Biological control of cabbage head rot (Sclerotinia sclerotiorum) by Coniothyrium minitans

Abstract

Background

Sclerotinia sclerotiorum is the causal agent of white mould, stem and head rot diseases on a wide variety, including cabbage in the field and storage. Control of this pathogen by using commercial disease management methods is extremely difficult. Therefore, this study was performed to identify the potential biological control agent.

Results

The antagonist fungal isolates, a mycoparasite of S. sclerotiorum, were isolated and described as KET Cm1, PAL Cm 2 and TDK Cm 3. They were subjected to morphological and microscopic observation. The growth of antagonist fungal isolates on different media revealed that PDA and half-strength Czapek dox agar medium showed the maximum mycelial growth (28.67 and 28.33 mm) and pycnidial production in the PDA medium. The studies on pH and temperature found that growth and sporulation of the antagonist fungal isolates were optimum at pH 5.5 (34 mm) and 20 °C. The molecular confirmation of antagonist fungal isolates was carried out using ITS 1 and ITS 4 primers, which target 18S-28S rRNA gene fragment. The isolates were partially sequenced and showed that more than 99% identity with Coniothyrium minitans already exist in the NCBI database. The accession numbers (MW093134, MW111282 and MW136938) were obtained for all the three isolates and also confirmed using specific primers Cm sp 1F and Cm sp 1R. Among the tested isolates, the antagonistic activity of C. minitans TDK Cm 3 showed the maximum mycelial growth inhibition of 48.8% S. sclerotiorum and was used for further studies. Penetration and formation of appressoria on the hyphae of S. sclerotiorum by C. minitans isolate TDK Cm 3 were observed through the scanning electron microscope. The ethyl acetate fractions of TDK CM 3 isolate were evaluated against S. sclerotiorum by agar well diffusion assay, paper disc assay and poison food technique in vitro. Through GC–MS analysis, the effective antifungal compounds were identified.

Conclusion

The present study focused on the identification and characterization of effective Coniothyrium isolates as eco-friendly, integrated disease management strategies against cabbage head rot disease.

Background

In general, control of the soil-borne diseases is difficult. Continuous use of fungicides leads to the development of resistance among the pathogen. Soil application of fungicides may imbalance microbial communities in the soil and also suppress the beneficial soil micro-flora (Sankar and Jeyarajan 1996). Indiscriminate use of fungicides leads to the environment and human health hazards. Biological control is the alternate and sustainable research for the control of plant diseases. Under field conditions, sclerotia are attacked and degraded by a number of mycoparasites such as Coniothyrium minitans and Sporidesmium sclerotivorum and they have been proposed as biocontrol agents for Sclerotinia sclerotiorum (Lib.) de Bary (Adams and Ayers 1981). C. minitans was discovered from sclerotia in harvested samples, suggesting that this mycoparasite has the potential in reducing the survival of sclerotia in the field (Huang et al. 2000). C. minitans and Gliocladium virens have shown a practical potential for biological control of S. sclerotiorum under field conditions (Budge et al. 1995).

C. minitans (Paraconiothyrium minitans) was firstly described for biological control of S. sclerotiorum in California (Campbell 1947). It is a sclerotial mycoparasite and is isolated from sclerotia in soil (Sandys-Winsch et al. 1993). It has a good ability to degrade the sclerotia in soil and has a potential to control S. sclerotiorum by decreasing carpogenic germination and viability of sclerotia (Jones and Whipps 2002). The mode of action of C. minitans includes mycoparasitism, production of antibiotics and secondary metabolites, competition for space, nutrients and induction of defence responses including systemic resistance responses in the plant and reduction in the viability of carpogenic germination of sclerotia. Currently, biological control is getting great attention due to eco-friendly management and low-cost technology.

In the present study, an attempt was made to isolate and characterize the antagonist activity of C. minitans from the S. sclerotiorum sclerotia against cabbage head rot diseases.

Methods

Isolation of Coniothyrium minitans

Coniothyrium minitans was isolated from baiting the sclerotia in the autoclaved sand (Sandys-Winsch et al. 1993). The mycelial suspensions of S. sclerotiorum were sprayed over the surface-sterilized sclerotia collected from the white mould-infected cabbage, because the antagonist was found as mycoparasite in the sclerotia of S. sclerotiorum. Then, the sclerotia was placed in Petri plates containing 10 g of autoclaved sand. The plates were incubated for 40–45 days at 20 °C. In order to maintain the adequate humidity, 8 ml of sterile distilled water was poured into the Petri plates at weekly intervals. Some of the sclerotia showed the minute growth of fungal mycelium along with pycnidial production on the sclerotial surface (Campbell 1947). The mycelium along with pycnidia were grown on the PDA medium and assumed to be C. minitans (Phaeosphaeria minitans). The isolated C. minitans isolates were subjected to morphological and molecular identification.

Standardization of cultural media and temperature for the growth of Coniothyrium sp.

