Volatile metabolites of endophytic Klebsiella aerogenes from Zingiber zerumbet rhizome and its antagonistic effect on soft rot causative Pythium myriotylum

Rhizomes of Zingiber zerumbet collected from their natural habitat and reported earlier to have high zerumbone content were selected for isolation of endophytes. Biochemical and molecular characterization using 16S rRNA sequencing of the endophytes identified the isolates as belonging to genus Klebsiella, Pantoea and Enterobacter. Isolate designated ZzKSD8 identified as K. aerogenes yielded maximal antagonistic activities against P. myriotylum determined as 83.5% ± 0.77. Volatile metabolites produced by ZzKSD8 caused absolute impairment of P. myriotylum hyphal growth compared to control (23.9 ± 0.37 cm). Volatile metabolites were extracted from 48 hour grown stationary phase ZzKSD8 cultures using absolute methanol, ethyl acetate and ethanol (60% v/v). GC-MS metabolite profiling detected alkanes and fatty acid methyl esters as the predominant constituents in the solvent extracts. Major constituents included methyl palmitate (31.37%), methyl stearate (18.57%) and cyclopropaneoctanoic acid, 2-hexyl-, methyl ester (17.05%) in methanol extract; alkanes like tetratetracontane (13.18%) and 2-methyloctacosane (12.10%) in ethyl acetate extracts and 4,22-sigmastadiene-3-one as major metabolite (22.51%) and stigmast-5-en-3-ol, (3.beta) (17.40%) in ethanol extract. Identified metabolites reported to modulate defense strategies in plants against phytopathogens makes ZzKSD8 a potential candidate for development of biological alternatives to control soil-borne soft-rot disease.


Introduction
Compared to foliar pathogens for which resistance is often encoded in plant genome, finding resistance factors against soil-borne pathogens is challenging. Roots and rhizomes with their nutrient reserves tend to attract microbes to rhizosphere (Shubin et al. 2014;Santoyo et al. 2016). As a consequence there exists increased competition and dynamics in the soil habitat (Raaijmakers et al. 2009) making it difficult to manage soil-borne pathogens. Rhizosphere dynamics is regulated by the metabolites produced from below-ground tissues like roots and rhizomes that create specific niches and thereby foster beneficial microbial associations (Shubin et al. 2014;Jia et al. 2016). Some of these microbes form mutualistic associations as endophytes benefiting growth and defense of host plants (Mendes et al. 2013;Adedeji & Babalola 2020). Host genotype is known to significantly influence endophytic colonization (Overbeek & Elsas 2008;Shubin et al. 2014) as in wheat cultivars wherein differences in microbial composition were observed based on varying resistance levels (El Arab et al. 2001). Thus endophytic microbes isolated from resistant taxon root/ rhizome with allelopathic potential can serve as candidates for development of biocontrol agents for plant protection.
Endemic plants with ethnomedicinal properties harbors diverse endophytic microbes making them ideal source for prospecting candidate endophytes with biocontrol potential (Strobel & Daisy 2003). Tropical medicinal plant, Zingiber zerumbet is thus ideal for prospecting endophytes due to its wide medicinal properties (Chien et al. 2008) and robust resistance to soft-rot causative Pythium species (Nair & Thomas 2013), a necrotrophic pathogen causing extensive economic loss due to its wide host range. Preliminary study on prospecting Z. zerumbet endophytes led to the isolation of fungal isolates of Fusarium genus with antagonistic activity to Pythium spp. (Keerthi et al. 2016). Compared to fungal endophytes, bacterial endophytes with plant growth promoting (PGP) traits (Gaiero et al. 2013) have been identified to hold greater potential for development of biocontrol agents and as soil amendments (Ryan et al. 2007;Chaparro et al. 2012). This is owing to higher efficacy and survival of bacterial endophytes with PGP traits in the competitive soil environment due to production of allelochemicals with antagonistic activity (Chaparro et al. 2012).
The principal secondary metabolite in Z. zerumbet namely zerumbone which contributes to antagonistic activity towards Pythium species (Keerthi et al. 2014) has been reported to vary based on geographical location with highest zerumbone content observed in accessions from Kasaragod District of Kerala (Baby et al. 2009). Such habitat induced variations in metabolite content can therefore influence the rhizosphere microbial composition (Aleklett et al. 2015). Against this backdrop, present study was undertaken to (i) isolate and characterize antagonistic bacterial isolates from the rhizome of Z. zerumbet accessions collected from Kasaragod and (ii) characterize the metabolites produced by selected isolate(s).

