Phylum Level Diversity of Plant Interior Bacteria in Seeds, Supernatant and Pellet Phases of Seed Suspension of Mustard Plant

Abstract

Our previous study explored the bacterial endophytic diversity in a certain quantity of mustard seeds using culture dependent method by development of new isolation strategies. No bacterial colony was initially observed in supernatant obtained after centrifugation of mustard seed suspension. This was later overcome by usage of surfactant whereas pellet part showed presence of bacterial colonies on media. In our present study, presence of bacteria was examined in supernatant and whether the diversity was similar to that of pellet and seeds by culture independent approach. In addition, growth of bacterial colonies on media is explained using GC–MS. In this study, Proteobacteria was the dominant bacterial phyla followed by Firmicutes in pellet, supernatant and seed. This indicated that bacteria were present in supernatant but may not be viable when plated on media. This is either due to antimicrobial compounds or oil-imposed difficulty during their isolation which might have hindered their survival. GC–MS study revealed the presence of compounds with antimicrobial property as suggested by previous literature. Our research conducts fundamental investigations to address the primary objective of understanding the bacterial diversity in mustard seed, pellet, and supernatant. Further research using workflows of metagenomics and metabolomics approaches can enrich our understanding ofthe diversity of microorganisms in seeds.

Graphical Abstract

Introduction

Microbes are present everywhere in air, soil and water. They can enter and colonize the plant interior and animal body. The microbes residing inside plants without causing any harm are known as endophytes. Study of endophytes and their role inside plants has recently gained interest because of their various applications in agriculture, healthcare and food industry [1]. Isolation of endophytes from their host plant has been possible with the help of two approaches i.e. culture dependent and culture independent method [2, 3]. The former involves isolating bacteria or fungi on media at laboratory and their identification using 16S rRNA sequencing. The latter involves isolation of plant DNA and performing metagenome sequencing using next- generation sequencing (NGS). India has a huge diversity of flora, and Assam is one among the many states contributing to the country’s varied flora [4]. In Assam, mustard cultivation among other crops is done on a larger scale. Rapeseed and mustard constituted 89.86% percent of total area for oilseed in 2011–2012 [5]. Assam contributed about 1.98% of total mustard and rapeseed production in India. Although several studies have reported endophytic diversity in various crops and their population, however, oilseeds are very less explored. Among the oilseeds, mustard is a very important commercial crop in India and all over the world due to its demand for oil, food and fuel. For our study, seeds of mustard were selected. Seeds have been an unusual choice for endophytic study as compared to other parts of a plant even though theyplay an essential role of forming the basis of emergence of an entire plant [6]. Microbes enter the seed from soil, colonizing its regions based on several factors such as availability of food, space and less competition from other microbes. Among these residing microbes, some may get vertically transferred to the next generation [7]. Isolation of endophytes in four varieties of mustard seed using culture dependent approaches was performed in our previous study where it was observed that in centrifugation based isolation approach, bacterial colonies did not appear on media plates containing supernatant obtained from centrifuged mustard seed suspension whereas on pellet containing media plates, bacterial colonies appeared [6]. It was assumed that there might be some compounds present in the supernatant which was inhibiting the bacterial colony growth. To overcome the research gap in our previous study, here, we conducted fundamental research using a single variety of mustard. For our study, mustard seeds of Toria variety (TS-46) obtained from agricultural land of Nagaon in Assam have been selected for studying endophytic bacteria (EB).

Toria (TS-46) var. is a newly recommended variety, grown mostly in Brahmaputra valley zones of Assam [6] and has been the preferred choice of our study as it is the most cultivated and easily available in this region. Previously, using culture based approach and Scanning electron microscopy; this variety showed presence of diverse bacteria [6] and this is the first time that this variety has been explored for study of endophytes using culture independent approach. This is a preliminary study and focuses on the questions raised while exploring diversity and population of seed endophytes in our previous study. Here, we tried to understand and elucidate the factors responsible for non-appearance of bacterial colonies on media plated with supernatant. We have performed experiments such as next generation sequencing and tried to investigate whether our supernatant contained any bacterial diversity (phylum level based bacterial distribution) as during our previous study using culture based approach, supernatant showed no bacteria. Two reasons were assumed i.e. either the interference of oil during isolation or the presence of antimicrobial compounds in supernatant. In our previous study, the former difficulty was overcome using surfactant, Tween 20 and therefore for this study we chose to investigate the latter assumption using gas chromatography-mass spectrometry (GC–MS) (Shimadzu, Japan). Here, similar isolation and study was conducted for pellet and seed as we wanted to understand if supernatant showed similarity with pellet and seed. We have conducted research that facilitates the primary objectives of the present study. Metagenomic and metabolomics study using workflows such as QIIME, KRAKEN, BRACKEN and MOTHUR were thus not performed and may be conducted in another study using different varieties of mustard seeds. The functional capabilities of our bacteria obtained from culture based approach has been explored in our another study and published [8]. Moreover, PICRUST based analysis can also be performed.

