Diffusible and volatile compounds produced by an antagonistic Bacillus subtilis strain cause structural deformations in pathogenic fungi in vitro

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Microbiological Research 160 (2005) 75—81

KEYWORDBacillus suDiffusiblecompoundVolatile coPathogenic

0944-5013/$ - sdoi:10.1016/j.

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Diffusible and volatile compounds produced by anantagonistic Bacillus subtilis strain cause structuraldeformations in pathogenic fungi in vitro

Bhaskar Chaurasiaa, Anita Pandeya,�, Lok Man S. Palnib, Pankaj Trivedia,Bhavesh Kumara, Niharika Colvinc

aG B Pant Institute of Himalayan Environment and Development Kosi-Katarmal-Almora, 263 643 Uttaranchal, IndiabState Biotechnology Programme, Biotech Bhavan, P.O.-Haldi, Pantnagar (US Nagar) 263 146 Uttaranchal, IndiacApollo Hospitals, Bilaspur, 495 001 Chhattisgarh, India

Accepted 30 September 2004

Sbtilis;

s;mpounds;fungi

ee front matter & 200micres.2004.09.013

ing author.ess: [email protected]

SummaryAn efficient antagonistic strain of Bacillus subtilis, originally isolated from therhizosphere of established tea bushes, was found to cause structural deformities in sixpathogenic fungi under in vitro culture conditions. This effect was attributed to theproduction of diffusible and volatile antifungal compounds. Out of the selected testfungi four were phytopathogenic, while the remaining two were of clinicalimportance. The bacterial strain successfully restricted the growth of all test fungiin dual cultures, and induced morphological abnormalities such as mycelial andconidial deviations. The inhibitory effect caused by volatiles was greater than that bydiffusible compounds.& 2004 Elsevier GmbH. All rights reserved.

Introduction

An important step in disease control programmesinvolves selection of effective biocontrol agents.Bacillus species including Bacillus subtilis areknown for their antifungal properties, hence theirimportance in the biological control of a number ofplant and animal diseases (Broadbent et al., 1977;

4 Elsevier GmbH. All rights rese

ic.in (A. Pandey).

Fravel, 1988; Weller, 1988; Milner et al., 1996;Pandey et al., 1997; Ryder et al., 1999; Whipps,2001). Three mechanisms of biocontrol involvecompetition, parasitism or predation, and antibio-sis (Baker, 1968). Upadhyay and Jayaswal (1992)had suggested the induction of morphologicalabnormalities and inhibition of conidiation inphytopathogenic fungi by an antagonistic bacterium

rved.

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B. Chaurasia et al.76

(Pseudomonas cepacia) as a possible mechanism ofbiological control.

Based on a long term study on the rhizospheremicrobiology of tea, involving a number of teagardens of Indian Himalayan region, several strainsof Bacillus spp. were isolated and screened for theirantifungal activity against a range of saprophytic andpathogenic fungi (Pandey and Palni, 1997; Pandey etal., 1997). This investigation characterises one of theselected strains of B. subtilis (Pandey and Palni,1997) with respect to antagonistic activity andinduction of structural abnormalities caused byproduction of diffusible and volatile compounds, ina number of fungi in culture.

Materials and methods

Microorganisms

The pathogenic fungi Alternaria alternata, Clados-porium oxysporum, Fusarium oxysporum, Paecilo-myces lilacinus, Paecilomyces variotii, and Pythiumafertile were isolates from soil samples collectedfrom temperate and alpine forests of UttaranchalHimalaya. The test fungi are known to causediseases (F. oxysporum: Fusarium wilt and rots,P. afertile: damping off of seedlings and Pythiumblight, A. alternata: leaf spot and leaf blight,C. oxysporum: fruit and crop rots, P. lilacinus andvariotii: various human diseases.) (Bilgrami et al.,1991; Dhindsa et al., 1995; Fletcher et al., 1998).B. subtilis (NRRL B-30408) was isolated from soilsamples collected from the rhizosphere soil ofestablished tea bushes (Pandey and Palni, 1997).Fungal and bacterial isolates were maintained at4 1C by repeated subculture on potato dextrose andtryptone yeast extract agar slants, respectively.

