Introduction

Macrophomina phaseolina (Tassi) Goid. is a widespread soil-borne phytopathogenic fungus. It is responsible for charcoal rot, basal stem rot and damping off disease in more than 500 species of plant kingdom (Khan et al. 2017). It mainly produces pycnidia or either microsclerotia in the soil (Li 1993). It is not only soil-borne but also a seed-borne pathogen and causes infections in plants both at seedling stage and at plant maturity (Purushotham et al. 2020). It is very hard to manage this pathogen through agronomic practices because of its diverse nature (Naseri et al. 2018). To combat the pathogens, chemical fungicides are the first choice for farmers as are easily available in market (Iqbal and Mukhtar 2020). However, long-term fungicides application has created toxic effects in environment, humans and animals so their usage should be discouraged (Aravind and Brahmbhatt 2018). Chemical fungicides should be replaced with biocontrol organisms because of the public concerns regarding negative impact of the synthetic agrochemicals (Rahman et al. 2018). During the past few decades, diverse biocontrol agents have received consideration in disease management programs in addition to their use in commercial enzyme production namely hemicellulases, cellulases, β-1,3-glucanase and proteases (Bischof et al. 2016; Deng et al. 2018).

Biological control of soil-borne pathogens by microorganisms has been considered as environmentally safe and virtuous supplement to the synthetic fungicides (Sohaliya et al. 2019). The widespread application of Trichoderma spp. such as T. asperellum, T. atroviride, T. gamsii, T. hamatum, T. harzianum, T. polysporum, T. virens and T. koningii as biocontrol agents has been exploited and reported against Phytophthora, Pythium, Aspergillus, Fusarium, Rhizoctonia and Macrophomina (Moosa et al. 2017; Javaid et al. 2018; Sharma and Prasad 2018; Ingale and Patale 2019). Akin to the most fungal biocontrol organisms, genus Trichoderma can be efficiently used in the form of conidia or spores which are more tolerant to harsh environmental conditions in comparison to mycelia for field use and product formulations (El-Mougy and Abdel-Kader 2018). The activation mechanism exerts biocontrol weapons either directly or indirectly, by competition for space, nutrients, mycoparasitism and antibiosis (Guzman-Guzman et al. 2017). Moreover, Trichoderma is a growth promoting fungus that can easily multiply in diverse soil types and establish persistent colonies for several months. Its commercial success rate depends upon the shelf life, easy and quick multiplication on host, bioefficacy and readily availability in market (Kamal et al. 2018; Rini et al. 2018). The aim of the present study was to assess in vitro antagonistic potential of T. pseudokoningii as biological control agents against M. phaseolina and to unearth the possible mechanisms involved in antagonism.

Materials and Methods

Molecular characterization of selected isolates

Pure culture of T. pseudokoningii was obtained from the First Culture Bank of Pakistan (FCBP), University of the Punjab Lahore, Pakistan. The fungus was multiplied on malt extract agar plates and its DNA was procured through cetyl trimethylammonium bromide (CTAB) method (Doyle and Doyle 1990). The isolated DNA was confirmed on molecular basis by using two sets of primer pairs namely ITS (ITS 1-F and ITS 4-R) and EF1 (EF1-728-F and EF1-986-R). Annealing temperature for all the primers was 60°C. The amplified single band PCR products were got sequenced at 1^st Base Sequencing Singapore Co., Ltd. (Fig. 1).

In vitro antagonistic activity of T. pseudokoningii

Biological activity of T. pseudokoningii was evaluated by direct co-culturing with the tested phytopathogenic fungus M. phaseolina. A mycelial plug (0.5 cm diameter) of T. pseudokoningii was prepared from the 7-day-old culture and uniformly inoculated in vertical direction at one end of the malt extract agar containing plates opposite to the pathogen mycelial plugs. A control was also prepared by only inoculating the pathogen in the center of malt extract agar (MEA) plates with three replicates of each and let them to grow for five days at 28°C. Percent hyphal growth inhibition of M. phaseolina was determined and calculated through a given below growth inhibition formula (Rini and Sulochana 2008).