We believe there may be bacteria present in the supernatant phase of centrifuged mustard seed suspension though dead. This may be either due to internal pressure of oil hydrophobicity not allowing the bacterial exposure to water and nutrients thus preventing their growth as reported previously [6]. Or there may be presence of some compounds inside the seed with antimicrobial property which may have moved in the supernatant part while centrifugation. It is known that mustard seed is beneficial for its healing properties and is known to consist of compounds having antioxidant and antimicrobial activity. This study will help us obtain answers or at least understand if the above may be a possibility for such observation.

This is a first kind of study in oilseeds. This novel attempt to solve a basic question on one of the basic characteristic of seeds provides a more detailed understanding of oilseeds for better isolation of endophytes and exploring their diversity and functional role. The objectives of this study were: (i) To resolve the mystery of non-appearance of bacterial colonies on media plated with supernatant using GC–MS.(ii) To investigate the existence of bacteria in supernatant along with pellet and seed by culture independent approach.

Methods

Collection of Seeds, Surface Sterilization and Centrifugation

Mustard seeds were collected fromRegional Agricultural Research Station (RARS), Shillongani of Assam Agricultural University and stored in air tight bags at 4ºC. Seeds were washed three times with sterile distilled water and sterilized using the technique as described by Sinha and Talukdar, 2022 [6].

The surface sterilized seeds were used further for DNA extraction. Another batch of seeds was surface sterilized and crushed using sterile distilled water. The suspension was centrifuged and the supernatant and pellet was separated for DNA extraction procedure [6]. The culture dependent approach used for isolation of EB from mustard seeds has been described in our previous study [6].

DNA Extraction from Supernatant

The supernatant is a cocktail of water and oil therefore the procedure for extraction of DNA is different than that of pellet and seeds. The protocol was followed as described by Consolandi et al. [10]. The supernatant was vacuum dried and used for further study. 0.017 g of the lyophilized tissue was taken in 2 ml eppendorf tube and 500 µl of hexane was added to it. The tube was vortexed for 10 min and centrifuged at 4 °C for 15 min at 12000 rpm. The hexane containing supernatant is separated from the pellet. Add 400 µl lysis buffer to pellet tube (Lysis buffer- TE buffer, 0.5% v/v Tween 20, 2 µl 100 mg/ml Proteinase K). Incubate the tube at 48 °C for 2 h. Centrifuge for 30 min at 4 °C, 12000 rpm. Two layers are formed. An oil layer on top and the suspension below is separated into 2 tubes very carefully without touching the interface. Heat both the tubes at 95 °C for 5 min to inactivate Proteinase K. 500 µl of cold isopropanol was added and gently mixed. Keep overnight at − 20 °C. Centrifuge for 30 min at 4 °C, 14000 rpm. DNA pellets are obtained. The DNA pellet was washed with 200 µl 70% v/v cold ethanol. Centrifuge for 30 min at 4 °C, 14000 rpm. Dry the tubes till no ethanol smell or drops are present. The pellets were dissolved in 50 µl TE buffer.

DNA Extraction from Pellet

Mustard seeds were surface sterilized, crushed and centrifuged. The supernatant and pellet were separated and vacuum dried. Lyophilized pellet was taken in 2 ml Eppendorf tube. DNA was extracted using QiagenDNeasy Plant Mini Kit (Cat. Nos.69104). DNA quality was checked by running at 0.8% Agarose gel electrophoresis. The final DNA concentration and purification were determined by NanoDrop 2000 UV–vis spectrophotometer (Thermo Scientific, Wilmington, USA) as performed by Garcia Alegria et al. [9]. The samples were sent for next generation sequencing (NGS) to Macrogen Inc. (Seoul, Korea). 4 µl DNA and 1 µl bromophenol dye was mixed and loaded into 30 ml of 0.8% Agarose gel. The DNA bands were observed.

