Introduction

Mung bean (Vigna radiata L.) is a standout amongst the most imperative and critical pulse crops of Pakistan. It belongs to the family Fabaceae1 and developed from the tropical to sub-tropical territories in the world2,3. There are in excess of about five hundred varieties of pulses that assume a helpful part in increasing the fertility of the soil by a relationship with nitrogen-fixing bacteria. Seeds of pulses are profitable nutritional sources and are thought to be contrasting options to meat as they contain proteins (20 to 30% of dry weight). Seeds additionally have a low-fat substance (about 5%), fibers, sugars, calcium, zinc, and folic acid4,5,6. The mung bean seeds contain 1.30% fats, 24.20% protein contents, 60.4% starches; phosphorus (P) 340 mg, and calcium (Ca) is 118 mg for each 100 g of seed7. Besides, in mung bean seeds the protein content is two times higher than in the seeds of maize, with the least storage protein content (7 to 10%)8,9. Mung bean is a significant measure of bioactive Phyto synthetic substances. With expanding clinical confirmation proposing that mung bean plants have different potential advantages for health, their utilization has been developing at a rate of 5 to 10% every year10. It is notable for its detoxification exercises and is utilized to invigorate mindset, mitigate warm stroke, and diminish swelling in the late spring. Mung bean was recorded to be valuable in the direction of gastrointestinal disturbance and skin motorization11. Additionally, the seeds and sprouts of mung beans are generally utilized as a new serving of mixed green vegetables or as regular sustenance food in Pakistan, India, Bangladesh, South East Asia, and western nations12. Mung bean is developed in the biggest pulse region in Pakistan just second to chickpea13. Pakistan imports a very high number of legumes to cover the breach in demand and supply of pulses.

Plant diseases reduce the yield and productivity of several crops all over the world including mung bean. Yield losses because of the absence of plant security measures change from 46 to 96% contingent upon any crop varieties. Biotic diseases harm plants in different life forms, viz., insects, weeds, nematodes, allelopathic chemicals, and so on. Among these, fungi and viruses are the biggest and the most critical gatherings influencing all parts of the plant at all phases of the development of the food legumes14. Fungi are the most harmful pathogens to mung beans and cause diverse infections like leaf spots (Cercospora leaf spots and Alternaria leaf spot etc.), Phytophthora stem blight, powdery mildew, and wilting15,16,17 etc.

Most plant pathogenic fungi have the capability to rupture the primary cuticles of host plants by developing appressoria which can be either single-celled assemblies or multiple appressoria, that mutually form structures recognized as infection cushions18,19. The appressorium is a specialized infection structure that is crucial for penetration into the host cell20. Appressorium development is a complicated procedure comprising various signals, including physical and chemical stimuli. The mitogen-activated protein kinase (MAPK) cascade is responsible for the morphogenesis, conidiation, appressorium establishment, and pathogenicity of numerous fungi, including Magnaporthe grisea21, Pyrenophora teres22, Colletotrichum spp.23, Botrytis cinerea24, and so on. Generally, three types of MAPK signaling cascades exist in the filamentous fungi which include; (i) Slt2-homolog, (ii) Hog1-homolog, and, (iii) Fus3/Kss1-homolog MAPK. The latter one is essential for pathogenicity and virulence25. In the budding yeast Saccharomyces cerevisiae, five MAPK pathways are known to regulate mating, invasive growth, cell wall integrity, hyper osmoregulation, and ascospore formation26. In the plant fungal pathogen model Magnaporthe oryzae (previously known as M. grisea), appressorium establishment is mediated by hydrophobic surface induction27. Ste12 is a homeodomain transcription factor and, is a key target of MAPK signaling pathway during invasive growth in the filamentous fungi28. Moreover, Ste12 is regulated by kinases included Fus3/Kss1 in the MAPK signaling cascade that regulate activation or repression of the mating pathways in filamentous fungi in response to pheromone and starvation29. Ste12 homologs in filamentous plant pathogens mainly regulate penetration, intrusive growth, and disease formation. On the other hand, different fungal pathogens are varied in their pathogenic mechanisms24. In true filamentous fungi, Ste12-like proteins play essential roles in sexual development and pathogenicity. Interestingly, Ste12 and Ste12-like factors are important for pathogenesis in all animal and plant pathogens tested so far, and further functional analyses revealed their importance in the setting up of a pathogenicity genetic program specific to the host. This indicates that Ste12 genes are required for these developmental processes, which accompany the invasive colonization of a new environment30.