For statistical analysis, standard errors of means of five replicates were calculated using MS Excel. To determine significant difference in growth of M. phaseolina between control and dual culture treatments up to 5 days growth, LSD test was applied at P ≤ 0.05 using software Statistix 8.1.

DNA cleavage bioassay

A DNA cleavage experiment was conducted to evaluate the antagonism potential of secondary metabolites produced by T. pseudokoningii. For this, malt extract (ME) broth containing flasks were inoculated aseptically with freshly prepared mycelial plugs of T. pseudokoningii and kept on an orbital shaker (150 rpm) for two weeks at 28°C. Subsequently, the broth was filtered through Whatman No. 1 filter paper and its concentration was considered as 100%. The resultant filtrate was evaporated in an electric oven at 40°C for the preparation of concentrated broth in order to construct five different concentrations viz., 200, 300, 400 and 500%. The isolated M. phaseolina ribosomal DNA (5 µL) was mixed in each concentration (5 µL) for 24 and 48 h at 37°C. Experiment was conducted in completely randomized design in triplicates. To determine the extent of pathogenic fungal DNA degradation, all the treatments were loaded on 1% agarose gel with a negative control. The gel was run for 45 min at 100 volts and visualized under UV illuminator (Katrahalli et al. 2019).

GC-MS analysis

For identification of bioactive compounds, T. pseudokoningii mycelial plugs (5 mm diameter) were added to 250-mL Erlenmeyer flasks containing 100 mL of sterilized ME medium. The flasks were then kept on an orbital shaker (150 rpm) for 15 days at 28°C. The resultant homogenate was passed through a filter paper and extracted with chloroform and ethyl acetate. The organic solvent layers were collected by using a separating glass funnel and were further subjected for GC-MS analysis.

Results

Molecular identification of T. pseudokoningii

Molecular characterization of the antagonistic T. pseudokoningii DNA was confirmed by sequencing of ITS and EF1 regions. The amplified PCR products with EF1 441 bp and ITS 542 bp showed 100% similarities with the already submitted sequences to NCBI, and deposited under accession No. MN736409 and MN721869, respectively.

Interactions of T. pseudokoningii with M. phaseolina

Antagonistic effect of T. pseudokoningii against the saprotrophic soil-borne phytopathogen M. phaseolina is shown in Fig. 2. It was observed that T. pseudokoningii successfully restricted the growth of the targeted pathogen. There was up to 62% inhibition in growth of M. phaseolina in dual culture treatment with T. pseudokoningii as compared to its growth in the control treatment after five days’ growth (Fig. 3).

DNA cleavage study

Secondary metabolites produced by the T. pseudokoningii rapidly degraded the M. phaseolina rDNA as shown in Fig. 4. The findings revealed that after the incubation of 24 h, pathogenic fungal DNA bands appeared in the form of smears at lower concentrations of 100%, 200% and 300%, whereas, at the higher concentrations of 400% and 500% the genomic DNA was fully degraded. At higher incubation time of 48 h, it was noted that all the concentrations successfully degraded the M. phaseolina DNA bands.

GC-MS analysis

Fig. 1: Trichoderma pseudokoningii (M): 1 kb DNA standard marker (1): Genomic DNA, (2): ITS1/ITS4 amplified PCR product (3): EF1_f/EF1_r amplified PCR product. 

Fig. 2: Interaction of Macrophomina phaseolina with Trichoderma pseudokoningii

A)- Pure culture of Macrophomina phaseolina (MP); B)- MP co-culture with T. pseudokoningii.

Fig. 3: Effect of Trichoderma pseudokoningii on growth of Macrophomina phaseolina in dual culture interaction. Vertical bars show standard errors of means of three replicates. Values with different letters at their top show significant difference (P ≤ 0.05) as determined by LSD Test. Values (%) given on bars of dual culture treatment show decrease in growth of M. phaseolina in dual culture over corresponding control treatment.

Fig. 4: DNA cleavage. A)- After 24 hours, B)- After 48 hours.