DNA Extraction from Seeds

Mustard seeds (1gm) in triplicates were surface sterilized and crushed. The suspension was used for DNA extraction using QiagenDneasy Plant Mini Kit (Cat. Nos.69104). DNA quality was checked by running at 0.8% Agarose gel electrophoresis. The final DNA concentration and purification were determined by NanoDrop 2000 UV–vis spectrophotometer (Thermo Scientific, Wilmington, USA) [9].

Identification of Bacterial Species by Next Generation Sequencing (NGS) and Raw Data Analysis

Metagenomic DNA of 12 seed samples among which 4 replicates were taken each from supernatant, pellet and seeds were sent to Macrogen Inc. (Seoul, Korea) for next generation sequencing (NGS). Prior to that, DNA was quantified. Amplicon-based metagenome sequencing of 16 s rRNA V3-V4 region was performed to analyze the diversity of endophytic bacteria. The raw data sequences were obtained in FASTQ format. These sequences in FASTQ file was uploaded to MG RAST (Metagenomic RAST). The first step performed was Quality Control wherein 3 steps; preprocessing, dereplication and screening were performed. The primers used during sequencing and barcode sequences were trimmed from the sequences. The low quality regions in FASTQ were removed. Other sequences having ambiguous bases, shorter length sequences (less than 100 bp), primer mismatch and an average Phred score of 25 and lower were removed. In the second step of data reduction, gene prediction and clustering were performed where consensus sequences were created. The last step was data analysis step where the sequences obtained were analyzed using SILVA and Greengenes database. Taxonomy was assigned with 97% homology match. Abundance of bacteria was obtained at phylum level from the server and pieplots and graphs were plotted using Excel Sheet.

GC–MS Analysis of Chemical Compounds in Methanolic Extract of Supernatant and Sediment of Mustard-Rapeseed

Chemical composition of supernatant and sediment separated during centrifugation of crushed mustard seed was studied using GC-MS (Shimadzu, Japan). 20 gm of dried seeds of mustard seeds (MS) were extracted by maceration in methanol. Extraction was repeated and the extract was concentrated under rotary vacuum evaporator (RVE) (Buchi Rota vapor). Another 20 gm of MS were crushed using water and centrifuged. Supernatant (85ml) was separated and extracted using ethyl acetate (EtOAc) (100ml) in separating funnel. The solvent fraction was again extracted using methanol and concentrated. Sediment from centrifuged seeds was also extracted using methanol (MeOH) at room temperature. The extracts were concentrated in RVE. The GC program was optimized for the detection of mustard chemical components. Helium was used as the carrier gas with a flow rate of 1.0 ml min^-1. The injection temperature was set at 300°C. The column oven temperature was set at 60°C with a hold time of 10 min and was subsequently increased to 300° C with a ramp of 5°C min^-1. The final hold was for 10 min. The ion source temperature was set at 280°C for MS using an interface temperature of 300°C. The mass range was selected as (m/z) 50-800 for the entire analysis. The chromatograms were analyzed with GC-MS solution software (version 4). For compound identification NIST 11 library database was used.

Results

Endophytic Bacterial Population and Diversity in Seed Suspension, Pellet and Supernatant

The data was obtained under order No. HN00104633 using Illumina Platform from M/s Macrogen Inc. (Seoul, Korea).The reads had significant homology with predicted rRNA sequences in the Ribosomal Database Project (RDP) resource.Based on Illumina based study, four bacterial phyla were most abundant in surface sterilized mustard seeds. Similarly, five bacterial phyla were found most abundant in pellet and supernatant forms of surface sterilized and centrifuged mustard seed suspension. Phylum level based distribution of bacteria in mustard seeds have been represented in Figs. 1 and 2. Pellet and supernatant showed the presence of Proteobacteria, Firmicutes, Cyanobacteria, Bacteriodetes and Actinobacteria.Likewise, only four phyla werefound most abundant in surface sterilized mustard seed suspension. They wereFirmicutes, Proteobacteria, Actinobacteria and Cyanobacteria (Fig. 1).