Eight culture media, viz. Potato dextrose agar, Oat meal agar, Carrot dextrose agar, Malt extract agar, Czapek dox agar, half-strength Czapek dox agar, Water agar and V8 Juice agar, were selected to identify the best media and temperature like 20 °C and room temperature (28 ± 2 °C) for growth of the Coniothyrium sp. All the media were prepared and sterilized at 15 psi for 30 min, and a 20 ml of sterilized warm medium was poured in sterilized Petri dishes and allowed for solidification. A 5-mm-diameter culture disc of 15-day-old culture of Coniothyrium sp. was placed at the centre of the plate and incubated at both 20 °C and room temperature (28 ± 2 °C). Three replications were maintained for each medium and radial growth of the fungus was measured at 10-day intervals up to 60 days after inoculation.

Effect of pH on the growth of Coniothyrium sp.

Sterilized PDA medium was distributed to 250-ml Erlenmeyer flasks at the rate of 100 ml per flask and the pH of the medium was adjusted to levels of 4.0, 4.5, 5.0, 5.5, 6.0 and 6.5 with 0.1 N HCl or 0.1 N NaOH and autoclaved at 1.4 kg cm⁻² for 20 min. Fifteen ml of the medium from each pH level was poured onto sterilized Petri dishes and allowed to solidify. A five mm culture disc of actively growing mycelium was placed at the centre of each Petri plate under an aseptic condition. The plates were incubated at 20 °C to observe the mycelial growth of the biocontrol agent.

Cultural and morphological characterization of Coniothyrium minitans

The isolates of Coniothyrium minitans were grown on Potato dextrose agar, Oat meal agar, Carrot dextrose agar, Malt extract agar, Czapek dox agar, half-strength Czapek Dox agar, Water agar, V8 Juice agar medium to study the cultural, morphological and pycnidial characters. From the 15-day-old culture plate, a 5-mm mycelial disc of fungus was cut by a sterilized cork borer and placed at the centre of each sterile Petri dish containing 20 ml of sterilized and solidified medium. The plates were incubated at 20 °C for 30 days. C. minitans mycelia, pycnidia and pycnidiospores characteristics were studied.

Molecular characterization of Coniothyrium sp. using ITS 1 and ITS 4 primers

The 20-day-old culture of Coniothyrium sp. was transferred into 250-ml Erlenmeyer flasks containing 150 ml PDB and incubated at 20 °C for 20 days. The mycelial mat was harvested and stored at -70 ͦ C. DNA was extracted by using CTAB buffer as described by Zhang et al. (1996). The extracted DNA was stored at -20 °C. DNA concentrations were quantified by using NanoDrop ND-1000 spectrophotometer (NanoDrop, Wilmington, DE). The fungal DNA was amplified using ITS 1 and ITS 4 Primers (sequence: 5′-TCCG ATGG TGAA CCT GCGG-3′) and ITS4 (sequence: 5′-TCCT CCGC TTATT GATA TGC-3′) according to White et al. (1990). The gradient PCR (Eppendorf Germany) was performed with 10 µl reactions. (5 µl Master mix (Bangalore Genei Ltd., India), 1 µl ITS1 and 1 µl ITS4 primers, 2 µl double sterile distilled water, 1 µl DNA (DNA Nanodrop value—843.9 ng/µl of raw DNA). The cyclic conditions were as follows: an initial step at 95 °C for 10 min; denaturation at 94 °C for 30 s; annealing at 52 °C for 1 min followed by 35 cycles; extension at 72 °C for 1 min; final extension at 72 °C for 10 min; and hold at 10 °C (White et al. 1990). A negative control was maintained using water instead of DNA. The PCR products were examined by 1% agarose gel electrophoresis using ethidium bromide staining and documented for amplification of appropriate 560 base pairs (Sambrook 1989). For identification, the PCR products were eluted using the QIA quick gel extraction kit (Qiagen, Inc., Chatsworth, California) and sequenced using the primer ITS1 and ITS4 on an ABI 3730 XL sequencer at Chromos Biotech Pvt. Ltd., Bangalore, India. The partial sequence of the 5.8S gene and the flanking internal transcribed spacer (ITS1 and ITS4) of the isolated strains were submitted to GenBank. The amplified PCR products were sequenced by Sanger’s dideoxy sequencing method and sequences were edited, aligned and the accession numbers of sequences were obtained.

Sequencing and phylogenetic tree construction

In order to study the variability, the sequences of three C. minitans isolates were compared with the other isolate P. minitans JX 496017 available in the NCBI database. The sequence identity matrix was constructed among the isolates with bio edit software version 7.2 (Kumar et al. 2016). Further, the phylogenetic tree was developed by using Mega 7.0 software including the sequence of P. minitans JX 496017 by maximum neighbour-joining method.

Molecular characterization of Coniothyrium sp. using specific primers

The ribosomal RNA gene of C. minitans was amplified by using specific primers Cm sp 1F (5′-CCCCAGGTGGTAA GGTGAAA-3′) and Cm sp 1R (5′-TACTAGAT GCAAAAAAGGTTTATCAG-3′) (Thompson et al. 1994). This was the species-specific primer of C. minitans and does not amplify the genome of C. sporulosum and C. fuckelii. PCRs were carried out in 10 µl reactions (5 µl Master mix, 1 µl Coniothyrium forward and 1 µl reverse primers, 2 µl Sterile distilled water, 1 µl DNA) using thermocycler. The cyclic conditions of PCR were an initial denaturation of 95 °C for 10 min; denaturation at 94 °C for 30 s; annealing at 52 °C for 1 min followed by 35 cycles; Extension at 72 °C for 1 min; final extension at 72 °C for 10 min and hold at 10 °C (White et al. 1990). Samples (5 µl) of the PCR products were separated on the 1% agarose gel in TBE buffer (90 Mm Tris-borate, 2 mM EDTA (pH 8.3) containing 0.5 μg of ethidium bromide per ml at 75 V for 2 h. The amplified PCR products were visualized with a UV illuminator and documented with an Alpha imager (Alpha Innotech Corporation, San Leandro, California).

