Diversity, Biocontrol, and Plant Growth Promoting Abilities of Xylem Residing Bacteria from Solanaceous Crops

Eggplant (Solanum melongena L.) is one of the solanaceous crops of economic and cultural importance and is widely cultivated in the state of Goa, India. Eggplant cultivation is severely affected by bacterial wilt caused by Ralstonia solanacearum that colonizes the xylem tissue. In this study, 167 bacteria were isolated from the xylem of healthy eggplant, chilli, and Solanum torvum Sw. by vacuum infiltration and maceration. Amplified rDNA restriction analysis (ARDRA) grouped these xylem residing bacteria (XRB) into 38 haplotypes. Twenty-eight strains inhibited growth of R. solanacearum and produced volatile and diffusible antagonistic compounds and plant growth promoting substances in vitro. Antagonistic strains XB86, XB169, XB177, and XB200 recorded a biocontrol efficacy greater than 85% against BW and exhibited 12%–22 % increase in shoot length in eggplant in the greenhouse screening. 16S rRNA based identification revealed the presence of 23 different bacterial genera. XRB with high biocontrol and plant growth promoting activities were identified as strains of Staphylococcus sp., Bacillus sp., Streptomyces sp., Enterobacter sp., and Agrobacterium sp. This study is the first report on identity of bacteria from the xylem of solanaceous crops having traits useful in cultivation of eggplant.


Introduction
Ralstonia solanacearum is a vascular wilt pathogen that belongs to the subdivision of the Proteobacteria [1] and is one of the most destructive plant pathogens causing bacterial wilt (BW) in many crop plants. It has broad host range and infects around 54 plant families and 450 plant species [2]. This pathogen also has a wide geographical distribution ranging from tropical, subtropical, and warm temperate regions of the world [3]. Cultivation of eggplant in the coastal state of Goa, India, is severely affected by BW leading to 30-100% crop loss [4]. The bacterium infects the plant through root cracks at the site of root emergence. Subsequently, the intercellular spaces of the root cortex and vascular parenchyma are colonized. Cell wall degrading exoenzymes disrupt the cell walls and facilitate its entry in the vascular system [5]. Inside the xylem vessels, the bacterial populations rapidly reach very high levels of 10 10 cells/cm of stem [6]. High cell density and production of high molecular weight exopolysaccharides by R. solanacearum lead to clogging of xylem vessels, wilting, and eventually death of plant.
Xylem of healthy plants has been reported to be colonized by endophytic xylem residing bacteria (XRB) at low population levels and has been isolated from xylem of various crops, namely, citrus [7], sugar beets [8], maize [9], alfalfa [10], grape [11,12], and Bermuda grass [13]. Several endophytic bacteria have been reported to originate from the rhizosphere soil, initially entering the host plant during germination and radicle development, through wounds or by colonizing the cracks formed in lateral root junctions when the endodermis and casparian strips are disrupted thus gaining an easy access to the stele [14,15]. After their initial entry, depending on the endophytic colonization ability, bacteria may remain localized in the roots [16] or colonize intercellular spaces and vascular system [11] and move to the stems [17]. Few endophytes have been reported to be able to migrate to aerial plant parts through the vascular system passively with the transpirational flow or through additional assistance by 2 International Journal of Microbiology production of cell wall degrading enzymes [18,19]. These systemically migrated endophytes have been isolated from leaves [20], inflorescence [21], fruits [22], and seeds [23].
Among the several methods of plant disease management, biocontrol plays an important role particularly in the control of soil borne diseases. Biocontrol agents may be used as an alternative pathogen management strategy or can be combined with other management practices. Biological control not only helps in suppressing the disease and increasing crop yield but also has importance in reducing the environmental pollution due to use of chemical pesticides [24]. Several studies have shown that endophytic bacteria can be used as biocontrol agents against plant pathogens. The capability of colonizing internal host tissues and ability to produce volatile and diffusible substances which inhibit pathogen, induction of systemic resistance in the plant, and directly or indirectly promoting plant growth have made endophytes a valuable tool in agriculture to improve crop performance [18]. Endophytic biocontrol agents isolated from potato [25], tomato, chilli [26] and eggplant [27] have been used for management of BW. However, the wilt prevention ability of xylem residing bacteria of solanaceous crops that share an ecological niche with the BW pathogen has remained unexplored. This study was undertaken to identify and screen bacteria isolated from the xylem of eggplant, chilli, and S. torvum for their biocontrol activities against R. solanacearum and growth promotion abilities in eggplant.  [7,28]. Briefly, after the surface sterilization of stem, one cm piece from each end was discarded. Epidermis and cortex from each end were removed and the vascular cylinder was fitted to sterile glass tubing attached in a rubber cork. To the other end of the stem piece a sterile plastic tubing was attached that could hold at least 500 L of 1 X phosphate buffered saline (PBS) (NaCl 8 g/L, KCl 0.2 g/L, Na 2 HPO 4 ⋅2H 2 O, 1.44 g/L, and KH 2 PO 4 0.24 g/L, pH 7.4). The cork with plant sample attached was then fitted onto a Buchner flask. For extraction of PBS through the xylem vessels a suction pressure 8 mbar was applied using a diaphragm pump MPC101Z (Ilmvac GmbH, Germany). A total of four successive infiltrations using 500 L of PBS were performed for each sample. The sap was collected directly in a sterile test tube placed inside the Buchner flask. Alternatively, maceration/trituration was performed for isolation of the XRB from young eggplant and chilli samples which had thin and soft stems. The epidermis and cortex from the surface sterilized stem piece were removed aseptically to expose the vascular bundles. The decorticated pieces were macerated in a sterile mortar and pestle using 2 mL of sterile 1X PBS.