(M): 1 kb DNA standard marker (1): Genomic DNA of M. phaseolina (2): Negative control (Genomic DNA of M. phaseolina + Malt extract broth) (3):  100% concentration (Genomic DNA of M. phaseolina + T. pseudokoningii fungal metabolite) (4): 200% concentration (Genomic DNA of M. phaseolina + T. pseudokoningii fungal metabolite)  (5):  300% concentration (Genomic DNA of M. phaseolina + T. pseudokoningii fungal metabolite) (6):  400% concentration (Genomic DNA of M. phaseolina + T. pseudokoningii fungal metabolite)  (7):  500% concentration (Genomic DNA of M. phaseolina + T. pseudokoningii fungal metabolite). Arrows indicate the presence or absence of DNA

The GC-MS analysis of chloroform and ethyl acetate fractions of T. pseudokoningii metabolites is given in Fig. 5, which presented 9 peaks in each of the two chromatograms. The compounds identified in chloroform fraction with their percent abundance and retention time are given in Table 1 and their structures are illustrated in Fig. 6. The compounds present in higher concentrations were undecane (36.47%) and decane (23.45%). On the other hand, moderately abundant compounds were benzene, 1,2,3-trimethyl (9.24%), 1-hentetracontanol (8.78%), n-dodecylpyridinium chloride (6.14%), octadecanoic acid, 9,10-dihydroxy-, methyl ester (5.74%), pentatriacontane (5.44%), tetracontane (4.43%) and benzene, 1-ethyl-2,3-dimethyl- (3.03%).

The details of identified compounds in ethyl acetate fraction with retention time and percent abundance is presented in Table 2 and their structures are given in Fig. 7. The most prevailing compounds were 9,12-octadecadienoic acid (Z,Z)- (23.48%), 9,12-octadecadienoic acid (Z,Z)-, methyl ester (20.84%), cyclohexanecarboxylic acid, 4-pentyl-, 4-ethoxyphenyl ester (11.45%), 7,8-epoxylanostan-11-ol, 3-acetoxy- (10.38%) and hexadecanoic acid, 2-hydroxy-1-(hydroxymethyl) ethyl ester (10.36%). The compounds in less abundance were gamma-sitosterol (7.66%), benzene, nitro- (7.03%), 1-(+)-ascorbic acid 2,6-dihexadecanoate (5.43%) and hexadecanoic acid, methyl ester (3.15%).

Discussion

The morphological characters of Trichoderma spp., can give misleading results so the accurate identification on molecular basis is necessary (Mokhtari et al. 2017). The amplification and sequencing of ITS and EF1 regions is Table 1: Compounds identified from chloroform fraction of culture filtrate of Trichoderma pseudokoningii through GC-MS analysis

Table 2: Compounds identified from ethyl acetate fraction of culture filtrate of Trichoderma pseudokoningii through GC-MS analysis

Fig. 5: GC-MS chromatograms of chloroform and ethyl acetate fractions of culture filtrate of Trichoderma pseudokoningii.

common and highly trusted to differentiate among the species (Li et al. 2020). Therefore, in the present study, molecular characterization of T. pseudokoningii was carried out with ITS and EF1 that gave accurate identification. Consequently, accession numbers were deposited in GeneBank.

In dual culture plate assay the inhibition in two interacting organisms’ radial growth has been attributed to the inhibitory substances produced by one or both of the organisms as a result of competition, hyper-parasitism,

Fig. 6: Structures of compounds identified in chloroform fraction of culture filtrate of Trichoderma pseudokoningii through GC-MS analysis

Fig. 7: Structures of compounds identified in ethyl acetate fraction of culture filtrate of Trichoderma pseudokoningii through GC-MS analysis

mycoparasitism or secretion of antibiotics (Deng et al. 2018). The present findings revealed that T. pseudokoningii significantly retarded the growth of M. phaseolina and is in confirmation of the recent work performed by Li et al. (2019) who reported that three antagonistic strains of Trichoderma namely T. pseudokoningii, T. harzianum and T. asperellum were very effective against the Fusarium oxysporum, the pathogen of Fusarium wilt in various economically important host plants. Previously, Nirwan et al. (2016) also evaluated the antagonistic potential of T. pseudokoningii against the root rot pathogen Ganoderma lucidum with promising results. Similarly, Akinbode et al. (2018) managed Fusarium verticillioides, a seed-borne fungal pathogen by using T. pseudokoningii and benomyl a synthetic chemical under field conditions. The results showed that T. pseudokoningii was found to be more efficient against the targeted pathogen in comparison with the fungicide. Ibrahim and Abdel-Azeem (2015) also effectively controlled the M. phaseolina by using T. pseudokoningii under field conditions.