Fig. 1.

Relative abundance of endophytic bacteria at phylum level among the different forms of surface sterilized mustard seeds

Fig. 2.

Phylum level based diversity analysis pieplots in seeds, pellet and supernatant of mustard

In pellet,Proteobacteria(38%) was the most abundant phylum. Firmicutes(24%) was the second most abundantphylum followed by Cyanobacteria, Bacteriodetes and Actinobacteria(Fig. 2).Similarly, Proteobacteriawas the most abundant representative phylumin both supernatant (57%) and seeds (76%) followed by Firmicutes(16%) in supernatant and Cyanobacteria (17%) in seeds. In comparison to supernatant and pellet, Bacteriodeteswere found negligible in seeds even though they constitute 9%in supernatantand 10% in pellet.This data showed that bacterial diversity existed in all forms of mustard seed: seed, supernatant and pellet.

GC–MS Analysis of Different Sources of Mustard Seeds

The GC–MS(Shimadzu, Japan) spectrum confirms the presence of different components with different retention times. The mass spectrometer analyzes the compounds eluted at different times to identify the nature and structure of the compounds. The large compound fragments into small compounds giving rise to appearance of peaks at different m/z ratios. These mass spectra are fingerprint of that compound which can be identified from the data library and available literature. There was a difference observed in the chemical composition of entire mustard seeds, supernatant and sediment (Fig. 3) which indicates their role in influencing the bacterial function on nutrient media. On the basis of similar retention time, a peak of compound ‘a’ was common in both seeds (ES) and supernatant (SUP) (Fig. 3A and B). Peaks of compounds ‘b’ and ‘c’ were observed in mustard seed (ES) and supernatant of mustard seed (SUP) respectively (Fig. 3A and B). But peak of compound ‘d’ was observed only in pellet (CRUS) (Fig. 3C).The compound ‘c’ may be present in ES in very less amount but its concentration in supernatant might have increased due to centrifugation and separation of supernatant from sediment of surface sterilized mustard seed (SSMS). Data showed the presence of non-polar components such as aromatic phenolic compounds, alcohols and alkenes. Major compounds identified were 2,2,5-Endo,6-exo,8,9,9,10 octachlorobornane, n-Hexadecanoic acid, 1,E-1,Z-13-Octadecatriene, 9-Tetradecenal,(Z)-, (Z)-4-Decen-1-ol,trifluoroacetate, Undecanoic acid,10-bromo, 10-undecyn-1-ol, Ethofumesate (Table 1). However the compound present in highest amount is 1, E-1,Z-13-Octadecatriene with 44.55% area in seeds and 3’, 5’-Dimethoxyacetophenone with 50.03% area in supernatant of mustard seeds(SUMS). 13-Tetradece-11-yn-1-ol was the only compound identified in pelletor sedimentof mustard seeds (SDMS) with 100% area. The compounds 9-Tetradecenal, (Z)-, Pentanoic acid, Cyclobutyl ester and 2-tert-Butyl-4, 6-bis(3,5-di-tert- butyl- 4-hudroxybenzyl phenol were also present in good amount. Undecanoic acid is common in both mustard seeds and SUMS but absent in SDMS. The compound 9-Tetradecenal is present in both mustard seeds and SDMS but not in SUMS. Table 1 also mentions the biological activities of the phytochemical compounds identified.

Fig. 3.

Chromatogram obtained from GC–MS analysis of surface sterilized mustard seed and its phases separated by centrifugation

Table 1.

List of phytochemical compounds obtained from seeds and their biological activity

