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 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 −} r₂)/r₁ * 100, where r₁ and r₂ 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 pre-weighed 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 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 10⁴ 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. 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 OD₆₀₀ of 0.68, equivalent to 10⁸ CFU/mL. The cells of R2 and spores of A. alternata (10⁴ 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 (10⁴ 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 (10⁴ 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 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.

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.

Table 1. Antifungal activity of the six best isolates against seven phytopathogenic fungi by dual culture plate assay

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 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. A clear and overlapping inhibition zone was supported by spots I, II and III for A. alternata, Helminthosporium sp. and F. moniliforme along with signs of early sporulation in hyphae immediately around the inhibition zone (Figs. 4a-c). However, in Colletotricum sp and M. grisea a hazy inhibition zone was observed corresponding to these biomolecules (Figs. 4f-g). On the other hand, in the case of F. oxysporum and R. oryzae, distinct and overlapping inhibition zones were observed corresponding to spots I and II and a hazy zone of inhibition was evident at spot III (Figs. 4d-e). The iturin-like biomolecules at spot IV afforded a hazy inhibition zone against A. alternata, F. moniliforme, Helminthosporium sp, F. oxysporum andno inhibition zone against R. oryzae. However, a clear zone of inhibition was observed corresponding to spot IV for Colletotricum sp and M. grisea.

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 degeneration 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). 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.

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.

Table 2. Germination of wheat grains into plants, after infection and treatment with APB of isolate R2 in comparison to those in control