Characterization of fungal antagonistic bacilli isolated from aerial roots of banyan (Ficus benghalensis) using intact-cell MALDI-TOF mass spectrometry (ICMS)

Authors


Correspondence

Hareshkumar Keharia, BRD School of Biosciences, Sardar Patel Maidan, Satellite campus, Vadtal road, Sardar Patel University, P.O.Box 39, Vallabh Vidyangar-388120, Anand, Gujarat, India. E-mail: haresh970@gmail.com

Abstract

Aims

To characterize fungal antagonistic bacilli isolated from aerial roots of banyan tree and identify the metabolites responsible for their antifungal activity.

Methods and Results

Seven gram positive, endospore-forming, rod-shaped endophytic bacterial strains exhibiting a broad-spectrum antifungal activity were isolated from the surface-sterilized aerial roots of banyan tree. The isolates designated as K1, A2, A4 and A12 were identified as Bacillus subtilis, whereas isolates A11 and A13 were identified as Bacillus amyloliquefaciens using Biolog Microbial Identification System. The antifungal lipopeptides, surfactins, iturins and fengycins with masses varying in the range from m/z 900 to m/z 1550 could be detected using intact-cell MALDI-TOF mass spectrometry (ICMS). On the basis of mass spectral and carbon source utilization profile, all seven endophytes could be distinguished from each other. Furthermore, ICMS analysis revealed higher extent of heterogeneity among iturins and fengycins produced by B. subtilis K1, correlating well with its higher antifungal activity in comparison with other isolates.

Conclusion

Seven fungal antagonistic bacilli were isolated from aerial roots of banyan tree, exhibiting broad spectrum of antifungal activity, among which B. subtilis K1 isolate was found to be most potent. The ICMS analysis revealed that all these isolates produced cyclic lipopeptides belonging to surfactin, iturin and fengycin families and exhibited varying degree of heterogeneity.

Significance and Impact of the study

The endophytes are considered as a potential source of novel bioactive metabolites, and this study describes the potent fungal antagonistic bacilli from aerial roots of banyan tree. The isolates described in this study have a prospective application as biocontrol agents. Also ICMS analysis described in this study for characterization of antifungal metabolites produced by banyan endophytic bacilli may be used as a high throughput tool for screening of microbes producing novel cyclic lipopeptides.

Introduction

Micro-organisms have been extensively used ever since they were discovered for various applications such as in fermentation, pharmaceutical, dairy and agriculture industries or in remediation of environmentally hazardous chemicals. Considering the vast diversity of microbes residing in various niches, a hunt for novel micro-organisms as source of novel antibiotics, therapeutics or enzymes is never going to end (Strobel et al. 2004). Among the microbial diversity, microbial endophytes are relatively less explored source for the wide variety of chemically novel and biologically active compounds for their application in agriculture as well as pharmaceuticals in comparison with microbes isolated from other biotic as well as abiotic sources. Endophytes are the microbes that colonize the internal plant tissue without causing much harm to the host plant and may either be beneficial to the host or both (host and endophyte) (Bacon et al. 2002). The symbiotic association of host plants with endophytes offers shelter and protection to the microbes against physiological and environmental conditions while in return microbes may provide several benefits to the host plant such as protection against pathogens, fitness by contributing functional metabolites for improvement of the plant's ability to withstand environmental stress (e.g. drought) or enhance N2 fixation (Sturz and Nowak 2000; Kloepper et al. 2004; Strobel et al. 2004; Naik et al. 2009; Li et al. 2008; Slimene et al. 2012). The microbial endophytes are expected to be ubiquitous in the plant kingdom (Stone et al. 2000); however, not many of these plants have been studied for the presence of endophytes with the potential to produce bioactive natural products. The knowledge of surrounding environment, age or natural history as well as ethno-botanical importance of a given plant is regarded valuable for search of antibiotics producing endophytes (Castillo et al. 2002). The banyan tree (Ficus benghalensis) is medicinally, socioculturally and ethnobotanically important Asian endemic plant with a very long life span. Various parts of the banyan tree like aerial roots, latex, stem, bark, leaves and fruits have been used in the preparation of traditional medicine for the treatment of various ailments like toothache, diarrhoea, dysentery, female sterility, leucorrhoea, rheumatism and skin disorders (Ayyanar and Ignacimutu 2009). Thus, isolation of endophytes from aerial roots of banyan tree was undertaken, and the present study describes characterization of antifungal activity of banyan endophytic bacilli employing intact-cell MALDI-TOF mass spectrometric analysis (ICMS).