Biological control is used as a technique for controlling fungal diseases as being environmentally friendly and non-lethal to the health of human beings, livestock, and wildlife; particularly now that the entire world is screaming for IPM methods of pest control. A number of researchers reported that many plants and microorganisms contain antifungal compounds31,32,33. Substances that are extracted from different parts of plants i.e. root, stem, leaves, bark, flower, fruit and seed, and essential oils (terpenes,) and by the microorganisms i.e. bacteria, fungi, etc. have antimicrobial properties32,33,34,35,36,37,38. Some species of fungi secrete secondary metabolites which possess the very specified activity and can be toxic to specific groups or groups of organisms. Fungi are known to have great potential as a biocontrol agent against pests since 196339,40. These days, fungal biological control is thought to be a quickly developing characteristic phenomenon in modern research for better plant yield41. Penicillium is a predictable source of bioactive metabolites. Penicillium species secrete an expanded range of extracellular active secondary metabolites, having effective mycotoxins as well as antibacterial and antifungal properties42,43. A number of Penicillium species have been reported to have antagonistic potential against many fungal pathogens hence, are used to control fungal diseases like root rot of Okra caused by Fusarium solani44, charcoal rot of Sorghum caused by Macrophomina phaseolina45, Cercospora leaf spot of Sugar beet caused by Cercospora beticola46, rice blast caused by Pyricularia oryzae47, charcoal rot of Mung bean caused by Macrophomina phaseolina48, etc. Few Penicillium species are well recognized because of their antagonistic activity against pathogens by producing antibiotics and persuading resistance in their hosts by triggering various defense signals49.

Keeping in view the problems of pathogens; the main objective of the present study was to evaluate the efficacy of extracellular secondary metabolites from some Penicillium species for their eventual use.

Results

Effect of metabolites of Penicillium species on fungal biomass production

Penicillium species metabolites significantly reduced the biomass production of the target pathogen. However, variability in the effect of metabolite extracts was observed.

Effect of metabolites of Penicillium janczewskii

The antifungal potential of P. janczewskii was evaluated against P. herbarum where the obtained data revealed a sharp reduction in fungal growth production with the increase in the concentration of P. janczewskii extract. The fungal growth and the extract concentrations demonstrated a nonlinear relationship with R2 = 0.8564. Overall, a 7–38% reduction in fungal biomass production was observed over the control (Figs. 1 and 2).

Figure 1
figure 1

Effect of metabolite concentrations of P. janczewskii on the growth of P. herbarum.

Figure 2
figure 2

Effect of different metabolite concentrations of P. janczewskii on the biomass production of P. herbarum. Vertical bars show standard errors of the means of three replicates.

Effect of metabolites of P. digitatum

Antifungal activity of various concentrations of P. digitatum on the biomass production of P. herbarum was evident from the results obtained as all the concentrations significantly retarded the growth of the targeted pathogen gradually. A non-linear relationship was recorded between fungal biomass and extract concentrations with R2 = 0.8096. The lowest concentration (10%) of P. digitatum extract proved very effective as it induced approximately 46% suppression in fungal biomass production while the highest concentration of 60% demonstrated about 57% biomass inhibition (Figs. 3 and 4).

Figure 3
figure 3

Effect of metabolite concentrations of P. digitatum on the growth of P. herbarum.

Figure 4
figure 4

Effect of different metabolite concentrations of P. digitatum on the biomass production of P. herbarum. Vertical bars show standard errors of the means of three replicates.

