Antifungal potential of Bacillus vallismortis R2 against different phytopathogenic fungi

The cash crops grown in an agro-climatic region are prone to infection by various fungal pathogens. The use of chemical fungicides over the years has resulted in emergence of resistant fungal strains, thereby necessitating the development of effective and environmental friendly alternatives. The natural antagonistic interactions among different microbial populations have been ex-ploited as an eco-friendly approach for controlling fungal pathogens resistant to synthetic chemicals. Morphologically distinct bacterial cultures (150), isolated from rhizospheric soils of wheat, rice, onion and tomato plants were screened for their antifungal potential against seven phytopathogenic fungi prevalent in the State of Punjab (India). The bacterial isolate R2, identified as Bacillus vallismortis, supported more than 50% inhibition of different phytopathogenic fungi ( Alternaria alternata, Rhizoctonia oryzae, Fusarium oxysporum, Fusarium moniliforme, Colletotrichum sp, Helminthosporium sp and Magnaporthe grisea ) in dual culture plate assay. The thin layer chromatography based bio-autography of acid-precipitated biomolecules (APB) indicated the presence of more than one type of antifungal molecule, as evidenced from zones of inhibition against the respective fungal pathogen. The initial analytical studies indicated the presence of surfactin, iturin A and fengycin-like compounds in APB. The antifungal activity of whole cells and APB of isolate R2 was evaluated by light and scanning electron microscopy. The wheat grains treated with APB and exposed to spores of A. alternata showed resistance to the development of black point disease, thereby indicating the potential application of R2 and its biomolecules at field scale level.


Introduction
Agriculture is the mainstay of economy in the State of Punjab (India) with wheat (Triticum aestivum), rice (Oryza sativa), onion (Allium cepa) and tomato (Lycopersicon esculentum) as the major cash crops cultivated in the State. The infestation of these crops by fungal pathogens, i.e. Alternaria alternata, Rhizoctonia oryzae, Fusarium oxysporum, Fusarium moniliforme, Colletotrichum sp, and Magnaporthe grisea, affects the overall yield thus causing significant economic losses to farmers (Mew, 1991;Roberts et al., 2005;Zhao et al., 2010). The black point or kernel smudge of wheat caused by different fungal pathogens, including A. alternatA, has emerged 2 having four different bacterial isolates streaked per plate, one isolate in each quarter. The plates were incubated at 30 °C and observed daily up to seven days. The fungal growth in presence of bacterial isolates was compared with that of growth in absence of bacterial isolates (control) to determine the percentage of inhibition, using the formula: (r 1 − r 2 )/r 1 * 100, where r 1 and r 2 represent the radius (mm) of fungal growth in absence and presence of bacterial isolate, respectively.
On the basis of these observations, the isolate R2 was selected for further studies as it showed best antifungal potential among all the screened isolates.

Identification of isolate R2
The identification of isolate R2 was carried out by routine biochemical tests and by amplifying the sequence of gene coding for 16S rRNA using universal primers 27F and 1492R (Cappuccino & Sherman, 1999;Frank et al., 2008). The sequence was aligned with already available sequences in GenBank database (NCBI) using ClustalW software version 1.81 (http://www.clustal.org/ clustal2/). The phylogenetic tree was constructed by neighbor-joining method using MEGA software version 5.1 (http://mega.software.informer.com/5.1b/) to identify the isolate on basis of percentage homology.

Preparation of acid-precipitated biomolecules (APB)
One litre of YME broth in a 3 L Erlenmeyer flask was inoculated with 0.1% (v/v) overnight activated cells of R2. The flask was incubated for 48 h at 30 °C in an orbital shaker (180 rpm). The broth was centrifuged at 10,000 rpm for 10 min and pH of the supernatant was lowered to 2.0 with concentrated HCl (11.6 N). This acidified supernatant was kept at 4 °C for 24 h to allow complete precipitation. The APB obtained were pelleted by centrifugation at 10,000 rpm for 15 min in a preweighed centrifuge tube. The pellet was dried and dissolved in methanol to prepare 1% (w/v) stock solution. The stock solution was filter-sterilized (Acrodisc 13 mm with 0.2 µm Supor Membrane, Pal Life Sciences, USA) and used for further studies.