In vitro screening of Coniothyrium minitans against S. sclerotiorum

(i) Effect of C. minitans on the mycelial growth of S. sclerotiorum by co-inoculation method

The antagonistic effects of C. minitans isolates (KET Cm1, PAL Cm2, TDK Cm3) were tested against S. sclerotiorum by dual culture technique (Dennis and Webster 1971). A 5-mm-diameter mycelial disc of 20-day-old culture of C. minitans was placed 1 cm away from the edge of the Petri dish containing solidified sterilized PDA medium on one side and on another side 7-day-old culture of S. sclerotiorum was placed at one cm away from the edge of the Petri plate just opposite to antagonist and the plates were incubated at 20 °C. Four replications were made for each treatment, and the observation of radial growth of mycelium was recorded and expressed as percentage inhibition over control. The pathogen-alone inoculated plates served as a control.

$${\text{PI}} = \frac{C - T}{C} \times 100$$

where PI is the percentage inhibition over control; C is the growth of test pathogen (cm) in the absence of the antagonist strain; and T is the growth of test pathogen (cm) in the presence of the antagonist strain.

(ii) Effect of pre-inoculation of Coniothyrium minitans isolates on the mycelial growth of S. sclerotiorum

Five-mm-diameter mycelial disc of C. minitans (KET Cm1, PAL Cm2, TDK Cm3) isolates were placed 1 cm away from the edge of the Petri plate containing sterilized solidified PDA medium. After 10 days of C. minitans inoculation, a 5-mm-diameter mycelial disc of 7-day-old culture of S. sclerotiorum was placed 1 cm away from the edge on the other side of the Petri plate. The plates were incubated at 20 °C. Control plates were maintained with pathogen alone from the 10 DAI (Days after Inoculation). Four replications were maintained for each treatment. The mycelial growth of the S. sclerotiorum was measured in cm and expressed as per cent inhibition over control.

Electron microscopic studies on hyperparasitism of C. minitans against S. sclerotiorum

Field emission scanning electron microscope (FESEM, SIGMA-5) was used for assessing the hyperparasitism of C. minitans on the hyphae of S. sclerotiorum. A 15 ml of PDA medium was poured into sterilized Petri plates and allowed to solidify. A 5-mm-diameter mycelial disc of S. sclerotiorum was placed at one end of the Petri dish and the 5-mm mycelial disc of antagonists was placed at the opposite end. The plates were incubated at 20 °C for five days. The mycelium from the zone of interaction between pathogen and antagonist was collected and the samples were processed for FESEM analysis.

The morphometric parameters of the interaction zone were viewed using scanning electron microscopy (SEM: Quanta 250, FEI, Hillsboro, OR, USA) with a large field detector (LFD). The SEM was operated in a vacuum of 10 kV with a spot size of 3.0 and a pressure of 60 Pa. The sample images were recorded at 5000 × and 10,000 × magnifications. For the analysis of hyperparasitized region, the mycelia from the interaction areas were fixed directly in carbon stubs. The source of electrons used in the FESEM was tungsten filament, and thermionic emission was used for the detection of the samples using FESEM.

Identification and characterization of secondary metabolites of C. minitans by gas chromatography mass spectrometry (GC–MS)

1. Extraction of antifungal antibiotic from C. minitans

A conical flask containing 250 ml of potato dextrose broth was autoclaved at 121.5 °C for 20 min, and then 5-mm mycelial disc of 20-day-old culture of C. minitans was inoculated into the broth. The flasks were incubated at 20 °C and allowed to grow into mycelial mat. After 30 days of incubation, the broth was separated using Whatman No.1 filter paper. The cultural filtrates were extracted three times with an equal volume of ethyl acetate and incubated overnight on a shaker. The solvent fraction of ethyl acetate was separated using a separating funnel. The extract was concentrated to about 150 ml by evaporation under diminished pressure using a vacuum flask evaporator, and the resulting solvent was air-dried in the sterilized Petri plate. The metabolites were scrapped using HPLC-grade methanol.

(i) Bioassay of crude antibiotics of fungal antagonist against S. sclerotiorum by agar well diffusion assay

The inhibitory effect of crude extracts of Coniothyrium isolates on the mycelial growth of S. sclerotiorum was studied in vitro by using Agar well diffusion method (Dhingra and Sinclair 1985). Using sterilized cork borer, a 5-mm-diameter well was made on the Petri plates containing solidified PDA medium on all four sides of the plate by leaving 1 cm away from the periphery. The 5-mm agar from the punched area was removed by using the sterilized inoculation needle. The actively growing 7-day-old culture of S. sclerotiorum was placed in the centre of the Petri plate. Extracted crude metabolites from the Coniothyrium isolates were poured into the well at the rate of 100 µl per well and incubated at 20 °C. The area of inhibition was recorded by tracing the surface area of inhibition in tracing paper and then plotted on the graph sheet and the zone of inhibition was measured. Methanol control was maintained to assess the effect of crude metabolites. All the treatments were replicated four times.

