between the three host plants in the seasonal variation are a signi cant subject of inquisition. Several endophytic bacterial phylum, families, and genera were present within the plant systems that were shifted between the hosts in the different seasonal variations (Figure S3, S4; Table 1,  2; Table S1, S2). The maximum fold increase found in the case of the relative abundance of Firmicutes (>9.5 folds) followed by Actinobacteria during the pre-owering season compared to the post-owering season (Table 1). Furthermore, the abundance of endophytic bacterial phyla were maximum in Macrosolen than in mango and Loranthus ( Table 2). The genera Bacillus resides in the mango and Loranthus sp. They were multiplied and disseminated in the three hosts ( Figure S3, Table S2). In contrast, Actinomycetospora resided in the Macrosolen plant in the pre-owering season disseminated in all three hosts during the post-owering season. Another genus, Streptococcus present in the Loranthus sp. during the pre-owering season and is disseminated in Loranthus sp. and Macrosolen sp. during the post-owering season. (Table S2). Similarly, the family Bacillaceae resides in all three hosts during the post-owering season in lesser abundance, increasing several-fold during the pre-owering season. Propionibacteriaceae, another family in mango as a primary reservoir in post-owering season, were disseminated in each host during the pre-owering season. In contrast, Pseudonocardiaceae resided in the Macrosolen sp. during the pre-owering season and were disseminated in Loranthus sp. and Macrosolen sp. (Table S1). Rest families and genera were found to be the host and season-speci c instead of having any shift in abundance between seasonal or host variations ( Figure S3, S4). From these observations, it can be stated that speci c endophytic populations were shifted between the three plants on seasonal variations from the respective reservoir hosts. Macrosolen acted as a reservoir of the endophytic bacterial community and Loranthus in both the pre-and post-owering season of mango. So, most bacterial communities were shifted from hemiparasites to host mango plants during their favourite seasons. The Venn diagram also showed that the endophytic bacterial community was disseminated throughout the three hosts during the pre-owering season of mango because maximum (45.5% families or 57.1% genera) endophytic bacterial taxon was present in all the three hosts ( Figure 5). The UPGMA showed that the endophytic populations were more similar between these two hemiparasites plants than mango, and that is why mango belongs to a separate clade with 100 bootstrap values ( Figure S5). This analysis also re ects the similar postulate that the hemiparasites commonly act as a reservoir of the endophytic community, and when the environment becomes favourable, they disseminate in mango plants.

Characterization of potential pathogens in endophytic communities
The pathogenic potency to develop pathogenicity in the host plants was estimated (Table 3) from the previously published literature. It was observed that many of the endophytic genera present predominantly in the hemiparasite plants as the reservoir system ( Figure S5). However, when the favourable condition occurs, such as summer and rainy season, they (Bacillus, Corynebacterium, Staphylococcus) were disseminated to the mango plant and potentially caused diseases such as wilt and rotting of fruits, roots, etc. ( . We observed the noticeable difference and higher relative abundance of bacterial community compositions in bacterial OTUs in mango and the hemiparasite plants with seasonal variation. As far we know, this is the rst report emphasizing the endophytic bacterial community dynamics between the three interconnected plants on a seasonal basis. In this study, Proteobacteria, Actinobacteria, Firmicutes, Cyanobacteria, and Bacteriodates were found to be the most abundant phyla present in all the hosts in both seasons. Previously Akinsanya et al. (2015) reported that Proteobacteria, Firmicutes, Actinobacteria, Bacteriodates are the major endophytic bacterial phyla present in Aloe vera. The endophytic communities of peony plants also possessed Proteobacteria, Firmicutes, Bacteroidetes, Acidobacteria, and Actinobacteria as the most dominant phyla with 86% relative abundance (Yang et al. 2017), whereas, in our study, 99.48% reads possessed major ve phyla ( Figure S2a, b). Most of the previous metagenomic analysis possesses many uncultured bacterial reads similar to our studies (Hong et al. 2019;Castañeda and Barbosa 2017). At genus-level, Bacillus was found to be the most abundant (32.46%) genus in all the three plants, followed by Acinetobacter (20.12%) and Corynebacterium (6.36%) (Fig. 2c). The earlier study also showed that the phyllospheric region of tomato also possessed Bacillus as an abundant genus (Romero et al. 2014). Despite this, Acinetobacter and Corynebacterium had higher relative abundance (next to Bacillus), but it was restricted during the preowering season of mango within each host ( Fig. 2; Table S2).
Plant-associated habitats are undulating due to the involvement of many factors in the dynamic environments that affect the species compositions in the microbial communities. Shen and Fulthorpe (2015) and Ou et al. (2019) reported that the endophytic bacterial community becomes ourished during the summer and rainy seasons. Similarly, in our study, the pre-owering season of mango (summer and rainy seasons) showed the highest beta diversity and dissemination of endophytic bacterial communities between the three plants, and the alpha diversity denotes the diversity of bacteria were shown higher in the post-owering season of mango plants (late winter). These observations of the endophytic community are highly variable, and the bacteria community act as a source in the hemiparasites, and when the favourable condition comes, these though out the three plants. In the account of core genera assessment, Ding  The pathogenicity development in the mango plants may cause production loss and leads to nancial disaster. In the post-owering season, the mango plants possess a lower number of endophytes due to developing a better immune system. However, in the summer and rainy seasons, i.e., pre-owering season, a higher abundance of endophytic community was observed in the mango plants, and these endophytic populations may develop pathogenicity and leads to crop loss. In this concern, it can be suggested that removal of these two hemiparasites from the mango plants before the summer may reduce the probability of disease induction during fruit setting and ripening of fruit by the potential quiescent pathogens that reside in the hemiparasite plants. A detailed study needs to elaborate on the interactions between mango, hemiparasite plants, and endophytic bacterial communities to improve the yield and quality of the economically important mango plants.

Conclusions
The endophytic communities in the mango (Mangifera indica L.) and its two hemiparasites, i.e., Loranthus sp. and Macrosolen sp. were shifted between the plant systems with the seasonal and physiological variations. However, this study was based on the three sample sets of a single mango plant containing hemiparasitic plants in two different seasons. To better assess the microbiome shift between the three plants, it will be essential to assess the microbiome community more frequently, and this work is going on. In this study, we were aware of knowing the dominant genera present within these plants as endophytic bacteria and can conceptualize the bene cial functions they belong to. This study reveals the overall interactions of endophytic bacterial community dynamics between plant and bacteria. To the best of our knowledge, this is the rst report to study the dynamics of endophytic populations between a host, and its hemiparasites.

Declarations Con ict of interest
The authors declare that they have no con ict of interest. Figure 1 Location map of the sampling site. The '*' mark indicates the speci c sampling site.

Figure 2
Heatmap of relative abundance of families and genera present within the host plants in different seasons. Here 'a' representing the relative abundance of families, and 'b' representing the relative abundance of genera. 'Man', 'Lor', and 'Mac' indicate mango, Loranthus and Macrosolen. All the data were represented as considering relative abundance ≥0.75% OTUs.   Supplementary Files