Analysis of APB by thin layer chromatography (TLC) and high performance liquid chromatography (HPLC)
The APB preparation was resolved on a TLC plate (silica gel 60 F 254, Merck, Germany) and compared with strategies to decrease the dependence on chemical fungicides and to address the problem of recurrent incidents of infestation by resistant fungal pathogens.
The studies over the last few years have reported the antifungal potential of biomolecules viz. lipopeptides, glycolipids, phospholipids, neutral lipids, polysaccharide-protein complexes and fatty acids produced by different bacterial isolates (El-Ghaouth, 1997;Szczech & Shoda, 2006;Romero et al., 2007, Tendulkar et al., 2007. The present study was taken up to isolate and screen the unexplored bacterial diversity of this region for its antifungal potential so as to develop biocontrol strategies as an alternative to chemical fungicides.

Phytopathogenic fungal strains and growth conditions
The fungal pathogens commonly causing disease to cash crops in the State of Punjab (India) viz. A. alternata, R. oryzae, F. oxysporum, F. moniliforme, Colletotrichum sp, Helminthosporium sp and M. grisea were obtained from the Department of Plant Pathology, Punjab Agricultural University (PAU), Ludhiana. The fungal strains were maintained on potato dextrose agar (PDA) medium by regular sub-culturing and preserved for long term at -80 °C as 25% (v/v) glycerol stocks.

Isolation
The soil samples from rhizosphere -defined by Hiltner (1904) as the zone in which plant roots interact with the surrounding soil-of wheat, rice, onion, and tomato plants were collected from agricultural fields located in and around the city of Amritsar (Punjab). One gram of each soil sample was serially diluted in 9 mL sterile saline (0.8% w/v NaCl), and 0.1 mL of the appropriate dilution was plated on yeast malt extract (YME) agar medium. The plates were incubated at 30 °C and observed daily for three days. Morphological distinct bacterial colonies were picked and repeatedly streaked to ascertain their purity.

Screening
The bacterial isolates were screened for their antifungal potential by dual culture plate assay. Fungal spores were inoculated on YME agar plates, which were incubated at 30 °C for seven days. Agar plugs of 5 mm diameter of each fungus were taken from these plates and placed in the centre of the YME agar plates 3 Antifungal potential of Bacillus vallismortis R2 against different phytopathogenic fungi The cells of R2 grown overnight in YME broth were pelleted and washed with 0.8% (w/v) saline. The pellet was suspended in 0.8% (w/v) saline to achieve an OD 600 of 0.68, equivalent to 10 8 CFU/mL. The cells of R2 and spores of A. alternata (10 4 spores/mL) were inoculated in 20 mL YME broth in 100 mL Erlenmeyer flask to evaluate the antagonistic effect of growing cells of R2 on fungal spores/hyphae. Similarly, the antifungal activity of APB of R2 was evaluated by supplementing YME broth (20 mL) with 0.1, 0.2 and 0.4 mg of APB respectively, followed by inoculation with spores of A. alternata (10 4 spores/mL). The YME broth without APB and inoculated with spores of A. alternata served as negative control, whereas the YME broth supplemented with methanol and inoculated with spores of A. alternata served as positive control. The flasks were incubated at 30 °C, 50 rpm in orbital shaker (Scigenics, India) for 48 h. The viability of A. alternata in flasks supplemented with different concentrations of APB was checked by transferring 10 µL of sample from each flask to the centre of YME agar plates. The plates were incubated at 30 °C for seven days and observed daily for the appearance of hyphal growth.
Further, 400 µL sample from each flask was centrifuged for 10 min at 10,000 rpm. The pellet was washed twice with sterile water and fixed on the surface of slide with 4% (v/v) glutaraldehyde for 12 h at 4 °C followed by washing with 0.1M sodium cacodylate buffer to remove excess of glutaraldehyde. This was followed by dehydration of fixed sample on slide with series of ethanol concentrations from 30% to 100% (v/v) and analyzed by using SEM-EVO LS-10 (Carl Zeiss, Germany).