Antagonism assays

For examining antagonism due to diffusible com-pounds, a fungal lawn of the test fungus was grownon potato dextrose agar plates. Discs of 7mmdiameter from the fungal lawns were cut andinoculated on carrot potato agar plates. A sterilisedpaper disc with 5mm diameter, dipped in yeastextract broth containing the bacterial culture(108 cfuml�1) was inoculated about 2.0–2.5 cm awayfrom the fungal disc. The plates were incubated ininverted position at 28 1C in the dark. The observa-tions were recorded after 24, 72, and 120h incuba-tion by measuring the growth of the fungus towardsand away from the bacterial colony. Inhibition offungal growth was calculated using the formula:

ðR1� R2=R1Þ � 100: R1 (a control value) representsthe largest radial distance grown by the fungus in thedirection of the antagonist and R2 represents thedistance on a line between the inoculation positionsof the fungus and the bacteria.

Antagonism due to volatile compounds wasevaluated by preparing a bacterial lawn on tryp-tone yeast extract agar plates. After incubation for24 h, the lid was replaced by a plate containingagar blocks of 7mm diameter with the test fungusgrown on carrot potato agar. The two plates weresealed by parafilm. Control sets were preparedwithout bacteria in the bottom plate. Petri disheswere incubated at 28 1C, and observations wererecorded after 24, 72, and 120 h. The % growthinhibition of the test fungus was calculated usingthe formula: ðr12r2=r1Þ � 100; where r1 (a controlvalue) represents the radial growth of the fungus incontrol sets without bacteria, and r2 represents theradial growth of the fungus in sets inoculated withthe bacterium. The experiments were conducted intriplicate.

Microscopic observations

After 120 h incubation, fungal samples were takenfrom areas showing inhibition due to antagonisticactivity of the bacterial strain, and stained withlactophenol-cotton blue, subjected to microscopicexamination to record structural abnormalities.Samples from control plates without bacterialinfluence were also stained and observed.

Enumeration of antagonised fungi. A 4mm diaagar block with fungal growth was taken from theantagonised (showing inhibition on plates in both‘‘diffusible’’ and ‘‘volatile’’ sets) and control platesafter 120 h incubation. The agar block with thefungus was serially diluted and plated on potatodextrose agar plates. The plates were incubatedat 28 1C and counts were recorded after threedays. The percent inhibition in colony formingunits (cfus) was calculated by using the formulaðCI� C2=C1Þ � 100 where C1 (a control value)represents the cfus in control plates and C2represents the cfus in antagonised plates. Experi-ments were conducted in triplicate.

Results and discussion

Table 1 presents the effect of diffusible and volatilecompounds produced by B. subtilis, evaluated interms of reduced radial growth of the test fungus.Bacterial suppression of fungal growth was evidentafter 24 h incubation. The values ranged from

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Table 1. Inhibition in radial growth of pathogenic fungi caused by diffusible or volatile compounds produced byBacillus subtilis

Pathogen Per cent inhibition in radial growth

24 h 72 h 120 h

Diffusible Volatile Diffusible Volatile Diffusible Volatile

Alternaria alternata 39.1070.72 20.0070.10 49.5070.19 60.0070.11 71.7070.22 65.2070.10Cladosporium oxysporum 27.7070.50 15.9070.14 46.0070.11 41.0070.08 53.3070.16 52.1070.98Fusarium oxysporum 17.5070.20 34.4070.04 45.3070.98 40.9070.24 66.1070.17 60.0070.12Paecilomyces lilacinus 35.0070.21 20.0070.11 50.0070.16 42.1070.22 67.0070.19 52.1070.22Paecilomyces variotii 26.5070.08 33.3070.16 48.1070.09 46.8070.25 59.2070.98 62.8070.19Pythium afertile 29.2070.05 30.0070.09 35.1070.11 84.0070.18 67.9070.18 84.0070.18

n=3, 7 SE

Figure 1. (A) Normal hyphae and conidia of A. alternata (control), (B) hyphal and conidial deformations in A.alternata, (C) normal structures of C. oxysporum, and (D) abnormal conidiophores of C. oxysporum (bar=10 mm).