(ii) Bioassay of crude antibiotics of fungal antagonist against S. sclerotiorum by poison food technique

The inhibitory effects of crude metabolites at different concentrations (0.25, 0.50, 0.75 and 1%) were evaluated on the growth of S. sclerotiorum by poisoned food technique (Nene and Thapliyal 1993). Each treatment was replicated four times. The PDA medium without metabolite of C. minitans was used as control. All the plates were incubated at 20 °C for twenty days. The mycelial growth of S. sclerotiorum was measured in cm and expressed as percentage reduction over control. The number of sclerotia produced in each treatment was expressed in numbers.

(iii) Bioassay of crude antibiotics of fungal antagonist against S. sclerotiorum by paper disc assay

The filter paper was made into a 5-mm-diameter disc by using a punching machine, and these paper discs were placed in the Petri plates and autoclaved it. The autoclaved paper discs were dipped in the metabolite suspension and placed on all four sides of the Petri plate containing sterilized solidified PDA medium and leaving one cm away from the periphery. A 5-mm mycelial disc of S. sclerotiorum was placed at the centre of the Petri plate and the plates were incubated at 20 °C. The paper disc dipped in water was maintained as control. Three replications were made for each treatment and the growth of mycelium and inhibition zone around the paper discs were observed and expressed in per cent reduction over control.

GC–MS analysis of crude antibiotics

The crude antibiotics of the effective isolates of C. minitans were analysed for the detection of active biomolecules responsible for the suppression of S. sclerotiorum through GC–MS (GC Clarus 500 PerkinElmer). The volatile components were identified by GC–MS using a column Elite-5MS (100% dimethylpolysiloxane), 30 mm × 0.25 mm × 0.25 μm df equipped with GC Clarus 500 PerkinElmer. The TurboMass Gold PerkinElmer detector was used. The carrier gas flow rate was 1 ml per min, split 10:1 and injected volumes were 3 μl. The column temperature was maintained initially at 110 °C at the rate of 10 °C/min-No hold was followed by increasing up to 280 °C at the rate of 5° C /min with 9 min (hold). The injector temperature was 250 °C and this temperature was held constant for 36 min. The electron impact energy was 70 eV, the Julet line temperature was set at 2000 °C, and the source temperature was set at 200 °C. Electron impact (EI) mass scan (m/z) was recorded in the 45–450 a MU range. Using computer searches on the NIST Ver.2005 MS data library and comparing the spectrum obtained through GC/MS, the compounds present in the crude sample were identified.

Results

Isolation and morphological characterization of Coniothyrium minitans

The biocontrol agent associated with sclerotia of the head rot pathogen was isolated. A total of three isolates were isolated and identified as C. minitans, based on the morphological characteristics and designated as KET Cm1, PAL Cm2 and TDK Cm3. The colony colour of C. minitans isolates was initially white and later changed to grey at the centre. The Coniothyrium isolates KET Cm1 and PAL Cm2 produced white to grey colonies, while TDK Cm3 isolate produced white colony with pale green concentric rings. The mycelia of Coniothyrium sp. showed a slow growth and lasted around 60 days to cover Petri plates. Under the surface of the Petri plate, it showed dark brown colour due to melanin accumulation. The pycnidia of C. minitans were 150–700 µm in diameter, ostiolate, globose, brown to black, sub-globose at maturity. Pycnidiospores were dark brown colour, ovoid to ellipsoid, nearly globose smooth to roughened (Tables 1, 2; Fig. 1a, b).

Table 1 Isolation of Coniothyrium species from isolates of Sclerotinia sclerotiorum

Table 2 Morphological characterization of Coniothyrium minitans in different media

Fig. 1

a Morphological characterization of Coniothyrium minitans in the PDA medium. b Morphological characterization of Coniothyrium minitans in different media. PDA, potato dextrose agar; MEA, malt extract agar; CZA, Czapek dox agar; H.CZA, half-strength Czapek dox agar; WA, water agar; OMA, oat meal agar; CDA, carrot dextrose agar; V8, V8 juice agar

Molecular characterization of Coniothyrium sp.

Agarose gel electrophoresis amplified the genomic DNA of three C. minitans isolates and yielded an amplicon size of approximately 560 base pairs. The rDNA homology sequences analysis using BLAST revealed that all the three isolates share more than 99% nucleotide sequence identity with the existing C. minitans isolates available in the NCBI database. A nucleotide sequence of three isolates was submitted to NCBI for getting accession number. The accession numbers of three Coniothyrium isolates are listed in Table 3, Figs. 2 and 3.