Materials and methods

Isolation of endophytes from hanging roots of banyan tree

Approximately 10–15 young aerial roots were collected from a single banyan tree located at Vadtal road, Anand, Gujarat, India, and processed within an hour for the isolation of endophytes. Bacterial endophytes were isolated from surface-sterilized budding aerial roots of banyan tree as per the method described by Pathak et al. (2009). The aerial roots were dipped into solution of 15% Savlon™ (v/v) for 15 min followed by treatment with 70% ethanol for 2 min. The ethanol-treated roots were further immersed in 0·1% HgCl2 (w/v) for 30 s followed by repeated (at least 6–8 times) washes with sterile distilled water to remove excess of HgCl2 from the surface of explants. The surface-sterilized root tissues were then cut into 8-mm pieces, which were placed on surface of sterile potato dextrose agar (PDA) and Luria agar (LA) plates. All these Petri plates were further incubated at 30°C up to 10 days. The bacterial growth near surface-sterilized aerial root explants on plates was subjected to further isolation of pure cultures by streak plate method. The pure cultures thus obtained were maintained by subculturing on Luria agar slopes and preserved at 4°C as well as in the form of glycerol stocks stored at −20°C. The surface sterilization of explants was ascertained by streaking a loop full of each water wash on sterile LA and (PDA) plates (dehydrated media were obtained from Himedia limited, Mumbai) as well as by rolling the surface-sterilized root pieces on the sterile LA and PDA plates. These plates were then incubated at 30°C and monitored for sterility up to 10 days.

Identification of banyan endophytes

The identification of bacterial endophytes was performed as per manufacturers manual using MicroLog 1 (bacterial identification system) employing GP2 plates procured from Biolog Inc., USA. The endophytes were grown on Biolog universal agar (BUG) media for 10 h at 30°C. The Biolog GP2 microplates (gram positive 2) were inoculated with 150 μl bacterial suspension of 29% (1·2 O.D.) turbidity measured using turbidity meter provided by Biolog Inc. USA and incubated at 30°C. The plates were read after 18 h for utilization of carbon sources, and identification was performed on the basis of carbon source utilization pattern from the gram positive database using Biolog Microlog software (version 4.2; Biolog, Inc., Hayward, CA, USA). On the basis of carbon source utilization pattern in Biolog GP2 microplates, the similarity coefficients among the isolates were determined following numerical taxonomy approach using the NTSYS PC Version 2.0 software. The identification of potential antagonist, endophyte K1, which was selected for further investigation, was also confirmed by 16S rDNA gene sequence analysis.

In vitro fungal antagonism

The antifungal activity of banyan endophytic isolates was investigated against following fungal cultures: Aspergillus niger 40211, A. niger 16404, A. niger 181, Aspergillus flavus, Chrysosporium indicum, Mucor indicus, Fusarium oxysporum f. sp. lycopersicii, F. oxysporum f. sp. gingiberi, F. oxysporum 1072, Candida albicans, Alternaria brunsii (1), A. brunsii (2), Cladosporium herbarum 1112, Sclerotia rolfsii and Lasiodiplodia theobromae ABFK1. All A. niger strains, C. indicum, M. indicus and C. albicans cultures are our own isolates and maintained in our laboratory from long time. The A. brunsii (1) and (2), cumin pathogens were obtained from Anand Agriculture University, Anand, Gujarat, India. The A. flavus and S. rolfsii strains (ground nut pathogens) were obtained from Prof. J.S.S. Mohan of our institute. F. oxysporum f. sp. lycopersicii and gingiberi strains are wilt-causing pathogens of tomato and ginger and were obtained from Prof. R. B. Subramanian of our institute. The fungal strains F. oxysporum 1072 and C. herbarum 1112 were procured from the NCIM, National Chemical Laboratory, Pune, India. L. theobromae ABFK1 was isolated from the bark of Adansonia digitata tree located near Anand by us and was identified at fungal identification service center, Agharkar Research Institute, Pune, India, and the isolate was deposited in its Fungal culture collection centre. All these fungal cultures are maintained in our laboratory and will be made available to anyone on request for academic purpose. The pure cultures of four endophytic isolates were spot inoculated in four sectors on sterile PDA plate (3 cm away from the center of the Petri dish) and incubated at 30°C for 48 h. After 48 h of incubation, 9-mm mycelial plug of each fungal pathogen mentioned above was placed on the centre of agar medium in the Petri plate and further incubation was continued for 5–7 days.