Isolation.
One hundred L of the vacuum in-filtered sap or macerate was plated onto TSA or medium 523 [29]. The plates were incubated at 28 ± 2 ∘ C for 5 days. Different colonies from isolation plates were selected based on differences in their shape, color, and texture and purified onto medium 523. Pure cultures of the xylem residing bacteria (XRB) thus obtained were maintained at −80 ∘ C, as glycerol stocks for long term, and 4 ∘ C for temporary storage.

Amplified rDNA Restriction Analysis (ARDRA).
ARDRA was performed to determine the genetic diversity of the XRB in the collection. Genomic DNA from the XRB was extracted as described by Wilson [30]. Quality and quantity of the DNA were measured using Nanodrop 1000 (Thermo Scientific, USA). 16S rRNA gene was amplified using universal primers 27F (5 -AGAGTTTGATCCTGGCTCAG-3 ) and 1492R (5 -GGTTACCTTGTTACGACTT-3  [27]. Briefly, single colony of R. solanacearum and XRB was grown in 5 mL CPG broth (Casein hydrolysate 1.0 g/L, Peptone, 10.0 g/L and Glucose, 5.0 g/L) and King's B broth (Peptone, 20.0 g/L, K 2 HPO 4 , 1.5 g/L, MgSO 4 ⋅ 7H 2 O, 1.5 g/L and Glycerol 10.0 mL/L), respectively, at 28 ± 2 ∘ C for 48 h with constant shaking at 140 rpm. One hundred and fifty microliters of R. solanacearum was seeded every 100 mL molten cooled King's B agar, mixed well, and poured into plates. After the plates solidified, three wells were made in each plate by removing a circular agar piece with the help of cork borer (8 mm diameter). Twenty-five L of culture broth of XRB containing 8.0 Log CFU/mL was added into each of the three wells. All the plates were incubated at 28 ± 2 ∘ C for 48 h. Plates were observed for inhibition of R. solanacearum.
Zones of inhibition were measured as radius in mm from the edge of the agar well. Strains that were found antagonistic to R. solanacearum were screened for in vitro production of antagonistic compounds and plant growth promoting substances and identified by 16S rRNA gene sequencing.