Sampling and microbial isolation
Rhizomes of healthy Z. zerumbet plants were collected from Kasaragod District, Kerala (Latitude: 11.7381° N, Longitude: 76.0740° E). Collected rhizomes were immediately sealed in plastic bags and transported under ice to laboratory. Soil and debris sticking to rhizomes were removed by washing under running tap water for 30 minutes. Washed rhizomes were successively treated with ethanol (70% v/v) for 1 min; rinsed with sterile water; treated with sterilization solution (NaOCl containing two drops of Tween 20; 20% v/v) for 5 min; rinsed with sterile water; treated with mercuric chloride (0.1% w/v) for 8 min followed by three washes with sterile water. Aliquot of last wash water (1 ml) was plated on LB medium to evaluate the effectiveness of surface sterilization procedure. Treated rhizome was cut into pieces and ground using sterile mortar and pestle (Zhang et al. 2018) and suspended in Luria Bertani (LB) broth. The obtained rhizome homogenate was plated (20 µl) on freshly prepared LB agar and incubated at 25°C for 24-72 hour. Endophytic isolates obtained were characterized and stored as glycerol stocks at -80°C.

Morphological and biochemical characterization of rhizome endophytes
For cultural and morphological characterization, isolates were grown overnight in LB plates and LB broth at 25 ± 3 o C. Cultures were observed to determine colony surface, margin and opacity. Further biochemical characterization like gram staining, motility, endospore staining, catalase activity, methyl red test, gelatin hydrolysis and citrate utilization ability of all the bacterial isolates were determined for taxonomic identification.

Molecular and phylogenetic characterization
Genomic DNA was extracted from overnight grown cultures following CTAB protocol (William et al. 2012). PCR amplification was carried out using 16rDNA universal primers, 16SF (5'-AGAGTTTGATCCTGGCTCAG-3') and 16SRev (5'-GGTTACCTTGTTACGACTT-3'). PCR reaction mixture (20 µl) contained 20 ng DNA, 10 pmol of each primer, 10 mM each dNTP, 10X PCR reaction buffer with 1.5 mM MgCl2 and 0.6U Taq DNA polymerase. Thermo-cycling was carried out in S1000 Thermal cycler (Bio Rad, USA) and consisted of initial denaturation step at 94°C for 3 min followed by 35 cycles of 94°C for 1 min, 60°C for 1 min, 72°C for 1 min and a final extension at 72°C for 5 min. PCR products were electrophoretically examined in 1.2% agarose gel, excised from the gel and purified using Wizard SV gel and PCR Clean-up System (Promega, WI, USA) following manufacturer's instruction prior to sequencing. Sequences were subjected to homology searches using BLASTN algorithm (http://www.ncbi.nlm. nih.gov/) in NCBI database and aligned using CLUSTAL W (Thompson et al. 1994) with homologous sequences retrieved from NCBI database. Phylogenetic relationship of endophytic isolates was inferred from the maximum likelihood (ML) (Felsenstein 1981) tree generated using MEGA 5 software (Kumar et al., 2018). Bootstrap analysis was carried out with 1000 datasets to check the robustness of the constructed phylogenetic tree.

