Seasonal Variation Imparts The Endophytic Bacterial Community Dynamics in Mango Plants and Its Hemiparasites Rajsekhar Adhikary University of Gour Banga Sukhendu Mandal University of Calcutta Faculty Council for Post-Graduate Studies in Science Vivekananda Mandal ( [email protected] ) University of Gour Banga https://orcid.org/0000-0001-6523-8069 Research Article Keywords: Bacterial ecology, Endophytic bacteria, Mango host, Microbial community dynamics, Seasonal variation Posted Date: July 26th, 2021 DOI: https://doi.org/10.21203/rs.3.rs-641910/v1 License: This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Page 1/18 Abstract 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. Introduction Endophytic bacteria reside in the plant system with a wide community structure without any disease syndrome and benets 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. (Choudhary and Johri 2009; Radhakrishnan et al. 2017; Sha et al. 2017). Moreover, apart from plant growth promotion or plant protection, bacterial endophytes produce many secondary metabolites that can be used as a source of bioremediation, waste management, pharmaceuticals and so on (Gouda et al. 2016; Sahoo et al. 2017). 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 signicant disadvantages of this method are there are mitochondrial, chloroplast, and ribosomal DNAs also amplied during the PCR amplication process (Lucaciu et al. 2019). To overcome this constrain, the locked nucleic acid (LNA) oligonucleotide-PCR clamping technique has been adopted to selective amplication 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). Page 2/18 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 earn67 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. (accepted name; synonym: Loranthus parasiticus (L.) Merr.) (Family: Loranthaceae) and Macrosolen colchinchinensis (Lour.) Tiegh. (Family: Loranthaceae) were collected from a selected mango plant with hemiparasites from the Malda district's mango orchard, West Bengal, India (24.9624 ºN, 88.1823 ºE) (Figure 1). Samples were collected during pre-owering (October 2018) and post-owering (April 2017) seasons of mango plants of two respective years (2017-2018). The samples were surface sterilized by 4% sodium hypochlorite solution and 80% ethanol solution (v/v, Adhikary et al. 2020) and stored in a sterile falcon tube at 4°C. DNA isolation and 16SrDNA amplicon-based Illumina library preparation 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 amplied using specic 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 Page 3/18 ltered and identied 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 classications 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 coecient 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 signicant 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 identied 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