Hydrogen Cyanide (HCN)
Production. Antagonistic strains were tested for HCN production ability in presence of glycine as described by Saraf et al. [31], a slight modification being the use of broth for the HCN test. Immediately after inoculation of strains in King's B broth containing 4.4 g/L of glycine, sterile filter paper strips dipped in picric acid solution were introduced taking care that the strips did not touch the medium and walls of the tube. The tubes were sealed with parafilm and incubated at 28 ± 2 ∘ C for 4 days with constant shaking at 140 rpm. The color change of the filter paper strips from yellow to brick red during incubation indicated the production of HCN.

Ammonia Production.
To detect ammonia production, antagonistic XRB were grown in peptone water (peptone 20.0 g/L, NaCl 5.0 g/L) with constant shaking at 140 rpm for 48 h at 28 ± 2 ∘ C. Ammonia production was determined using Nessler's reagent as described by Marques et al. [32].

Acetoin Production.
Acetoin production by antagonistic isolates was tested in Voges Proskauer broth (peptone 7.0 g/L, K 2 HPO 4 5.0 g/L, dextrose 5.0 g/L pH 7.0). After incubation for 30 h at 28 ± 2 ∘ C at 140 rpm, one mL each of 5% napthol and 40% KOH were added to the culture and mixed well. Appearance of red coloration indicated production of acetoin [33].

Siderophore
Production. Antagonistic XRB were tested for siderophore production on a medium containing chrome azurol S (CAS) [34]. Isolates producing orange haloes on the blue green colored medium after incubation at 28 ± 2 ∘ C for 48 h were positive for siderophore production.

Production of Growth Promoting Substances by
the Antagonistic XRB 2.5.1. Indole Acetic Acid (IAA) Production. Antagonistic strains were tested for their ability to produce phytohormone IAA in presence of tryptophan as described by Gordon and Paleg [35]. Briefly, strains were grown in nutrient broth amended with 100 mg/L of tryptophan for 30 h at 28 ± 2 ∘ C at 140 rpm. The supernatants were obtained by centrifugation at 6200 g for 10 min. One mL of supernatant was mixed with one mL of Salkowsky's reagent (50 mL 35% perchloric acid, 1 mL 0.5 M FeCl 3 ). The mixture was allowed to stand at room temperature for five minutes and the absorbance was read at 530 nm. A standard curve was prepared using analytical grade IAA and the concentrations of IAA in the culture supernatants of XRB were estimated based on the curve.

1-Aminocyclopropane-1-carboxylate (ACC) Deaminase
Activity. Antagonistic strains were tested for their ability to produce enzyme ACC deaminase as per the method described by Godinho et al. [36]. Strains were streaked on Dworkin and Foster's DF salts agar containing 3.0 mM ACC and incubated at 28 ± 2 ∘ C for 7 days. Ability of the strains to grow on the medium containing ACC as a sole nitrogen source was indicative of ACC deaminase production.

Phosphate
Solubilization. Antagonistic strains were tested for phosphate solubilization by a method described by Godinho et al. [36]. All strains were spot inoculated on Pikovskaya's agar plates (Hi Media Laboratories, Mumbai).
Plates were incubated at 28 ± 2 ∘ C for 48 h. Transparent zones around the growth of XRB on the opaque white medium were indicative of solubilisation phosphate.   Antagonism to R. solanacearum tested in vitro as described in Section 2.

Isolation of
BCE (biocontrol efficacy) and GPE (growth promotion efficacy) were determined under greenhouse conditions in eggplant as described in Section 2. VI: Vacuum infiltration, M: maceration.

Production of Plant Growth Promoting Substances by
Antagonistic XRB. Results of the screening of antagonistic XRB for in vitro production of several plant growth promoting compounds is presented in Table 3. Majority of the antagonistic strains produced the phytohormone IAA with concentrations ranging from 15.91 g/mL to 645.91 g/mL. XB202 was found to be the best ACC deaminase producing strain based on its luxuriant growth on DF salts medium supplemented with 3.0 mM ACC as sole nitrogen source. Other ACC deaminase producing strains include XB1, XB62, XB86, and XB140. Scarce growth of XB165 and XB200 was observed on DF salts medium. 64.28% of the strains produced phosphate solubilizing organic acids as indicated by clear haloes on Pikovskaya's agar plate.