Effect of metabolites of P. verrucosum

The results obtained from the biomass assays of P. herbarum in different metabolites concentrations of P. verrucosum exhibited a similar pattern of growth inhibition as depicted by P. digitatum (Fig. 5). The fungal biomass showed a nonlinear relationship between biomass and extract concentrations with R2 = 0.7974. The lowest concentration i.e. 10% of P. verrucosum extract exhibited a sharp decline of approximately 47% in fungal biomass production. The higher concentrations (20–50%) resulted in growth inhibition in the range of 47–55% with some insignificant differences. While the maximum arrest of about 58% in fungal biomass production was evidenced at the highest concentration (60%) of the employed extract (Fig. 6).

Figure 5
figure 5

Effect of metabolite concentrations of P. verrucosum on the growth of P. herbarum.

Figure 6
figure 6

Effect of different metabolite concentrations of P. verrucosum on the biomass production of P. herbarum. Vertical bars show standard errors of the means of three replicates.

Effect of metabolites of P. crustosum

Data pertaining to the effect of different concentrations of P. crustosum on the biomass production of P. herbarum depicted that the growth of the targeted pathogen was found to be retarded gradually with the increase in metabolites concentrations. The relationship between fungal biomass and the employed extract concentration is nonlinear with R2 = 0.9114. The lower concentrations i.e. 10–30% demonstrated the suppression in biomass production in the range of 27–33% while the higher concentrations of 40–60% caused approximately 40–68% reduction in fungal biomass production (Figs. 7 and 8).

Figure 7
figure 7

Effect of metabolite concentrations of P. crustosum on the growth of P. herbarum.

Figure 8
figure 8

Effect of different metabolite concentrations of P. crustosum on the biomass production of P. herbarum. Vertical bars show standard errors of the means of three replicates.

Effect of metabolites of P. oxalicum

Metabolite extract obtained from P. oxalicum reduced the growth of P. herbarum in all concentrations in an almost similar manner as depicted by other species of Penicillium. A nonlinear relationship was displayed between the biomass of the target fungus and extract concentration with R2 = 0.9696. The lowest concentration (10%) induced aba out 21% decline in fungal growth production. The effect of 20–30% concentrations of P. oxalicum was significantly higher than this i.e. in the range of 32–38%. However, 40 and 50% concentrations showed an insignificant reduction in biomass production among each other but significant with respect to control and the lower concentrations treatments. Conversely, the maximum reduction of about 51% was observed at the highest concentration i.e. 60% (Figs. 9 and 10).

Figure 9
figure 9

Effect of metabolite concentrations of P. oxalicum on the growth of P. herbarum.

Figure 10
figure 10

Effect of different metabolite concentrations of P. oxalicum on the biomass production of P. herbarum. Vertical bars show standard errors of the means of three replicates.

Determination of kinetic constants of inhibition for P. herbarum

The metabolic extracts of Penicillium species were found to be very effective and highly significant against fungal pathogens. To find the inhibition constants regression equation was used and determined the regression of fungal biomass production versus various concentrations of metabolite extracts of Penicillium species. From the regression equation, the reduced fungal biomass by 50% of the control was determined by all the concentrations of metabolite extracts of Penicillium species (Figs. 11, 12, 13, 14 and 15). Calculated K.I values of the pathogen were presented in Table 1. The K.I results based upon all treatments provided a range of 22.54–57.64. The metabolite extracts of all species of Penicillium (P. janczewskii, P. verrucosum, P. crustosum, P. digitatum, and P. oxalicum) showed the K.I values 22.54, 23.56, 44.70, 45.64, 57.64, respectively. The metabolite extract concentration of P janczewskii depicted a minimum K.I value than the other 4 species which demonstrated that the fungal growth of P. herbarum was statistically most significantly inhibited by P. janczewskii.

Figure 11
figure 11

Kinetic constants of inhibition of P. herbarum by metabolites of P. janczewskii (3 replicates).

Figure 12
figure 12

Kinetic constants of inhibition of P. herbarum by metabolites of P. digitatum (3 replicates).

Figure 13
figure 13

Kinetic constants of inhibition of P. herbarum by metabolites of P. verrucosum (3 replicates).

Figure 14
figure 14

Kinetic constants of inhibition of P. herbarum by metabolites of P. crustosum (3 replicates).

Figure 15
figure 15

Kinetic constants of inhibition of P. herbarum by metabolites of P. oxalicum (3 replicates).