Profile of growth, antifungal activity as well as emulsifying activity of potential fungal antagonist banyan endophytic bacterial strain Bacillus subtilis K1

For inoculum preparation, cells from a single colony of B. subtilis K1 were inoculated into 50 ml of sterile Luria broth (LB) in 250-ml Erlenmeyer flask and incubated at 30°C for 12 h (O.D. 1·9–2·0) on orbital shaker (150 rev min−1). For investigation of antifungal as well as emulsifying activity, the culture was inoculated into 100 ml of sterile LB in 250-ml Erlenmeyer flasks to an initial O.D.600 nm ~0·05. The flasks were incubated on orbital shaker (150 rev min−1) at 30°C for 96 h and at regular intervals of 24 h, one flask was harvested, cells were separated by centrifugation (~10 062 g for 20 min.), and supernatant was collected for the determination of antifungal as well as emulsifying activity. The antifungal activity was assayed by agar well diffusion method. The test plates were prepared by seeding 100 μl of spore suspension (1 × 107 spores ml−1) of A. niger 40211 into 4·5 ml of molten soft agar (1% agar, w/v) and over-layered on sterile PDA plates and allowed to solidify. The plate was divided into four sectors, and four wells were bored, one each in centre of each sector using sterile cup borer. To each well, 100 μl of methanolic antifungal extract obtained from different cultures was added and allowed to diffuse in medium. The plates were incubated for 48 h at 30°C. Upon incubation, the diameter of zone of inhibition was measured and arbitrary antifungal activity units (AAU) were determined. One arbitrary unit of antifungal activity was defined as the amount of antifungal active metabolites that yielded 13-mm zone of inhibition on PDA plates seeded with A. niger 40211. The emulsifying activity (E.A) was determined by using modified emulsification assay described by Navon-Venezia et al. 1995. The 1-ml aliquot of culture supernatant was added to 6·5 ml of 20 mmol l−1 TM buffer (20 mmol l−1 Tris-HCl buffer [pH-7], 10 mmol l−1 MgSO4) followed by addition of 0·1 ml of 1 : 1 (v/v) mixture of 2-methyl naphthalene and hexadecane. The samples were vigorously mixed for 2 min. and allowed to stand for 1 h at 30°C before measuring turbidity at 600 nm. One unit of emulsifying activity was defined as an amount of emulsifier that yielded an A600 nm of 0·1 in the assay mixture. The cell growth was determined by measuring O.D at 600 nm of fermentation broth harvested at regular interval of 24 h using spectrophotometer.

Intact-cell MALDI mass spectrometry

Intact-cell MALDI mass spectrometric analysis of the seven endophytic bacilli was carried out on Ultraflex TOF/TOF MALDI mass spectrometer (Bruker Daltonics, Billerica, MA, USA and Bremen, Germany). The 72-h-old single colony of each isolate grown on LA (at 37°C) was suspended into 20 μl of methanol/water (1 : 1, v/v) and 1 μl of the cell suspension thus prepared for each culture was separately mixed with equal amount of α-cyano-4-hydroxycinnamic acid saturated in acetonitrile/water (1 : 1 v/v) containing 0·1% (v/v) trifluoroacetic acid and spotted on the target plate. The mass spectra were acquired within the mass range of 550–3000 m/z in positive ion and reflector mode (Pathak et al. 2012).

Extraction of cyclic lipopeptides and its antifungal activity

The antifungal compounds were precipitated by lowering the pH of culture supernatant to 2 using 6N HCl followed by incubating it for 2 h at 4°C. The precipitates from the acidified broth were harvested by centrifugation at ~10 000 g for 20 min. The pellet thus obtained was then solubilized in pure methanol. The insoluble matter in methanolic extract was separated by centrifugation and discarded, while the clear methanolic supernatant was further concentrated by rotary vacuum evaporation (Buchi, Switzerland), and the yellow gummy extract thus obtained was used for further characterization. The crude concentrate thus obtained was dissolved in methanol to a final concentration of 1 mg ml−1 and its antifungal activity was assayed against several test fungi by agar well diffusion assay. The pure methanol was used as a solvent control.