Suppression of Bacterial Wilt by Antagonistic XRB.
Ability to suppress BW was assessed as the difference in the percentage of wilt in XRB treated plants with respect to wilt in untreated control and was expressed as the biocontrol efficacy (BCE) of the antagonists. BCE of 16 strains with values ranging from 28.6 to 100% is presented in Figure 1.   Inhibition zones are mean of three replications and showing standard deviation. Inhibition zone was measured as radius from the outer edge of well. All the experiments were conducted at 28 ± 2 ∘ C. + indicates presence of trait; − indicates absence of trait in plate based assays. a Levels of ACC deaminase activities based on growth on plate based assay denoted as + for less growth, +++ for moderate growth, +++ for luxuriant growth, and ++++ for highly luxuriant growth; b IAA was estimated in culture filtrate and expressed as g/mL using analytical grade IAA as standard; values indicate mean and standard deviation.   Table 4. Based on the identification, phylum Proteobacteria consisting of Gram-negative bacteria of subdivisions Alpha Proteobacteria (12.73%), Beta Proteobacteria (3.64%), and Gamma Proteobacteria (25.45%) were predominant (41.81% strains identified), followed by phyla Firmicutes (29.09%), Actinobacteria (25.45%), and Bacteroidetes (3.64%) (Figure 2(a)).

Statistical Analysis.
The statistical analysis of percentage wilts and shoot length of eggplant was performed using Web Agri Statistical Package (WASP) version 2.0 (http://www.icargoa.res.in/wasp2.0/index/php).