Trichoderma species are used worldwide as lucrative potent biocontrol agents against a diverse range of phytopathogens due to their prominent antimicrobial activities under in vitro and in vivo conditions (Vinale et al. 2008). Undoubtedly, it has been documented that novel secondary metabolites produced by the genus Trichoderma are the efficient source of bioactivities (Khan et al. 2019). To understand the mechanism of antagonism, a DNA cleavage experiment was carried out to evaluate the potential of T. pseudokoningii towards the M. phaseolina. This study clearly demonstrated that disintegration of the pathogen’s DNA is possibly the key mechanism of T. pseudokoningii as biocontrol agent against M. phaseolina. Previously, DNA cleavage studies were generally carried out to study the mechanism of nanoparticles (Jadhav et al. 2018). Application of silver nanoparticles fully deteriorated the gram-positive Staphylococcus warneri bacterial DNA (Dong et al. 2017). Likewise, plant extracts of Euphorbia prostrata showed excellent results against the bacterial DNA structure of E. coli. Furthermore, the nanoparticles prepared from E. prostrata plant extracts when used alone and in combinations actively degraded the DNA (Dashamiri et al. 2018). Similarly, Katrahalli et al. (2019) tested a naturally occurring compound benzothiazol-2-yl-malonaldehyde against a gram-negative bacterium Pseudomonas aeruginosa DNA with promising results.

GC-MS analysis of chloroform and ethyl acetate fractions of T. pseudokoningii secondary extrolites was undertaken to evaluate the bioactive compounds. Among the identified constituents, undecane was a predominant compound in chloroform fraction and was previously isolated from a medicinal plant Equisetum arvense with potent antibiotic, antioxidant, viricidal and anti-inflammatory properties (Altameme et al. 2015). Moreover, Moya et al. (2018) also identified this compound from the secondary metabolites produced by T. harzianum and found it effective against a pathogenic fungal strain Pyrenophora teres. 9,12-Octadecadienoic acid (Z,Z)- was another predominant compound present in ethyl acetate fraction. Bruno et al. (2019) recently identified this compound as an antimicrobial agent by testing it against bacterial pathogens Staphylococcus aureus and Bacillus subtilis as well as against a fungal pathogen F. oxysporum with promising results. Likewise, an important compound 7,8-epoxylanostan-11-ol,3-acetoxy- identified in the present study was previously found in ethanolic extracts of Rhus muelleri with strong antimicrobial potential (Rodriguez et al. 2015). Similarly, Soni et al. (2014) reported the antimicrobial, pesticide and pharmaceutical properties of gamma-sitosterol. Hexadecanoic acid, 2-hydroxy-1-(hydroxymethyl) ethyl ester was also identified from the methanolic extracts of Melia azedarach with anthelmintic, anti-inflammatory and antioxidant properties (Al-Marzoqi et al. 2015). Arora and Kumar (2018) identified 9,12-octadecadienoic acid (Z,Z)-, methyl ester having hypocholesterolemic, anti-histaminic, aepatoprotective and antieczemic activities. Likewise, decane and benzene, 1,2,3-trimethyl are known for their antifungal and antibacterial activities (Mohammed et al. 2015; Adomi 2017). 1-Hentetracontanol also possess antimicrobial properties (Ravi et al. 2018).

Conclusion

Findings of this study revealed that T. pseudokoningii is an effective biocontrol agent that can efficiently arrest the growth of M. phaseolina. T. pseudokoningii extracted secondary extrolites can effectively cleave the pathogenic fungal DNA. The literature survey showed that chloroform and ethyl acetate fractions are rich in antifungal compounds such as undecane, 9,12-octadecadienoic acid (z,z)- decane and gamma-sitosterol, which could be responsible of control of pathogen growth.

Author Contributions

Iqra Haider Khan did experimental work and wrote a part of paper. Arshad Javaid supervised the work and also contributed in paper writing.

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