Control of black point disease of wheat grains by APB of R2
The wheat grains were surface-sterilized by dipping them in 70% (v/v) ethanol for 5 min followed by 1 min exposure to 1% (v/v) sodium hypochlorite in distilled water and finally rinsed five times with sterile distilled water. Thirty grains were transferred to each flask (100 mL Erlenmeyer flasks) designated A, B and C. The grains in flasks A and B were inoculated with 100 µL spore suspension of A. alternata (10 4 spores/mL), followed by addition of 1 mL of APB (50 mg/mL) to flask A and 1 mL of sterile water to flask B (positive control). The grains in flask C received only 1.1 mL of sterile distilled water (negative control). The flasks were shaken properly so as to allow even absorption of respective suspension by the grains. The flasks were incubated at 30 °C for seven days and grains in respective flasks were observed daily for appearance of disease related symptoms. After the standards of iturin A and surfactin (Sigma, USA) using a mixture of water-methanol-chloroform (1/4/5 v/v) as developing solvent. The resolved components were visualized by exposing the TLC plate to iodine vapours. The plates were also sprayed with distilled water to ascertain the presence of lipopeptides as described by Li et al. (2007). The crude APB and standards of iturin A and surfactin (Sigma, USA) were further analyzed by HPLC using C18 reverse phase column (250 mm × 4.6 mm × 1/4 Microsorb VIV 100-5, Varian, USA) at a constant oven temperature of 30 °C (TCC-100, Dionex, USA). The sample (20 μL), was injected and eluted using solvent system of acetonitrile-water (70/30) containing 0.05% (v/v) trifluoroacetic acid, at a flow rate of 1 mL/min. The eluant was read at 225 nm using photo diode array detector (UVD340U, Dionex, USA).

TLC agar overlay assay
The TLC-based bio-autographs were developed as described by Kaur et al. (2006). The YME agar supplemented with 10 4 spores/mL of the respective test fungus was poured on TLC plates with resolved APB. The TLC plate developed without APB in solvent system as described above and overlaid with YME agar containing spores of respective fungus served as control. The TLC plates were placed in 200 mm glass Petri dishes and incubated at 30 °C to observe the clear zones or variation in hyphal growth pattern.

Microscopic evaluation of antifungal potential of R2 cells and their APB on A. alternata growth and morphology
The variation in morphology of A. alternata grown in presence and absence of cells and APB of R2 was observed by light microscopy and scanning electron microscopy (SEM). In light microscopy studies, an agar plug of A. alternata taken from well grown lawn of seven days old fungus was kept in the centre of fresh YME agar plate having four overlapping streaks of R2 cells, each at a distance of 1.5 cm from circumference of the plate. The control plate had fungal agar plug in the centre without any bacterial streaks. The plates were incubated for seven days at 30 °C and observations related to size and appearance of fungal growth were recorded daily. The hyphal growth on respective plate was observed using inverted microscope (Olympus CKX41, Japan).
The effect of whole cells of R2 and APB on growth of A. alternata was evaluated by shake flask studies. 4 7 days of incubation, 5 grains from each flask were sown in 15-well (1 well: 3 × 4 cm) seedling trays with 1 grain per well of tray. After 14 days, the shoot length, root length and dry weight (oven dried at 70 °C for three days) of plantlets were determined to evaluate the effect of different treatments on grains.

Statistical analysis
Data were subjected to one way analysis of variance (ANOVA) and the means were compared using Tukey's HSD at a significance level of p ≤ 0.05 using AS-SISTAT.

Isolation and screening
The results presented in Table 1 show the observations of initial screening in which six bacterial isolates supported best antifungal potential against different fungal pathogens. The highest among all the bacterial isolates screened was isolate R2, which supported more than 70% inhibition of all the tested fungi, except R. oryzae for which 56% inhibition was observed. In light of this broad spectrum activity the strain R2, isolated from rhizosphere of wheat, was selected for further studies.

Identification of isolate R2
The colonies of R2 on YME agar plates appeared off-white, round, wrinkled and mucoid. The cells are gram-positive, endospore former, rod-shaped with rounded ends. The cells were motile as observed under the microscope by hanging drop method. The isolate was positive for catalase and Voges-Proskauer (VP) test and negative for methyl red (MR) test. The isolate tested positive for starch and casein hydrolysis and negative for lipid hydrolysis. These morphological and biochemical properties are typical features reported for genus Bacillus (Cappuccino & Sherman, 1999). The comparison of partial sequence of R2 gene coding for 16S rRNA with already known sequences in GenBank database identified the strain as Bacillus vallismortis, as it showed 100% similarity with strain DSM11031 (Fig. 1). The nucleotide sequence obtained was submitted to GenBank, under accession no. KC283030.