Table 3 Molecular identification of Coniothyrium minitans isolates

Fig. 2

Molecular identification of C. minitans isolates using ITS 1 and ITS 4 primers. PCR amplicons of ITS regions of C. minitans on 1.2% agarose gel electrophoresis. M – 100 bp DNA ladder; Lane 1, KET Cm 1; Lane 2, PAL Cm 2; Lane 3, TDK Cm 3; Lane 4, Positive control

Fig. 3

Molecular identification of C. minitans isolates using CM sp 1F and CM sp 1R specific primers. PCR amplicons of ITS regions of C. minitans on 1.2% agarose gel electrophoresis. M – 100 bp DNA ladder; Lane 1, KET Cm 1; Lane 2, PAL Cm 2; Lane 3, TDK Cm 3; Lane 4, negative control

The identification of fungus C. minitans was further confirmed by molecular characterization using ITS and species-specific primers. In the present study, PCR and agarose gel electrophoretic reaction yielded an amplicon size of approximately 560 bp corresponding to ITS region. The fungus was further confirmed through species level through PCR with a specific primer, and an amplicon size of 170 bp size was recorded. The amplified products of ITS and species-specific primers were partially sequenced and identified as Coniothyrium minitans by comparing sequences available in the NCBI database. Species-level confirmation of C. minitans isolates was performed by PCR assay using species-specific primer Cm sp1F and Cm sp 1R. The genomic DNA of three isolates of C. minitans yielded approximately 170 bp size of amplicon in agarose gel electrophoresis assay. This has confirmed that all the three Coniothyrium isolates belonged to C. minitans (Fig. 3).

Phylogenetic analysis of Coniothyrium minitans isolates

The phylogenetic analysis was carried out using MEGA7.0 software in order to analyse the genetic diversity among the other isolates of Coniothyrium. From this study, the result revealed that the two major clads were formed. In clad 2, isolate TDK Cm3 was out-grouped from the major clad 1 which showed diverse genetic bases from other isolates. The major clad 1 was further divided into two sub-clads. In sub-clad 2, the isolate KET Cm1 was shown 100 per cent genetically similar to the isolates JX 496017 and PAL Cm2 which was clustered together as sub-clad 1(Fig. 4).

Fig. 4

Phylogenetic tree generated from a neighbour-joining (NJ) analysis using 5.8S rRNA sequence of Coniothyrium minitans using MEGA 7 software. Number besides branches represents the percentage of congruent cluster in 1000 bootstrap trails. The bar indicates 1 per cent sequences dissimilarity

The phylogenetic analysis of C. minitans revealed that the two major clads were formed. In clad 2, isolate TDK Cm3 was out grouped from the major clad 1 which showed diverse genetic bases from other isolates. The major clad 1 was further divided to two sub-clads. In sub-clad 2, the isolate KET Cm 1 was shown 100% genetically similar to the existing isolate JX 496017 available in GenBank and PAL Cm 2 which was clustered together as sub-clad 1.

Standardization of culture media and temperature for the growth of Coniothyrium minitans

All the culture media evaluated for the growth of C. minitans supported the mycelial growth at 20 °C and room temperature 28 ± 2 °C. Among two temperatures tested, 20 °C supported the mycelial growth of C. minitans more than room temperature. At 20 °C PDA recorded the maximum mycelial growth (28.67 mm), followed by H.CZA (28.33 mm), WA (26.67 mm), V8 juice agar (25.67 mm), OMA (24.17 mm) and MEA (24.83 mm). The lowest mycelial growth of 8.45 mm was observed in the CDA medium. At room temperature (28 ± 2 °C), the maximum mycelial growth was observed in PDA (26.33 mm), followed by V8 juice agar (24.33 mm), CDA (24.00 mm), OMA (19.95 mm), MEA (19.16 mm) and H.CZA (18.48 mm). The lowest mycelial growth of 10.68 mm was recorded in WA (Tables 4 and 5; Figs. 5a–h and 6 a–h).

Table 4 Mycelial growth of Coniothyrium minitans TDK Cm3 in different media at 20 °C

Table 5 Mycelial growth of Coniothyrium minitans TDK Cm3 in different media at room temperature (28 ± 2 °C)

Fig. 5

a Mycelial growth of Coniothyrium minitans in the PDA medium at 20 °C at different days interval. b Mycelial growth of Coniothyrium minitans in malt extract agar medium at 20 °C at different days interval. c Mycelial growth of Coniothyrium minitans in Czapek Dox agar medium at 20 °C at different days interval. d Mycelial growth of Coniothyrium minitans in half-strength Czapek Dox agar medium at 20 °C at different days interval. e Mycelial growth of Coniothyrium minitans in water agar medium at 20 °C at different days interval. f Mycelial growth of Coniothyrium minitans in oat meal agar medium at 20 °C at different days interval. g Mycelial growth of Coniothyrium minitans in carrot dextrose agar medium at 20 °C at different days interval. h Mycelial growth of Coniothyrium minitans in V8 juice agar medium at 20 °C at different days interval