Effect of antifungal metabolites on conidia germination and morphology of mycelia

The conidia (1 × 103 spores ml−1) of a A. niger 40211 were incubated with various concentrations of cell-free culture (CFC) supernatant or methanolic antifungal extract, for 10 h at 30°C. Upon incubation, the conidiospores were stained with 1% lactophenol blue and observed using light microscopy under oil immersion lens (×100) (Lawrence & Mayo, Kolkata, India). Each experiment was performed in triplicate. A conidium was considered as germinated if the germ tube was more than half of the diameter of conidium (Chitarra et al. 2003).

Results

Isolation, identification and characterization of banyan bacterial endophytes

The surface-sterilized tender aerial root tips of the banyan tree were cut into small pieces and placed on LA as well as PDA plates and incubated at 30°C up to 10 days. On the fourth day of incubation, bacterial growth was observed at the edges of surface-sterilized banyan aerial root pieces. The bacterial growth thus obtained was then subcultured on fresh sterile LA plates for isolation of pure cultures. Seven isolates based on the variation in their colony morphology were purified and designated as K1, A2, A4, A11, A12, A13 and A32. All the seven isolates were found to be motile, gram positive, spore-forming bacilli.

All bacterial isolates except A32 could be identified on the basis of carbon source utilization profile employing GP2 plates of Biolog. The isolates designated as K1, A2, A4 and A12 were putatively identified as B. subtilis with similarity coefficients of 0·86, 0·68, 0·77 and 0·65, respectively. The isolates A11 and A13 were putatively identified as Bacillus amyloliquefaciens with similarity coefficients of 0·78 and 0·74, respectively. The isolate A32 produced highly mucoid colonies preventing the preparation of dense homogenous cell suspension, which is a prerequisite for identification using Biolog. The GP2 plate contains 95 different carbon substrates, among which 48 substrates were not utilized by all the six isolates, while these isolates exhibited significant variation in utilization of remaining 47 carbon sources (Table S1). The dendrogram on the basis of carbon source utilization profile for above six cultures grouped them into two clusters sharing more than 80% similarity. One of the clusters consisted of all four isolates, identified as B. subtilis, among which K1 and A2 exhibited about 90% similar carbon utilization profile. The other cluster consisting of two isolates, A11 and A13 identified as B. amyloliquefaciens, also exhibited high degree of similarity (90%) (Fig. 1). On the basis of carbon source utilization profile, it was thus possible to differentiate all the six isolates from each other.

Figure 1.

Dendrogram based on similarity coefficients calculated from carbon source utilization profile of six endophytic bacilli. The carbon source utilization profile was determined by employing GP2 plates of Biolog.

The endophytic isolates were then investigated for antifungal activity against 14 different fungal cultures (Fig. S1). The bacterial cultures K1, A13, A2 and A4 were found to inhibit the growth of all the 14 test fungal cultures, while A32 inhibited only 5 fungal cultures among 14 tested. The isolates A11 and A12 were also able to inhibit 13 of 14 test fungal cultures; they did not inhibit S. rolfsii (Table 1).

Table 1. Spectrum of antifungal activity of methanolic extracts of banyan endophytes monitored by agar diffusion assay
Sr. No.Fungal culturesDiameter of zone of inhibition (mm)
K1A11A12A13A2A4A32
1Aspergillus niger 402113636343622–2426
2A. niger 18132–343432–34382022
3A. niger 16404323034362224
4 Aspergillus flavus 30283130282826
5Alternaria brunsii (1)32223020243020
6A. brunsii (2)28–30243020223424
7 Chrysosporium indicum 34–3830–3636–4032–36283022–24
8Fusarium oxysporum(1072)322824202824
9 F.  oxysporum lycopercisi 302426243228
10 F. oxysporum gingiberi 3226–2824242620
11Cladosporium herbarum111230282525–2828–302825–27
12Lasiodiplodia theobromae ABK1322824202824
13 Sclerotia rolfsii 30152025
14 Mucor indicus 241822182624

Among all the banyan endophytic isolates, B. subtilis K1 was found to be most potent fungal antagonist based on in vitro inhibition assay and thus was selected for further characterization. The B. subtilis K1 was also putatively identified on the basis of full-length 16S rRNA gene nucleotide sequence (accession number EU056571) (Pathak et al. 2012).