Discussion
Eggplant and chilli not only are of economic and cultural importance but also are common ingredients in the cuisine throughout India. In the coastal state of Goa, R. solanacearum has been reported to be a destructive pathogen in cultivation of eggplant and chilli [4]. Isolation of biocontrol agents against the BW pathogen has been commonly restricted to endophytic tissue and plant rhizosphere [26,27]. Studies on xylem colonizing endophytes were undertaken because we speculated existence of interactions between the XRB and vascular wilt pathogen R. solanacearum during xylem colonization. Our study reveals the diversity, biocontrol potential, and identity of endophytic xylem colonizers from solanaceous crops cultivated in Goa, India. A total of 167 bacteria were isolated from the xylem of eggplant, chilli, and S. torvum with Gram-negative bacteria (59.28%) predominating in the collection. Congruent to our observation, Gardner et al. [7] and Bell et al. [11] have earlier reported isolation of more number of Gram-negative rod shaped bacteria from xylem of citrus and grapevine using vacuum infiltration. Scholander pressure bomb was found to be useful in extraction of diverse bacterial genera from xylem tissues in contrast to trituration methods that yielded higher number of Gram-positive rod shaped bacteria [12]. Though, Scholander pressure bomb was not used in this study, a combination of vacuum infiltration [7,11] and trituration of decorticated stems [10,12] was employed with an aim to isolate diverse XRB from xylem tissues. This is the first study reporting the use of vacuum infiltration and trituration methods for isolating xylem residing bacteria from eggplant, chilli, and S. torvum.
Traditionally bacteria have been characterized and grouped based on colony morphology and biochemical   [14]. However, factors that determine the selection of xylem colonists or the ability of XRB to colonize eggplant, chilli, and S. torvum in this study remain unknown. Interestingly, the structure of endophytic community in Nicotiana attenuata a member of Solanaceae family is shown to be influenced by soil composition and ethylene homeostasis [40]. Earlier evidence has shown that colonization by endophytic bacteria is also governed by plant genotype as well as root exudates [42]. Only 16.77% XRB out of 167 were antagonistic to R. solanacearum based on in vitro assays. Antagonistic XRB produced volatile and diffusible inhibitory compounds, namely, HCN, ammonia, and acetoin and siderophores. These substances have been long known to be involved in disease suppression and indirect growth promotion in plants [43][44][45][46]. These mechanisms possibly played a role in the evident biocontrol effect against BW exhibited by the XRB in the greenhouse screening. Endophytic bacteria have been known to have plant growth promoting traits, namely, production of IAA, ACC deaminase, and phosphate solubilization [25,33,36]. These traits were detected in the majority of antagonistic XRB tested in this study and may have resulted in the observed increase in shoot length of eggplant in our greenhouse experiments. In contrast, strains XB20, XB196, and XB203 suppressed growth in eggplant under greenhouse conditions; however no visible symptoms of disease were observed. Vascular plugging and production of certain metabolites toxic to plant cells, but not cell viability, may have resulted in stunted shoot in eggplant [11,47].
Evaluation of efficacy of antagonistic organisms to suppress the plant diseases under greenhouse conditions is one of the key steps for selecting a potential biocontrol agent for disease management [48]. Endophytic strains from BW susceptible varieties of eggplant have been shown to prevent wilt and promote growth in eggplant earlier [27]. Our greenhouse screening shows that 38.46% of antagonistic XRB with biocontrol efficacies greater than 40% were isolates from BW resistant varieties of eggplant. This raises a question whether the bacteria from resistant varieties are involved in BW resistance and whether BW resistant varieties are able to selectively influence xylem colonization by antagonistic bacteria? Presence of higher number of endophytes with antagonistic abilities was reported in BW resistant varieties of tomato as compared to susceptible varieties, and their role in resistance to BW was proposed [49]. Similar observations on correlation of resistance of potato to soft rot and endophytic bacteria have been reported [50]. Thus BW resistant varieties can be considered a better host for isolating potential biocontrol strains for management of bacterial wilt.
Identification of 55 XRB strains by 16S rRNA gene sequencing revealed the presence of 23 diverse genera of bacteria belonging to 4 phyla of Eubacteria. Strains belonging to phyla Firmicutes, Actinobacteria, and subdivision of Proteobacteria were the major xylem colonists identified in this study. Several genera of bacteria belonging to these phyla have also been reported to be present in endophytic tissues and xylem of a variety of other agricultural and horticultural plant species [14,51]. In addition, the majority of the antagonists identified belonged to Enterobacter sp., Pseudomonas sp., Bacillus sp., and Staphylococcus sp. Congruent to our results, several researchers have reported bacteria isolated from solanaceous crops and belonging to similar genera to be antagonistic to R. solanacearum [26,[51][52][53]. However, Flavobacterium sp.and Janibacter melonis identified in this study have never been previously reported to be inhibitory to R. solanacearum. Large population of the xylem inhabiting bacterial flora accounting for 83.23% exhibited no antagonism towards R. solanacearum. Nonantagonistic XRB were identified predominantly as Microbacterium sp. Endophytic persistence and nematicidal activities of Microbacterium sp.
have been reported [54,55]. Therefore the collection of XRB isolated in this study can be screened for inhibitory activities against other important agricultural pests.
Though few strains, namely, XB86, XB169, and XB177 exhibited plant beneficial properties in this study their usefulness in plant disease control remains to be seen. XB86 has been identified as Agrobacterium tumefaciens, the crown gall disease pathogen, and its deployment as biocontrol agent is uncertain. XB169 (Staphylococcus gallinarum) and XB177 (Bacillus cereus) are reported as opportunistic animal pathogens and thus unsuitable for field applications. Streptomyces sp. has earlier been reported as antagonistic to R. solanacearum and tested for management of wilt in potato and tomato [56,57]. XB200 (Streptomyces sp.) is one of the XRB high BCE and GPE; it could be explored further for biocontrol of bacterial wilt after additional characterization and field evaluation.

Conclusion
This study is the first report on the identity of novel and diverse XRB colonizing the xylem of eggplant, chilli, and S. torvum. XRB particularly from BW resistant varieties were found to protect eggplant from bacterial wilt and enhanced growth in eggplant in the greenhouse screening. Therefore the repertoire of XRB reported in this study may be useful for cultivation of eggplant in BW affected areas.