Fig. 6

a Mycelial growth of Coniothyrium minitans in the PDA medium at room temperature (28 ± 2 °C) at different days interval. b Mycelial growth of Coniothyrium minitans in malt extract agar medium at room temperature (28 ± 2 °C) at different days interval. c Mycelial growth of Coniothyrium minitans in Czapek Dox agar medium at room temperature (28 ± 2 °C) at different days interval. d Mycelial growth of Coniothyrium minitans in half-strength Czapek Dox agar medium at room temperature (28 ± 2 °C) at different days interval. e Mycelial growth of Coniothyrium minitans in water agar medium at room temperature (28 ± 2 °C) at different days interval. f Mycelial growth of Coniothyrium minitans in oat meal agar medium at room temperature (28 ± 2 °C) at different days interval. g Mycelial growth of Coniothyrium minitans in carrot dextrose agar medium at temperature (28 ± 2 °C) at different days interval. h Mycelial growth of Coniothyrium minitans in V8 juice agar medium at temperature (28 ± 2 °C) at different days interval

Standardization of pH for the growth of Coniothyrium minitans

The effect of different pH levels of PDA medium on the mycelial growth of C. minitans was tested with a range from 4.0, 4.5, 5.0, 5.5, 6.0 and 6.5, and the results are presented in Table 6. The results revealed that among the different pH levels tested, pH 5.5 exerted maximum mycelial growth of 34 mm. This was followed by pH 6.5 (33 mm), pH 6.0 (32 mm), pH 5.0 (27 mm) and pH 4.5 (23 mm). The lowest pH 4.0 recorded the least mycelial growth of 14 mm (Figs. 7, 8). The pH of cultural media had also influenced the hyphal extension and pycnidial formation in C. minitans. Though hyphal extension was observed in different pH, cultural medium with 5.5 pH recorded maximum mycelial growth.

Table 6 Influence of different pH level of PDA on the mycelial growth of TDK Cm3 isolate

Fig. 7

Mycelial growth of Coniothyrium minitans TDK Cm3 in different pH levels on PDA medium

Fig. 8

Influence of different pH levels on the mycelial growth of C. minitans on PDA medium

In vitro screening of Coniothyrium minitans against S. sclerotiorum

1. Co-inoculation

The effect of concurrent inoculation of C. minitans and S. sclerotiorum was tested in vitro using the dual plate method (Table 7). Among the three Coniothyrium isolates tested, the C. minitans TDK Cm 3 isolate showed maximum mycelial inhibition of 28.8% over control. This was followed by KET Cm1 and PAL Cm2 which recorded an inhibitory effect of 27.7% and 25.5% reduction over control, respectively (Fig. 9).

Table 7 In vitro screening of Coniothyrium minitans isolates against S. sclerotiorum NAJ SS5 at 20 °C (co-inoculation and pre-inoculation method)

Fig. 9

In vitro screening of Coniothyrium minitans against Sclerotinia sclerotiorum NAJ SS5 at 20 °C (co-inoculation)

2. Pre-inoculation

The effect of C. minitans against the mycelial growth of S. sclerotiorum was also studied by pre-inoculation of the former one. The results revealed that the maximum inhibition of mycelial growth was recorded at 48.8% in pre-inoculated Coniothyrium TDK Cm3 isolate over control. The isolate KET Cm1 was on par with TDK Cm3 in reducing the mycelial growth of S. sclerotiorum. The least mycelial growth inhibition was recorded in PAL Cm2 with a 44.4% reduction over control (Table 7; Fig. 10).

Fig. 10

In vitro screening of Coniothyrium minitans against Sclerotinia sclerotiorum NAJ SS5 at 20 °C (pre-inoculation)

SEM analysis of C. minitans against S. sclerotiorum

The hyperparasitic region of S. sclerotiorum was studied through SEM analysis. The results revealed that the hyphae of S. sclerotiorum showed abnormal swelling with malformations. Some parts of the hyphae became disintegrated due to the lysis action of the enzymes secreted by C. minitans. The hyphae of C. minitans formed appressoria on the surface of S. sclerotiorum hyphae. The above abnormalities observed in S. sclerotiorum hyphae and the formation of appressoria by C. minitans hyphae confirmed the hyperparasitic nature of C. minitans (Fig. 11). The FESEM analysis of the hyphae of S. sclerotiorum excised from the lytic zone around the C. minitans revealed that hyphal lysis, formation of appressoria and malformation of hypha.

Fig. 11

FESEM images of hyphal interaction between C. minitans TDK Cm3 and S. sclerotiorum NAJ SS5

Antifungal activity of secondary metabolites of C. minitans against S. sclerotiorum

1. Agar well diffusion assay

The effect of secondary metabolites secreted by C. minitans isolates on the mycelial growth of S. sclerotiorum was studied using agar well diffusion method (Table 8). The results revealed that KET Cm1 and TDK Cm3 isolate significantly reduced the mycelial growth of S. sclerotiorum (88.8%) than control. This was followed by isolate PAL Cm 2 which recorded mycelial growth inhibition of 83.3% reduction over control. Sterilized water control did not inhibit the mycelial growth of S. sclerotiorum and this recorded higher mycelial growth of 9 cm (Fig. 12).

Table 8 In vitro assay for crude metabolites of Coniothyrium minitans isolates against Sclerotinia sclerotiorum NAJ SS5 using agar well diffusion and paper disc method

Fig. 12

In vitro assay for crude metabolites of Coniothyrium minitans isolates against Sclerotinia sclerotiorum NAJ SS5 using agar well diffusion method

2. Paper disc assay

The effect of crude secondary metabolites secreted by C. minitans isolates against mycelial growth of S. sclerotiorum was also studied by paper disc assay (Table 8). The results revealed that the mycelial growth of S. sclerotiorum was inhibited by all the three C. minitans isolates. The inhibition percentage ranged from 37.7 to 40.0%. Among the three different isolates, maximum inhibition was exerted by the isolate TDK Cm 3 (40.0%) with an inhibition zone of 1 mm. The isolate KET Cm1 was on par with PAL Cm2 in reducing mycelial growth (Fig. 13).