Profile of growth, extracellular antifungal activity as well as emulsifying activity of B. subtilis K1

The extracellular emulsifying activity in the fermentation broth of B. subtilis K1 was found to increase with growth, reaching maximum in mid-logarithmic growth phase at about 33 h of incubation and then onwards it started decreasing up to 50 h of incubation (Fig. 2). The emulsifying activity was found to increase slowly with further incubation up to 84 h before reaching plateau at the time, when further increase in biomass ceased. In contrast to emulsifying activity, the extracellular antifungal activity in the fermentation broth of B. subtilis K1 could not be detected up to 31 h of incubation. The antifungal activity started appearing after 33 h of incubation, that is, approximately in the mid-logarithmic growth phase and increased up to 51 h of incubation (i.e. late logarithmic growth phase). The antifungal activity then sharply decreased and remained constant up to 96 h of incubation (Fig. 2). This suggests that emulsifying and antifungal activities of B. subtilis K1 are independent of each other and may be attributed to different metabolites with varying production profiles.

Figure 2.

Profile of growth and production of extracellular antifungal as well as emulsifying activity by Bacillus subtilis K1. (image_n/jam12161-gra-0001.png) Growth; (image_n/jam12161-gra-0002.png) Antifungal activity and (image_n/jam12161-gra-0003.png) Emulsifying activity.

Antifungal activity spectrum of methanolic extracts from endophytic bacilli

The methanolic extracts of isolates K1, A2, A4, A11, A12 and A13 exhibited antifungal activity against almost all the test fungi, while A32 inhibited only A. flavus, A. brunsii (1 and 2), C. indicum and C. herbarum 1112. The methanolic extracts obtained from all seven bacilli exhibited similar spectrum (Table 1) of activity against all the test fungi as demonstrated by in vitro co-culturing antagonism assay of endophytic bacilli against test fungal cultures (Fig. S1). Among the seven isolates, methanolic extract of B. subtilis K1 exhibited maximum antifungal activity, hence was selected for further studies.

Influence of antifungal extract on germinability and morphology of A. niger conidiospores and mycelia

To determine the effect of lipopeptides produced by B. subtilis K1 on germination and morphology of conidiospores of A. niger 40211, the conidiospores were incubated with different dilutions of CFC supernatant made in distilled water. The addition of conidiospores to 10, 25 and 50% (v/v) of CFC supernatant resulted in inhibition of A. niger conidiospores germination by 80, 89 and 96%, respectively. The treatment of A. niger mycelia with 50% (v/v) CFC supernatant for 3 h resulted in the extensive damages in the mycelia, and upon prolonged incubation for 8 h, complete destruction of mycelia was observed.

Intact-Cell MALDI-TOF mass spectrometry of banyan endophytic bacilli

In this study, MALDI-TOF mass spectrometry technique was applied to investigate the secondary metabolites produced by all seven endophytic bacilli using intact cell as a target. The ICMS of all seven endophytic bacilli showed mass peaks ranging from m/z 551·0 to m/z 2047·3 (Fig. 3, Tables 2-4). The surfactins (m/z, 979 to 1096·8), iturins (m/z, 1014·5–1123·5) and fengycins (m/z, 1422·2–1558·2), which represent the well-known families of cyclic lipopeptides produced by Bacillus sp. (Vater et al. 2002; Pabel et al. 2003; Pyoung et al. 2010), could be assigned putatively in the mass spectra of banyan endophytic isolates. On the basis of mass spectral profile, five isolates viz., B. subtilis K1, B. subtilis A2, B. subtilis A4, B. amyloliquefaciens A11 and B. subtilis A12 seemed to produce higher proportion of iturin homologues in comparison with fengycins and surfactins. All these five isolates produced fengycin homologues, but the intensities of fengycin m/z peaks were significantly lower in comparison with the intensity of iturin peaks (Fig. 3) (Tables 2-4). Similarly on the basis on MALDI-TOF MS data, isolate A32 seemed to produce higher proportion of fengycins in comparison with surfactins and iturins (Tables 2-4). Furthermore, peaks at m/z 1220·9, 1234·0 and 1248·0 observed in ICMS spectra of isolates B. subtilis A2 (ionization intensity, 200–600), B. amyloliquefaciens A13 (ionization intensity, 0–400) and Bacillus sp. A32 (ionization intensity, 0–50) differed from each other by 14 Da, suggesting that the corresponding metabolites belonged to the same family varying from each other in mass by multiples of 14 Da. The peak at m/z 1270·0 was putatively assigned as sodium adduct of m/z 1248·0. The mass peaks at m/z 1220·9 to 1270·0 could not be assigned as compounds with similar masses have not been reported in the literature from Bacillus sp. analysed by ICMS. The mass peaks at m/z 551·0, 614·7 and 660·8 in ICMS of A13 (ionization intensity, 100–500) also could not be assigned due to similar reasons. These unassigned m/z peaks may belong to new molecules produced by the strains of endophytic bacilli, but owing to their low intensity, it was difficult to select and fragment them further for structural elucidation. On the basis of ICMS profile, the similarity coefficients among these isolates were determined and used to construct a dendrogram (Fig. 4). The similarity coefficients of B. subtilis A2, B. subtilis A4, B. amyloliquefaciens A11, B. subtilis A12, B. amyloliquefaciens A13 and Bacillus sp. A32 with B. subtilis K1 were calculated to be 0·64, 0·55, 0·50, 0·51, 0·64 and 0·54, respectively.