Deformations in pathogenic fungi in vitro 77

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17.5070.20% (F. oxysporum) to 39.1070.72% (A.alternata) due to the secretion of diffusiblecompounds, and from 15.9070.14% (C. oxysporum)to 34.4070.04% (F. oxysporum) due to the produc-tion of volatile compounds. The inhibition of fungalgrowth continued to increase with longer incuba-tion time. Fungal inhibition after 120 h incubation,due to diffusible compounds of B. subtilis, rangedbetween 53.3070.16% (C. oxysporum) and71.7070.22% (A. alternata), and for volatilesubstances between 52.1070.98% (C. oxysporumand P. lilacinus) and 84.0070.18% (P. afertile).

Microscopic observations revealed that diffusibleas well as volatile substances induced similarmorphological abnormalities in fungal structures.

Figure 2. (A) Normal hyphae and macroconidia of F. oxysporuF. oxysporum, (C) normal structures of P. afertile, and (D) vafertile (bar=10 mm).

Deformation in mycelial, hyphal or conidial struc-tures was common in all fungi tested. Thetransverse as well as longitudinal septae comple-tely disappeared in A. alternata, and the conidiabecame thick walled and spherical or irregularin shape (Figs. 1A, B). In various instancesconidia formation was arrested, and only vegeta-tive mycelium developed. In C. oxysporum con-idiophores became vegetative and stunted;formation of normal conidia was also hampered inthis case (Figs. 1C, D). Lysis of fungal hyphae, andvacuolisation as well as granulation in mycelialstructures was observed in F. oxysporum. In thiscase, the conidia became swollen and thick-walled(Figs. 2A, B). Similarly in P. afertile, lysis of fungal

m, (B) swelling of hyphal tip and deformation of conidia inacuolation and swelling in hyphae and sporangium of P.

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Figure 3. (A) Normal conidiophores of P. lilacinus, (B) deformed conidiophores of P. lilacinus, (C) normal conidiophoresof P. variotii, and (D) prominent swelling seen in the conidiophores of P. variotii (bar=10 mm).


hyphae, vacuolisation, and granulation of mycelialstructures were observed too. Formation of normalsporangia and oogonia was found to be suppressedin P. afertile (Figs. 2C, D). Morphological abnorm-alities were also recorded in both species ofPaecilomyces; swollen and broad conidiophoresdue to mycelial vacuolisation were common toboth, P. lilacinus and P. variotii (Figs. 3A–D). Adetailed account on normal and deformed fungalstructures is presented in Table 2.

Evaluation of antagonised fungi

Experiments on cfus developed from antagonisedfungal growth revealed that volatile compounds

caused greater inhibition than the diffusibles(Table 3). While the inhibition in cfus ranged from23.80% (C. oxysporum) to 78.37% (A. alternata) dueto diffusible compounds, it ranged between 31.74%(C. oxysporum) and 99.15% (P. lilacinus) due tovolatile compound(s).

Antagonism is known to be mediated by a varietyof compounds of microbial origin, e.g., bacterio-cins, enzymes, toxic substances, volatiles, andothers. The effect of volatile compounds hasreceived only limited attention in comparison tothe antagonism affected by diffusibles. In thepresent study, both diffusible as well as volatilecompounds produced by B. subtilis were found toinduce qualitatively similar morphological abnorm-alities in the fungal structures; quantitative effectsvaried.

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Table 2. Comparative account of structures of normal and antagonised pathogenic fungi

Pathogen Structure Normal Antagonised

Alternariaalternata

Mycelium Somatic and fertile hypha welldistinguished

Not distinguished

Somatic hypha Septate, dia. 2.5–3.5mm Irregularly septate, dia4.0–10.0 mm

Fertile hypha Septate and dark in colour, dia.3.0–8.0mm

Irregularly septate, dia.4.0–10.0 mm

Conidia Narrowly ellipsoid to ovoid orelongated with or without shortconical beak, 10–30� 5–12 mm,septate, 3–7 transverse and 1–5longitudinal septa