Part of seed	List of phytochemical compounds	Mass/charge ratio	Biological activity
Pellet (PEL)	13-Tetradece-11-yn-1-ol (d)	55.05	Antibacterial, antifungal and antioxidant, antidiabetic, anti-inflammatory [17, 21–23]
Supernatant (SUP)	3’,5’-Dimethoxyacetophenone (c)	180.20	Antioxidant, antimicrobial [18]
	Undecanoic acid (a)	60.05	Antifungal [25]
	Pentanoic acid, Cyclobutyl ester	85.10	Antibacterial, antimicrobial [24]
	2-tert-Butyl-4,6-bis(3,5-di-tert- butyl- 4-hudroxybenzyl phenol	57.15	Compounds having di-tert butyl phenol display antioxidant activity along with other biological activity [26, 31]
Seeds (ES)	2,2,5-Endo,6-exo,8,9,9,10 octachlorobornane	460	Toxic chemical and organic pollutant, pesticide and insecticide [27]
	n-Hexadecanoic acid	73.10	Antioxidant, Antidiabetic[22, 23]
	1,E-1,Z-13-Octadecatriene (b)	67.10	Antimicrobial (when exposed to O2) [28]
	9-Tetradecenal,(Z)-	55.10	Sex pheromone [29, 31]
	(Z)-4-Decen-1-ol,trifluoroacetate	55.10	Food additive, Pharmaceuticals, pesticides with low to moderate toxicity [32]
	Undecanoic acid,10-bromo (a)	60.05	Anti fungal. Anti-biofilm activity [25]
	10-undecyn-1-ol	67.05	Antimicrobial[30]
	Ethofumesate	207.20	Herbicide [33]

Discussions

Mustard seed is primarily used for deriving edible oil for human consumption globally.Adding to that, the entire plant can be utilized as food, fuel and substrate. In our previous study, direct isolation of the endophytes using serial dilution technique was unsuccessful. Therefore different methods based on literature were followed but the results were found to be unreliable. This led to development of two novel culture dependent approaches for isolation of EB from mustard seeds [6]. One of them was the centrifugation method where pellet and supernatant were spread separately on Nutrient media. Pellet reported bacterial colonies but supernatant plates were devoid of any bacterial colony growth. Based on the existing literature about internal components of mustard seed and mustard bomb [10], it was assumed that some chemical componentshaving antioxidant, antimicrobial propertymay have caused hindrance in the bacterial isolation from supernatant. In this study, DNA was extracted from seeds and its forms; pellet and supernatant obtained after centrifugation of seed suspension. GC–MS (Shimadzu, Japan) was performed with seeds, pellet and supernatant for determining the presence of chemical compounds of biological activity. NGS was performed and diversity of endophytic bacteria was obtained at phylum level. The NGS result in this study confirmed the presence of endophytic bacteria in supernatant as well as in pellet and seeds of mustard. This is a first kind of study where mustard seeds were explored for bacterial endophytic diversity using culture independent approach. Different forms of mustard seed i.e. seed suspension, pellet and supernatant were obtained after crushing and centrifugation and further used for our study. The method used for DNA extraction from mustard supernatant was similar to the method of DNA extraction from olive oil [11]. DNA of good quality was extracted. Clear bands were observed on Agarose gel. Based on our present objective, only phylum based analysis were performed. Deep analysis of microbial diversity and study of their role in metabolomics have not been performed. In-depth analysis may be considered for future investigations using more varieties of mustard seeds. This can be supported by the fact that phylum based composition of seed endophytic bacteria is an important indication of overall plant endophytic phyla [34]. NGS analysis showed the presence of bacterial phyla; Proteobacteria, Firmicutes, Cyanobacteria, Actinobacteria and Bacteriodetes. Proteobacteria was the most dominant bacterial phylum observed in seeds, supernatant and pellet followed by Cyanobacteria and Firmicutes. Bacteriodetes were present in supernatant and pellet but negligible in seeds. Generally, Bacteriodetes are rarely characterized as seed endophytes where as Proteobacteria followed by Firmicutes and Actinobacteria are most predominantly found in seeds [35].Using culture dependent approach, Firmicutes were found to be the most abundant bacterial phylum followed by Proteobacteria and Actinomycetes in mustard seeds [6]. In another work using culturable methods, Choi et al. [36] have reported Actinobacteria, Firmicutes, Alpha and Gammaproteobacteria as major bacterial phyla in mustard seeds. Bacteriodetes suppresses pathogens and enable the plants to respond to biotic and abiotic stress [12].Their presence in mustard seed is confirmed as their abundance was observed in supernatant and pellet. It may be assumed that centrifugation of seed suspension led to their exposure and revealed their presence in both the separated phases of centrifuged seed. NGS study revealed bacterial diversity in supernatant. But in media plates, no bacterial colonies appeared. It is known that dead bacteria cannot grow in media but both live and dead bacteria can be detected by NGS as this method cannot distinguish between the live and dead.