Table 2. Putative assignment of mass peaks to iturins from intact-cell MALDI-TOF mass spectrometric analysis (ICMS) spectra of banyan endophytic bacilli cells
Putative assignments of cyclic lipopeptideMass peak (m/z)Banyan endophytic bacilli
K1A2A4A11A12A13A32
  1. Ionization intensity of ions belonging to putative iturins in ICMS spectra: 1000–6000 (K1), 1000–5000 (A2), 100–400 (A4), 2500–12 500 (A12), 200–800 (A11), 100–500 (A13), 100–600 (A32).

C12 Iturin [M+H+]1014·6+
C13 Iturin [M+H+]1028·9++
C14 Iturin [M+H+]1043·6+++
C15 Iturin [M+H+]1057·6++++
C16 Iturin [M+H+]1071·7++
C17 Iturin [M+H+]1084·7++
C14 Iturin [M+Na+]1065·6++
C15 Iturin [M+Na+]1079·7++
C17 Iturin [M+Na+]1107·7+++
C18 Iturin [M+Na+]1121·7+++
C19 Iturin [M+Na+]1134·7+
C20 Iturin [M+Na+]1150·8++
C21 Iturin [M+Na+]1165·9+++
C 15 Iturin [M+K+]1095·7+++++
C 17 Iturin [M+K+]1123·8+++
Table 3. Putative assignment of mass peaks to surfactins from intact-cell MALDI-TOF mass spectrometric analysis (ICMS) spectra of banyan endophytic bacilli cells
Putative assignments of cyclic lipopeptideMass peak (m/z)Banyan endophytic bacilli
K1A2A4A11A12A13A32
  1. Ionization intensity of ions belonging to putative surfactins in ICMS spectra: 200–1000 (K1), 250–1000 (A2), 100–350 (A4), 200–1000 (A12), 200–600 (A11), 100–400 (A13), 100–400 (A32).

C11 Surfactin [M+H+]979·6+++
C12 Surfactin [M+H+]995·5 ++++
C13 Surfactin [M+H+]1008·6++++
C14 Surfactin [M+H+]1022·9+++
C15 Surfactin [M+H+]1036·7+++
C20 Surfactin [M+H+]1106·6+++
C11 Surfactin [M+Na+]1002·5++
C12 Surfactin [M+Na+]1017·6+++
C13 Surfactin [M+Na+]1030·5++++
C14 Surfactin [M+Na+]1044·9+++
C15 Surfactin [M+Na+]1059·0+++
C18 Surfactin [M+Na+]1102·9++
C14 Surfactin [M+K+]1060·6++
C15 Surfactin [M+K+]1074·9++++
Table 4. Putative assignment of mass peaks to fengycins from intact-cell MALDI-TOF mass spectrometric analysis (ICMS) spectra of banyan endophytic bacilli cells
Putative assignments of cyclic lipopeptideMass peak (m/z)Banyan endophytic bacilli
K1A2A4A11A12A13A32
  1. Ionization intensity of ions belonging to putative fengycins in ICMS spectra: 200–2000 (K1), 200–1250 (A2), 20–80 (A4), 200–800 (A12), 200–1000 (A11), 20–80 (A13), 200–1200 (A32).

Fengycin [M+H+]1422·2+
Fengycin [M+H+]1436·1++
Fengycin [M+H+]1450·1++++++
Fengycin [M+H+]1464·1+++++++
Fengycin [M+H+]1478·2+++++++
Fengycin [M+H+]1492·2++++++
Fengycin [M+H+]1506·2++++++
Fengycin [M+Na+]1472·1++
Fengycin [M+Na+]1500·1+++
Fengycin [M+Na+]1514·1++
Fengycin [M+Na+]1528·6++
Fengycin [M+K+]1488·0++
Fengycin [M+K+]1502·6+++
Fengycin [M+K+]1516·1++++
Fengycin [M+K+]1530·2+++++
Fengycin [M+K+]1544·4++++
Figure 3.