Spherical to irregular, dia.7–16 mm, irregularly septate orseptae absent

Cladosporiumoxysporum

Mycelium Somatic hypha septate, dia.2.0–4.0mm

Somatic hypha septate, dia.2.0–4.0mm

Conidiophore Branched, brush like, dia.3.0–4.0mm

Branching irregular, dia.3.0–4.0mm

Conidia Spherical, dia. 1.5–3.0mm, conidiain chains

Spherical, dia. 1.5–3.0 mm, chainsformed abruptly

Fusariumoxysporum


Somatic hypha septate, dia.2–4mm

Microconidia Ellipsoid, curved or straight,3–4� 2–2.5 mm, 1-2 celled

Broader and irregular,6–8� 4–5mm

Macroconidia Gradually curved and pointedtowards the ends, 12–18� 2–4 mm,3–4 celled

Broader and irregular,12–20� 5–10 mm, reduced orsometimes dissolved

Paecilomyceslilacinus


Septation irregular, dia.3.0–6.0mm

Conidiophore Branched, brush like, dia. 2–4 mm Branching abnormal, dia. 3–7 mmPhialides Flask-shaped with small beak, no.

2–7Irregular, no. 2–3

Conidia Spherical, dia. 2–3 mm Spherical, dia. 2–3 mm

Paecilomycesvariotii


Somatic hypha septate, dia.3.0–8.0mm

Conidiophore Branched, dia. 3–7mm Branched, dia. 3–8 mmPhialides Swollen at the base and gradually

narrowed into a long beak,8–12� 2–2.5 mm, no. 2–4.

Broader at the base withshortened beak, 3–8� 2–4 mm, no.1–3.

Conidia Ovoid to spherical, dia. 2–.5mm Ovoid to spherical, dia. 2–3.5mm

Pythium afertile Mycelium Somatic hypha coenocytic, dia.2.0–8.0mm

Somatic hypha coenocytic, dia.5.0–10.0 mm

Sporangium Spherical, dia. 6–20 mm Irregular, 10–13� 20 35 mmOogoinum Spherical, dia. 10–30 mm Spherical to irregular,

10–13� 20–35 mm


Rhizosphere associated microorganisms are con-sidered ideally suitable for biocontrol. Establishedtea (Camellia sinensis) has been reported to exert anegative rhizosphere effect and to harbour largepopulations of antagonists (Pandey and Palni, 1996,2002–2003). Bacillus species, B. subtilis in parti-cular, have been found to be dominant bacteriaassociated with tea roots (Pandey and Palni, 1997).The present study further confirms the importance

of established natural rhizospheres for isolation,screening and selection of efficient biocontrolagents. Such agents exhibit antagonistic effectson various pathogenic fungi and could be useful fordisease management programmes. The bacterialstrain used in the present study has already proveneffective in biological hardening of plants, raised intissue culture, mainly by controlling fungal wiltcaused by F. oxysporum (Pandey et al., 2000, 2002).

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Table 3. Comparative account of colony forming units developed from the control and antagonised pathogenic fungi

Pathogens Control Diffusible Volatile

cfu from controlplates

cfu fromantagonisedplates

% Inhibition cfu fromantagonisedplates

% Inhibition

Alternaria alternata 37.0072.35 8.0070.81 78.37 3.5070.23 90.54Cladosporiumoxysporum

126.0072.82 96.0070.94 23.80 86.0072.82 31.74

*Fusarium oxysporum 34.0070.94 12.0073.29 64.70 2.5070.32 92.64Paecilomyces lilacinus 237.5071.17 102.0072.82 57.05 2.0070.47 99.15Paecilomyces variotii 15.0071.41 8.0070.94 46.66 2.0070.74 86.66Pythium afertile 13.5070.23 3.0070.23 77.77 1.5070.23 88.88

*cfu at n� 105 cells=ml; in all other cases cfu ¼ n� 103 cells=ml; n ¼ 3; 7SE.


Acknowledgements

The Department of Biotechnology, the Council ofScientific and Industrial Research and the UnionMinistry of Environment and Forests, Govt. of India,New Delhi are thanked for financial support.

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Diffusible and volatile compounds produced by an antagonistic Bacillus subtilis strain cause structural deformations in pathogenic fungi in vitro