Dual culture assay of endophytic isolates
Dual culture assay was conducted to evaluate the antagonistic effect of endophytic bacterial isolates against soft-rot causative Pythium myriotylum strain (RGCBN14), which was obtained from Rajiv Gandhi Centre for Biotechnology (RGCB), Trivandrum, Kerala. The isolates were streaked on one side of the PDA plate (pH-6.5) and the Pythium hyphal disc (5 mm) was placed at opposite side. The plates were incubated at 25 ± 3°C for 7 days and antagonistic activity determined as percentage of inhibition (PoI) was determined as: PoI = [(R1-R2)/R2] x 100 (Keerthi et al. 2016).

Evaluation of VOCs produced by KSD8 isolate against P. myriotylum
Effect of VOC produced by ZzKSD8 was determined by double plate technique. For the same, bacterial lawn was prepared on LB agar medium at 25 ± 3°C. The lid of the petri-plate was replaced with lid of another plate that was inoculated with P. myriotylum (RGCBN14) hyphal disc (5 mm) on PDA medium. The plates were sealed with parafilm to prevent leak of bacterial volatile compounds and incubated at 28 ± 3°C in dark for five days. The diameter of P. myriotylum hyphal growth was measured after the incubation period and PoI was calculated. Assays were performed three times with three replicates.

Metabolite profile of KSD8 isolate by GC-MS
Metabolite(s) produced by endophytic bacterial isolate, ZzKSD8 that exhibited significant antagonistic activity was evaluated. For the same, bacterial growth curve was determined for a period of 2 hours to 8 days. Metabolites were subsequently extracted using three solvents namely, absolute methanol, ethyl acetate and ethanol (60% v/v) from the stationary phase cultures. For solvent extraction, cultures were centrifuged at 10,000 rpm for 5 minutes to sediment the bacterial cells. Pelleted cells were suspended in pure solvent (1 ml/ 5 ml culture volume), shaken overnight and the suspension was sonicated the next day. The homogenate was centrifuged at 10,000 rpm for 15 min at 10°C and the supernatant was filtered (0.22 µm filter) and subjected to GC-MS analysis.
GC-MS analysis was done using high resolution Agilent GC 7890A (injector temperature 250°C) coupled to Agilent 5975C mass detector in the split mode of 50:1. Separation were carried out in a DB 5 MS column with dimensions 30 m × 0.25 mm × 0.25 µm. Carrier gas used was helium with a flow rate of 1.0 mL min -1 . Initial temperature of the oven was 40°C with an increase of 5°C min -1 to 280°C. Peaks obtained were identified by matching the mass spectra in Wiley and National Institute of Standards and Technology (NIST) Mass Spectral Library.

Results and discussion
Endophytic bacterial isolates from Z. zerumbet rhizome Z. zerumbet rhizomes were collected from their natural habitat in Kasaragod, reported earlier to have the highest zerumbone content (Baby et al. 2009). Our earlier endophyte isolation procedure from Z. zerumbet had used sterilized rhizome slices (Keerthi et al. 2016) and thus differs present procedure wherein ground rhizome was used as it ensures sampling from the internal plant tissue. Morphologically distinct bacterial colonies were selected and characterized for color, shape, elevation, texture and margins. The morphological features of the ten isolates obtained were tabulated (Table 1). Biochemical characterization of isolates revealed all to be gram negative except isolate ZzKSD1 which was also a spore producer; rod-shaped; motile except three isolates namely, ZzKSD2, ZzKSD5 and ZzKSD8 and all isolates were also catalase positive. Isolates also yielded positive results for methyl red test and fermentation of sugars, glucose, fructose and sucrose. Isolate ZzKSD8 was positive for indole test and citrate utilization. Gelatin liquefaction was positive only for ZzsKSD9 while all isolates were negative for sulphide test (Fig 1). PCR amplification using 16S rRNA yielded amplicons of ~1.4 kb which were eluted, sequenced and subjected to homology searches using BLASTn algorithm at NCBI (Table 1). Following multiple sequence alignment using CLUSTALX, phylogenetic analysis was carried out and the ML tree clustered the sequences with homologs which was supported by high bootstrap values (Fig. 2) (Martinez et al. 2003;Mendes et al. 2013) like maize wherein they have been shown to enhance plant growth and were distinct from clinical isolates lacking virulence factors (Dong et al. 2003). Furthermore, diazotrophic Klebsiella spp. are of agricultural interest and constitute excellent model organisms for study of nitrogen fixation (Hoover 2000). Epiphytic and endophytic Pantoea spp. and Enterobacter spp. exhibit plant growth promoting traits with excellent biocontrol potential (Mishra et al. 2011;Macedo-Raygoza et al. 2019). Bacillus spp. are ubiquitous endophytes in diverse plants and have been reported as potential candidate BCAs (Nigris et al. 2018).