Intact-cell MALDI-TOF mass spectrometric analysis analysis of (a) Bacillus subtilis K1, (b) B. subtilis A2, (c) B. subtilis A4, (d) B. subtilis A12, (e) Bacillus amyloliquefaciens A11, (f) B. amyloliquefaciens A13, (g) Bacillus sp. A32 and (h) isolated from banyan aerial roots. The ICMS spectra of each of these bacilli are expanded in two clusters, m/z 900–1350 and m/z1400–1620.

Figure 4.

Dendrogram based on similarity coefficient of intact-cell MALDI-TOF mass spectrometric analysis analysis of seven endophytic bacilli.

Discussion

Most of the studies on bacterial endophytes have been focused on agriculturally and medicinally important plants (Naik et al. 2009; Bhagat et al. 2012; Slimene et al. 2012), while the literature on bacterial endophytes from woody trees is sparse (Wang et al. 2006). To search for endophytic bacteria with their potential to produce bioactive metabolites, aerial roots of the banyan tree were selected for the isolation of endophytic flora with the ability to produce bioactive compounds. The present study describes characterization of banyan aerial root endophytic isolates and their antifungal metabolites. Seven endophytic bacilli identified as B. subtilis K1, B. subtilis A2, B. subtilis A4, B. amyloliquefaciens A11, B. subtilis A12, B. amyloliquefaciens A13 and Bacillus sp. A32 were isolated from the aerial roots of banyan tree. In previous study, presence of rod-shaped bacterial cells in the parenchymatous as well as vascular tissues of aerial roots of banyan tree were demonstrated by both transmission electron microscopy and light microscopy using tetrazolium vital staining (Pathak et al. 2009). Among all isolates, B. subtilis K1 was found to exhibit antagonistic activity against all the test fungi used in this study, viz. A. niger 40211, A. niger 16404, A. niger 181, A. flavus, C. indicum, M. indicus, F. oxysporum f. sp. lycopersicii, F. oxysporum f. sp. gingiberi, F. oxysporum 1072, C. albicans, A. brunsii 1, A. brunsii 2, C. herbarum 1112, Srolfsii and L. thoebromae ABFK1. In the antagonism assay of endophytic bacilli against susceptible fungi especially A. niger strains, the precipitation line was observed around the inhibitory zone between bacterial and fungal growth. This precipitation line might be a result of antibiotic products secreted by endophytic bacilli to inhibit the fungal growth. Similar observations have been documented by Cornea et al. (2003) in their in vitro antifungal assays of Bacillus sp. B209 against Sclerotinia sclerotiorum. Similar broad-spectrum antifungal activity was also reported for a bacterial endophyte B. amyloliquefaciens ES-2, isolated from Scultellaria baicalensis Georgi, against A. niger, A. flavus, Aspergillus ficuum, Aspergillus oryzae, Mucor wuntungkiao, Fusarium culmorum, F. oxysporum, Maganporthe grisea and Botryodiplodia theobromae (Sun et al. 2006). The most potent bacterial antagonist, B. subtilis K1, was selected for further characterization of fungal antagonism. The culture supernatant of B. subtilis K1 displayed antifungal activity as well as emulsifying activity.

In presence of culture supernatant of B. subtilis K1, germination inhibition of A. niger 40211 conidiospores occurred, and upon prolonged incubation, even germinated mycelia were found to be completely destroyed. These observations confirmed the ability of B. subtilis K1 to secrete functionally stable antifungal metabolites. To identify the secretary metabolites produced by all the endophytic bacilli, ICMS was employed. From the ICMS analysis, all seven endophytes were found to produce cyclic lipopeptides, surfactins (m/z 979 to 1096·8), iturins (m/z, 1014·5–1123·5) and fengycins (m/z, 1422·2–1558·2). The cyclic lipopeptide produced by Bacillus sp. exhibits microheterogeneity in the peptide moiety as well as in the fatty acid tail (Ongena et al. 2007). The similarity coefficient values derived on the basis of ICMS profile for all seven bacilli suggests that it can be used as an important tool in differentiating strains of bacilli from each other. The isolates B. subtilis K1, B. subtilis A2, B. subtilis A4, B. amyloliquefaciens A11, B. subtilis A12 and B. amyloliquefaciens A13 exhibited higher heterogeneity as well as intensity for mass peaks corresponding to iturins and fengycins, in comparison with isolate A32, which may be correlated with their spectrum as well as potency of antifungal activity. The Bacillus sp. A32, which produced more of fengycins and surfactins, exhibited relatively weaker antifungal activity with narrow spectrum. The intensity of mass peaks assigned as surfactins, iturins and fengycins in ICMS of B. subtilis K1 was significantly higher in comparison with the intensity of corresponding peaks in ICMS of other six isolates, which again correlates well with its higher potency as well as the spectrum of antifungal activity.