Analysis of acid-precipitated biomolecules (APB) by TLC and HPLC
The acid precipitation of biomolecules in cell free supernatant of R2 yielded 0.9 g/L of APB. The APB resolved into five spots on thin layer chromatogram with Rf value of 0.33-(I), 0.43-(II), 0.55-(III), 0.693-(IV) and 0.877-(V) (Fig. 2). These spots appeared white when the plates were sprayed with distilled water, indicating to all being different lipopeptides. The Rf values of biomolecules IV and V corresponded to standards of iturin A and surfactin (Sigma, USA) respectively. The HPLC analysis of APB preparation showed four peaks with different retention time (Rt) (Fig. 3). The peak with Rt 2.813 min matched with standard of iturin A (Rt 2.867), whereas peaks with Rt 3.077 min and 3.48 min matched with standard of surfactin (which has three peaks, each corresponding to different isoform of surfactin). An additional peak at 6.87 min was also observed which might be representing biomolecules I to III.

TLC agar overlay assay
It is evident from the bioautographs presented in Figs. 4a-g that for different fungal strains the level of

Microscopic evaluation of antifungal potential of R2 cells and their APB on A. alternata growth and morphology
Different morphological deformities viz. shrunken hyphal tips, disorganization of hyphae, swollen hyphae, excessive branching of hyphae and cell content degen-inhibition and appearance of the inhibition zones varied. The spots I to IV supported antifungal activity against all the fungi tested, whereas no antifungal activity was observed for surfactin like biomolecules at spot V.

Bacillus safensis XFB-Y (KC429628)
Bacillus pumilus T1 (  Antifungal potential of Bacillus vallismortis R2 against different phytopathogenic fungi The delay in fungal growth on YME agar plates after exposure to APB was directly proportional to increasing concentration of APB, as inoculum from flask treated with 0.1, 0.2 and 0.4 mg of APB showed mycelial growth on YME agar plate after 4, 5 and 7 days of incubation respectively, in comparison to samples taken from the positive and negative controls which showed mycelial growth after 2 days of incubation. eration were evident in plate assay where fungal hyphae were grown in presence of R2 cells as compared to control (Fig. 5). Scanning electron micrographs of fungal hyphae grown in presence of whole cells of R2 and different concentrations of APB showed shrunken, collapsed, empty hyphae, and large depressions and loss of turgidness in comparison to the regular morphological features in positive and negative control (Fig. 6).

Control of black point disease of wheat grains by APB of R2
The ability of APB to inhibit spore germination of A. alternata was evidenced as the wheat grains in flask A treated with APB and inoculated with fungal spores were healthy, resisted development of the disease and appeared similar to grains in flask C which were neither exposed to fungal spores nor to APB (Fig. 7). Further, these grains germinated into healthy plants with no significant variation in their shoot length, root length and dry weight as compared to plantlets of control grains (Table 2). However, flask B grains inoculated with fungal spores and not treated with APB showed disease symptoms after 2 days of incubation as evidenced by the characteristic discolouration starting from the embryonic end. The whole grains turned black after 4 days of incubation due to fungal growth and when sown in soil did not germinate.