The bacterial isolates identified in the seeds [6] were screened for their antimicrobial property in another study [8]. Results showed that some of the bacteria present in mustard seeds had antimicrobial property.Oilseed endophytic study is very scanty and to the best of our knowledge, only cultivable data of EB in oilseed is available in the literature. In a culture independent study by Rathore et al. [13] in root and shoot of oilseed rape, the bacterial phyla Proteobacteria, Actinobacteria and Bacteroideteswere thedominated ones among the other members of bacteria. Other crops such as rice showed Proteobacteria as the most abundant one followed by Actinobacteria, Bacteriodetes and Firmicutes [14]. Tween 20 surfactant was considered for bacterial DNA isolation from supernatant because of its emulsifying properties and its non-harmful effect on microbial growth at all concentration [15]. The use of Tween 80 is not considered as this surfactant has shown to affect bacteria and microbial growth [16]. Tween 20 separated the oil and water from the suspension and helped bacterial DNA isolation [11].

To understand the events, in this study, GC–MS(Shimadzu, Japan) was performed to find whether the supernatant had any antimicrobial component which may have caused the death of microbes in the supernatant or in the seed suspension from which supernatant was separated. GC–MS analysis revealed the presence of antimicrobial and antioxidant compounds in supernatant, pellet and seeds. Undecanoic acidwas found in both supernatant and seeds. This compound showed antifungal property and is used to treat skin infections such as dermititis and Athlete’s foot disease [17]. In pellet, 13-Tetradece-11-yn-1-olwas present having antibacterial, antifungal, antioxidant, antidiabetic, anti-inflammatory property [18]. 3’,5’-Dimethoxyacetophenonewas clearly visible in high quantity in supernatant and is antimicrobial and antioxidant in nature [19]. The presence of such phytoconstituents with antimicrobial property may be the reason for the bacteria to not grow and develop colonies in media. If any bacteria was alive, due to mixture of oil with bacteria, the latter’s growth may have been inhibited as their exposure with the media is restricted due to hydrophobicity of oil.In a study by Khan et al. [20], components such as thiocyanate glycosides, Sinalbin A and Sinalbin B were revealed in mustard seeds by GC–MS. Another study byLietzow [21], states that glucosinolates concentration is highest in seeds and their breakdown products such asisothiocyanates are volatile, water soluble, reactive and toxic for microbes. The products may obstruct the microbial growth on media by interrupting with the bacterial mechanisms required for their growth thus killing them.

Conclusions

The study has some limitations such as during the metagenomic study in-depth analysis of the seed lots was not performed.The identified compounds that were suggested as antimicrobial by literature reports were not tested during our experiments though we did perform a minor experiment. We combined the supernatant with some bacterial colonies and it was observed that no bacterial growth occurred on media which led us to further proceed with our research. Therefore screening of phytochemical compounds for antimicrobial activity can be further considered for another study. This was because our primary objective was to only understand if there was any bacterial diversity present in the supernatant as compared to pellet and seed. This phylum-based diversity study was performed in continuation with our previous study where no bacterial colony appeared on media plates with supernatant but plates containing pellet and seed suspension showed appearance of bacterial colonies. Besides, unavailability of information on mustard seed endophytes and difficulty in their isolation process encouraged us to pursue our research using mustard seeds. To the best of our knowledge, this is a first phylum-based diversity study of mustard seed bacterial endophytes using culture independent approach. Additionally, for the first time, the reason for the difficulty in endophytic isolation process in mustard seeds has been studied. Though culture dependent study reported Firmicutes as most abundant phylum, culture independent approach revealed Proteobacteria as the most abundant bacterial phylum in mustard seeds. Study of genes, metabolites and metabolic pathways of mustard seed endophytes and their role remains to be explored.

Author Contributions

TS: Writing–Conceptualization, Methodology, Data curation, Software, Data Analysis, Visualization, Validation and Writing original draft and preparation. NCT: Conceptualization, Validation, Writing–Review & Editing and Supervision.

Funding

This research was financially supported by The Council of Scientific and Industrial Research (CSIR) File No: 09/835(0018)/2017-EMR-1.

Declarations

Conflict of interest

The authors declare that they have no relevant financial or non-financial interests to disclose.

Ethical Approval

The experiments performed did not involve any animal or human participation.