Fig. 13

In vitro assay for crude metabolites of Coniothyrium minitans isolates against Sclerotinia sclerotiorum NAJ SS5 using paper disc assay

3. Poison food technique

The effect of secondary metabolites produced by the elite C. minitans isolates, TDK Cm3 was tested under the poison food technique (Table 9). Secondary metabolites secreted by TDK Cm3 showed an inhibitory effect on the mycelial growth of S. sclerotiorum. Among the four concentrations of secondary metabolite tested, low concentrations of 0.25 and 0.50% did not show any inhibitory effect on mycelial growth and sclerotial productions. High concentration of secondary metabolites like 0.75 and 1% showed a significant reduction in the mycelial growth and cent per cent inhibition of sclerotial productions. The maximum inhibition of mycelial growth was recorded in 1% concentration of crude metabolites of C. minitans (55%). Though less inhibition (11.1%) was observed at 0.75% concentration, the inhibition of sclerotial production was cent % (Fig. 14).

Table 9 In vitro assay for crude metabolites of Coniothyrium minitans TDK Cm3 isolate against Sclerotinia sclerotiorum NAJ SS5 using poison food technique

Fig. 14

In vitro assay for crude metabolites of Coniothyrium minitans TDK Cm 3 isolate against Sclerotinia sclerotiorum NAJ SS5 using poison food technique

The effect of NVOM produced by the effective isolate of C. minitans TDK Cm3, was analysed using agar well diffusion assay, paper disc assay and poison food technique. In all the three assays, the NVOM effectively inhibited the mycelial growth of S. sclerotiorum. The inhibitory effect of NVOM may be due to the antimicrobial properties of bioactive compounds present in non-volatile fraction.

Metabolic profiling of non-volatile organic compounds produced by Coniothyrium minitans TDK Cm3 isolate

The secondary metabolites with antimicrobial compounds produced by C. minitans TDK Cm 3 isolate were analysed through GCMS (Table 10). A total of 40 compounds was detected through GCMS analysis, out of these 10 compounds were selected based on relative abundance and peak area percentage. The results revealed that the secondary metabolites contained the biomolecules with antifungal activity. The biomolecules detected were decane, benzothiazole, tetradecanoic acid, 1-nonadecene, phytol acetate octadecynoic acid, dimethyl palmitamine, n-hexadecenoic acid, 1-heneicosylformate and 1-heptatriacotanol (Fig. 15).

Table 10 Non-volatile organic metabolites (NVOM) associated with the secretome of Coniothyrium minitans TDK Cm 3 isolate

Fig. 15

GC–MS chromatogram of Coniothyrium minitans TDK Cm 3 isolate

The GC MS analysis of non-volatile organic compounds secreted by C. minitans isolate TDK Cm 3 revealed that the ethyl acetate fraction of culture filtrate contained 10 different organic compounds with antifungal, antibacterial and antioxidant activities. The antifungal compounds included decane, benzothiazole, tetradecanoic acid, 1-nonadecene, phytol acetate, octadecynoic acid, dimethyl palmitamine, n-hexadecenoic acid, 1-heneicosylformate and 1-heptatriacotanol. Apart from the seven antifungal compounds, the compounds with antibacterial activity were also recorded in GC MS analysis. These compounds with antifungal activity may be responsible for the reduction in mycelial growth under in vitro conditions.

Discussion

Coniothyrium minitans was firstly described for the biological control of S. sclerotiorum in California by Campbell (1947). It was isolated from the sclerotia of S. sclerotiorum from more than 30 countries on all continents (Sandys-Winsch et al. 1993). This fungus is ecologically fastidious mycoparasite on sclerotia of S. sclerotiorum, Botrytis spp. and S. cepivorum. (Wang et al. 2008). C. minitans displayed a good ability to infect and degrade the sclerotia present in the soil by decreasing carpogenic germination and viability of sclerotia. Ojaghian (2009) isolated C. minitans from sclerotia of S. sclerotiorum infecting potato on PDA medium. It was described that C. minitans was slow growing with non-aerial mycelium and produced pycnidia 3 weeks after inoculation. C. minitans fungal colony was initially white and became grey after two weeks, without aerial mycelium. Its hyphae were 3 to 6 µm diameter, smooth, and simple with numerous septa (Whipps 1992). The pycnidia were 150–700 µm in size, ostiolate, brown to black, sub-globose which produced pycnidiospores of dark brown, and ovoid to ellipsoid are globose pycnidiospores (Punithalingam 1982). In the present study, three isolates of C. minitans were isolated and characterized morphologically. The fungal colony characters, mycelial characters, pycnidial and pycnidiospores were in accordance with characters of C. minitans described by earlier workers. Earlier the identity of C. minitans was molecularly confirmed by Muthumeenakshi et al. (2001) by using ITS 1 & ITS 4 primers and specific primers. Similarly, Muthumeenakshi et al. (2001) studied the intraspecific diversity and phylogenetic position of C. minitans. The result of the above findings showed that two major clad were formed; from major clad 1, the sub-clad 2 includes C. minitans and C. sporulosum which were clustered together and showed 100% similarity with each isolate.