Discussion
The rhizospheric soil is known to support higher number of bacteria due to release of root exudates and the resulting interactions viz. symbiotic, parasitic, antagonistic etc. among the microbial population improve the chances of isolation of potential biocontrol agents (Whipps, 2001;Lugtenberg et al., 2002;Villacieros et al., 2003;Singh et al., 2004). Different bacterial strains isolated from rhizospheric soil samples of wheat, rice, onion and tomato plants were screened for their antifungal potential against common phytopathogenic fungi prevalent in Punjab. The isolate R2, identified as B. vallismortis, achieving more than 50% inhibition of all the fungal strains in dual culture plate assay, was used for further studies. The APB of R2 resolved into five spots (I to V) as observed by exposing the TLC chromatogram to iodine vapours. The TLC and HPLC analysis of APB further indicated the presence of iturin A (IV) and surfactin (V) like compounds as compared   Antifungal potential of Bacillus vallismortis R2 against different phytopathogenic fungi infectoria as major species associated with this disease among the other Alternaria spp. (Perello et al., 2008). In light of this, the potential of APB to control spread of black point disease in wheat grains was explored. The wheat grains treated with APB of R2 resisted development of disease when challenged with spores of A. alternata and retained their ability to grow into healthy plants as compared to wheat grains in positive control which failed to germinate due to severe damage inflicted by the fungal pathogen. The biomolecules of endophytic isolate B. vallismortis strain ZZ185, have been reported to control disease symptoms of root rot in wheat seedlings caused by A. alternata and Fusarium graminearum. The active biomolecules belonged to iturin family and were identified as Bacillomycin D (n-C14 and iso-C15) (Zhao et al., 2010). Similarly, the biocontrol potential of B. vallismortis strain EXTN-1 against Phytophthora capsici and Pectobacterium carotovorum has been related to presence of seven iturin A analogs out of which only two analogs showed activity against fungal pathogens (Park et al., 2014). As per reported literature and to the best of our knowledge, this is the first report related to evaluation of the ability of biomolecules of indigenous B. vallismortis to control the black point disease of wheat grains. These first time observations indicated the presence of at least three different types of cyclic lipopeptides (CLPs) similar to surfactin, iturin and fengycins from isolate B. vallismortis. As per earlier reports mentioned above, only a CLP belonging to the family iturin has been identified from B. vallismortis. Further studies with purified molecules could provide insights into their synergistic interactions for improved antifungal potential. These basic studies could pave way for their future use as biocontrol agents along with other conventional chemical fungicides against fungal pathogens causing economic losses to diverse crops like wheat, rice, tomato, onion, sugarcane etc.
to standard molecules. The spots I, II and III might be isoforms of fengycins as their Rf values on TLC and HPLC profile (Rt 6.87 min) were similar to those earlier reported in literature (Yanez-Mendizabel et al., 2012;Grover et al., 2010). These amphiphilic cyclic lipopeptide (CLP) compounds have been reported for their wide spectrum antifungal activity due to their interaction with biological membranes (Magnet-Dana & Peypoux, 1994;Stein, 2005). The TLC-based bioautography studies indicated varied antifungal potential of these biomolecules with the respective fungal pathogen. The clear zone may be due to the inhibition of spore germination and/or inhibition of hyphal growth by antifungal biomolecules. The hazy zones indicated an intermediate inhibition and induction of early sporulation due to activation of stress response in presence of APB. These variations in inhibition patterns may be attributed to interaction of lipopeptides with sterol molecules in cell membrane, which vary in different groups of fungi (Hu et al., 2007). This was further confirmed by light and scanning electron microscopy, as Fig. 6b shows detachment of cell membrane from cell wall of fungal hyphae due to leakage of cytoplasmic fluid from the pores formed in the membrane by amphiphilic cyclic lipopeptides. Severity of deformities caused was directly proportional to the concentration of antifungal biomolecules in both agar plates and shake flask studies. The deformities observed were similar to those reported earlier, caused due to formation of pores by interaction of biomolecules with the fungal cell membrane resulting in shrunken and empty hyphae (Romero et al., 2007;Makovitzki et al., 2007). The formation of pores further causes an increased Ca 2+ influx which has been reported to induce excessive branching of hyphae (Thrane et al., 1999;Mortel et al., 2009). The appearance of bulbous morphology may also be attributed to the effect of APB on the cytoskeletal elements, actin and tubulin leading to collapsed cytoskeleton and un-polarized growth at the tip of hyphae (Tendulkar et al., 2007). The other cellular responses observed in bioautographs viz. inhibition of mycelial growth and early sporulation around the inhibition zone may be attributed to the induction and cross interaction between different signalling pathways viz "two component" histidine kinase, HOG1-mitogen activated protein kinase and SKN7-mediated ROS detoxification in fungal strains, which play an important role in regulating sporulation and other metabolic events in fungi (Kuang-Ren Chung, 2012).
In the State of Punjab, the incidence of black point disease is frequent, causing significant loss of wheat production. The incidence of this disease has also been reported in other parts of the world, i.e. Australia, North America and Europe with A. alternata and A.