Iturin, a cyclic heptapeptide with β-amino fatty acid, is known for its strong antifungal and haemolytic activity, while fengycin, a cyclic depsipeptide with 10 amino acids and β-hydroxy fatty acid tail, possesses a strong antifungal activity specific to filamentous fungi with very limited haemolytic activity (Winkelmann et al. 1983; Vanittanakom et al. 1986). The fengycins are classified into two classes, fengycin A (Ala6) and fengycin B (Val6), based on variation in amino acid residue 6. Surfactin is a cyclic heptapeptide with β-hydroxy fatty acid as lipid moiety, which is known for its excellent surface activity and other biological activities such as antiviral, antitumor, mosquitocidal (Sing and Cameotra 2004; Geeta et al. 2010). According to the literature, most strains of bacilli have been reported to produce cyclic lipopeptides of a single family (Vanittanakom et al. 1986; Bais et al. 2004). Nevertheless, there are reports of Bacillus sp. producing mixture of lipopeptides belonging to two different families such as surfactins + iturins (Ohno et al. 1995) or iturins + fengycins (Pryor et al. 2007) or fengycins + surfactins (Sun et al. 2006; Ongena et al. 2007). However, reports of bacilli co-producing lipopeptides of surfactin, iturin as well as fengycin families, with high degree of microheterogeneity, are sparse (Arguelles-Arias et al. 2009; Pyoung et al. 2010; and Nihorimbere et al. 2012). More significantly such strains have been found to exhibit a broader range as well as a higher potency of antifungal activity, suggesting synergism among members of different families of cyclic lipopeptides towards antifungal activity (Thimon et al. 1992; Ongena et al. 2007). The intensity of mass peaks assigned as surfactins, iturins and fengycins in ICMS of B. subtilis K1 was significantly higher in comparison with the intensity of corresponding peaks in ICMS of other six isolates, which again correlates well with its higher potency as well as the spectrum of antifungal activity.

Bacillus subtilis K1 was found to inhibit all test fungi used in this study. The detail mass spectrometric characterization of crude antifungal extract of B. subtilis K1 revealed the presence of seven iturins, seven surfactins and 82 fengycins. Among the 82 fengycin sequences, 62 new fengycins belonging to three new classes, fengycin A2, fengycin B2 and fengycin C, were discovered. Based on the variation of Glu/Gln at position 8, a new subclass of fengycin was also identified using high-resolution mass spectrometry (Pathak et al. 2012). It is noteworthy to mention here that all the banyan endophytic bacilli exhibiting antifungal activity were found to be co-producers of surfactins, iturins and fengycins. This implies that these organisms must be playing a definite biological role while residing as endophytes in the banyan aerial roots, which would be worth investigating. The aerial roots of banyan tree have been investigated for isolation and community studies of fungal endophytes by Suryanarayanan and Vijaykrishna (2001); however, to the best of our knowledge, this is the first report on isolation and ICMS analysis of bacterial endophytes, exhibiting potent antifungal activity, from aerial roots of the banyan tree.

Conclusion

The present work describes the fungal antagonistic properties of banyan endophytic bacilli. The ICMS approach used in this study provided quick identification of antifungal metabolites produced by all seven isolates without any requirement of downstream processing. The broad-spectrum antifungal activity of B. subtilis K1 suggests its prospective application as potent biocontrol agent against fungal plant pathogens.

Acknowledgements

The authors are thankful to University Grants Commission (UGC), New Delhi, Government of India, for funding this work. We are thankful to Prof. P. Balaram, Molecular Biophysics Unit, Indian Institute of Science, Bangalore, for providing MALDI-TOF mass spectrometer facility for intact-cell MS analysis of banyan endophytes.

Conflict of Interest

The authors declare that they have no conflict of interest.

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