Identification of isolates with antagonistic potential
Amongst the 10 Z. zerumbet endophytic isolates evaluated by dual culture or confrontation assays for antagonistic activities against P. myriotylum, ZzKSD8 yielded highest percentage of inhibition (83.5 ± 0.77) (Fig. 3) and thereby constitute potential candidate for soft-rot disease control. With Pythium species gaining entry through rhizomes, the endophyte characterized from rhizome constitute ideal candidates for developing BCAs to prevent soil-borne softrot disease. Root associated endophytes have been reported to mediate antagonistic activities against phytopathogens in various plants by de novo synthesis of metabolites that interfere with pathogenicity factors (Jia et al. 2016). These associations have been investigated in various plants such as maize, wheat, oat, barley, peas, canola, soy, potatoes, tomatoes, lentils and cucumber (Ryan et al. 2007). With ZzKSD8 showing homology to K. aerogenes, identification of volatile metabolites will have implications in both phytopathology and human health.

Volatile secondary metabolites of ZzKSD8 isolate
Volatile secondary metabolites constitute a major class of organic compounds produced by soil bacteria besides the soluble metabolites (Tyc et al. 2017). Biocontrol activity of many biocontrol endophytes has been attributed to production of volatile metabolites. Present experiments following double plate technique revealed that the volatile metabolites produced by ZzKSD8 caused impairment in P. myriotylum hyphal growth compared to control (39.12± 0.37 mm) experiments yielding 100% PoI (Fig. 4).
Double plate experiments thus reveal absolute antagonistic activity towards P. myriotylum ( Fig.  4 inset A) by ZzKSD8 (Fig. 4 inset B). Secondary metabolites are known to accumulate during stationary growth phase when there is nutrient depletion (Ruiz et al. 2010). Growth curve analysis of ZzKSD8 isolate was carried out to determine the time stationary phase is attained. Experiments revealed the stationary phase of ZzKSD8 isolate at 48 hour and subsequently volatile metabolite extraction was carried out from 48 hour old cultures using absolute methanol, ethyl acetate and ethanol (60% v/v) for GC-MS analysis. Predominant metabolites identified in the solvent extracts [cut-off Area (%) ≥ 10] were fatty acid methyl esters followed by sterols and alkanes. Fatty acid methyl esters like methyl palmitate (31.37%), methyl stearate (18.57%) and cyclopropaneoctanoic acid, 2-hexyl-, methyl ester (17.05%) were detected in the methanol extract; sterols like 4,22-sigmastadiene-3-one (22.51%) and stigmast-5-en-3-ol, (3.beta) (17.40%) were detected in ethanol extract and alkanes like tetratetracontane (13.18%) and 2-methyloctacosane (12.10%) were the major constituents in ethyl acetate extracts (Table 2). Fatty acid methyl esters like methyl palmitate and methyl stearate have been reported to possess nematicidal activity (Lu et al. 2020). Alkanes like tetratetracontane have been previously identified as volatile metabolites produced by endophytes with plant growth promoting and antimicrobial properties (Jishma et al. 2017