Coniothyrium minitans is a promising biocontrol agent of S. sclerotiorum both in glasshouse and in field trials (Budge and Whipps 1991). Culturing of C. minitans for large-scale production requires standard culture media, with suitable pH and temperature. In the present study, eight different cultural media were tested for their support in the growth of C. minitans and its pycnidial production. Among the different cultural media tested, PDA recorded maximum mycelial growth than other cultural media. The result of the present study was supported by the findings of Muthumeenakshi et al. (2001). McQuilken et al. (1995) reported that PDA supported conidial germination, mycelial growth and pycnidial production of C. minitans. Papavizas et al. (1984) stated that PDA medium supported hyphal extension of C. minitans when compared to molasses yeast broth. Similarly, the effect of temperature on the growth of C. minitans was studied. Muthumeenakshi et al. (2001) stated that both room temperature (28 ± 2 °C) and 20 °C supported the growth of C. minitans. However, high mycelial growth was recorded at 20 °C in various cultural media. A high temperature of 28 ± 2 °C had recorded lesser mycelial growth in different cultural media tested. Mcquilken et al. (1997) studied the temperature range for the mycelial extension of C. minitans and found that the temperatures 20 to 25 °C were optimal for mycelial extension in different isolates. They also observed that 20 °C was the optimum temperature for pycnidial production. Similar reports were made by Sandys-Winsch et al. (1993). The result of the present study also is in agreement with the report of the above-said workers. The results on the effect of pH agreed with the report of Mcquilken et al. (1997) who stated that cultural media pH between 4.5 to 6.2 supported conidial germination, hyphal extension and pycnidial production of C. minitans. The effect of C. minitans isolate on the mycelial growth of S. sclerotiorum tested under dual cultural technique as co-inoculation and pre-inoculation. The C. minitans isolates reduced the mycelial growth and sclerotial production of S. sclerotiorum under two different methods of inoculation. The effectiveness of co-inoculation of C. minitans isolates and S. sclerotiorum was comparatively less than pre-inoculation method. Approximately 90% increased reduction in mycelial growth was observed in the pre-inoculation method than the co-inoculation. Though the growth rate of C. minitans strains was slower than that of S. sclerotiorum, the colonies were coalesced and an inhibition zone was observed at the point of contact. Jones et al. (2014) studied the effect of different C. minitans on the mycelial growth of S. sclerotiorum. In dual culture, using both co-inoculation and pre-inoculation method, they observed that pre-inoculation reduced the mycelial growth comparatively than co-inoculation method. They also observed mycoparasitism of C. minitans around the S. sclerotiorum. They concluded that C. minitans LU112 reduced apothecial production and viability of sclerotia. Similarly, Hoes and Huang (1975) studied the mycoparasitism of C. minitans against S. sclerotiorum using a dual culture technique. They observed that the lysis of hyphae as well as sclerotia. The parasitized sclerotia decayed and became soft as a result of hyphal disintegration. This may be due to the production of exo- and endoglucanase by the C. minitans. Jones et al. (1974) observed the disintegration of S. sclerotiorum hypha due to lytic enzymes secreted by C. minitans. Jones and Watson (1969) described the production of endo- and exoglucanase by C. minitans involved in the breakdown of the cell wall of S. sclerotiorum. Boosalis (1956) showed different modes of parasitism by C. minitans on the P. vermiculatum and R. solani. Cell wall lysis and coiling of host hyphae by antagonist T. harzianum were observed by Inbar et al. (1994). Similar findings were reported by Vinodkumar et al. (2017) who observed the colonization of T. asperellum—NVTA2 on sclerotial bodies of S. sclerotiorum through SEM.

Choudary et al. (2007) observed the metabolite produced from T. harzianum, which completely inhibited the mycelial growths of F. oxysporum, S. rolfsii and R. solani, respectively. Similar reports were made by Nagamani et al. (2017) who observed a reduction in the mycelial growth of F. oxysporum f. sp. ciceri by non-volatile organic metabolites of Trichoderma isolates. Vinodkumar et al. (2017) found a good inhibitory effect of cell-free crude extract from T. asperellum NVTA2 on the mycelial growth of S. sclerotiorum. Maddila et al. (2016) reported the antibacterial and antifungal activity of Benzothiazole compounds.

Derbalah et al. (2012) confirmed the antifungal activity of Tetradecanoic acid. The antifungal activity of phytol acetate was reported by Aziz et al. (2019). The results of the present findings are also in agreement with the report of the above-said workers.

Conclusion

The present study mainly focused on the Coniothyrium minitans, a sclerotial mycoparasite of Sclerotinia sclerotiorum, and its morphology, molecular characterization, mode of action and its mechanisms against the pathogen, in vitro evaluation of effective isolates for the management of head rot pathogen. This provided the basic information for further studies to develop the formulations and in vivo evaluations for the management of head rot pathogen.