Table 1 Kinetic constants for fungal biomass inhibition by Penicillium metabolites.

Effect of Penicillium metabolites on the expression of STE12 gene

The test pathogen (P. herbarum) has the ability to form appressorium. Thus, for the development of appressorium to penetrate into the host tissue STE12 gene is required. P. herbarum was grown in malt extract employed with varied concentrations of metabolites of the most potent antagonist, P. janczewskii to evaluate the effect of Penicillium metabolites on the transcript level of STE12 using Real-time reverse transcription PCR (qPCR). The STE12 expression level was compared with the expression of the housekeeping gene, partial Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) coding gene. In order to optimize the annealing temperature for selected STE12 amplifying primers and housekeeping gene, a number of PCRs were conducted with a range of annealing temperatures from 50 to 65 °C using fungal genomic DNA as template. Optimum amplification of both pairs of genes was achieved at 60 °C (Fig. 16).

Figure 16
figure 16

Agarose gel electrophoresis of GAPDH and STE12 genes amplified using DNA of P. herbarum.

RNA of the pathogen grown in different concentrations of P. janczewskii metabolites was isolated using GeneAll® biotechnology kit and its concentration was determined. cDNA was synthesized from the extracted RNA from all the treatments of the pathogen and the concentration of cDNA was also measured using a NanoDrop® spectrophotometer.

Quantitative gene expression analysis by real-time PCR

qPCR results clearly demonstrated the expression of STE12 as well as GAPDH in all treatments of P. herbarum. However, different levels of expression were observed for the STE12 gene in different treatments. The threshold (CT) value remained similar in all treatments which is a clear indication that an equal amount of cDNA was used for each reaction mixture. ∆∆Cq was calculated to check the relative expression of STE12 genes with the GAPDH gene that showed unchanged expression by metabolites. ∆∆Cq values were used to calculate the % Knockdown (KD) of gene expression of the quantification cycle that indicated the increase or decrease in gene expression. An increase in the % Knockdown value means decreased expression level.

The expression pattern of the STE12 gene as determined by the % Knockdown value in P. herbarum grown in various concentrations of Penicillium janczewskii metabolites is recorded in Table 2. It was revealed from the results that there is a decrease in expression Knockdown values with the increase in metabolite concentrations. About 51.479% Knockdown of STE12 encoding gene was noticed when the P. herbarum was grown in 10% metabolites stress. At 20% metabolites concentration, the % Knockdown value was decreased to 43.224% hence increase in expression of the STE12 gene was recorded. The results exhibited a further decrease in % KD values 40.67, 38.01, 35.97, and 33.41 in P. herbarum employed with 30, 40, 50, and 60% metabolite concentrations, respectively.

Table 2 % Knockdown values of real-time PCR of Phoma herbarum treatments against Penicillium janczewskii.

Figure 17 also displayed a similar pattern of STE12 gene expression. At 0% concentration, the gene was amplified and detected. It was observed that as the concentrations were increased from 10 to 60% (maximum), the % Knockdown values decreased accordingly.

Figure 17
figure 17

Gene expression of STE12 Gene in P. herbarum against Penicillium janczewskii.

In-silico tools predicted direct involvement of Ste12 in MAPK signaling pathway

The results depicted MAPK signaling pathway cascade prevalent in filamentous fungi that were predicted through the in-silico tool, KEGG database. It was revealed that the transcription factor Ste12 has direct involvement in this pathway in response to pheromones and under low nutrient conditions (starvation) as shown in Fig. 18.

Figure 18
figure 18

MAPK signaling Pathway in filamentous fungi developed by using KEGG pathway database50 with permissions from Kanehisa Laboratories (KEGG orthology entry: K11215): The cascade of MAPK is activated through kinases and phosphatases in all eukaryotes. In fungi, it maintains the survival rate during various stress conditions through adaptations. Ste12 a fungal transcription factor involves in response to stimulation through pheromones and low nutrient conditions to endure with increased mating and filamentation ability respectively. Source: https://www.genome.jp/dbget-bin/www_bget?sce:YHR084W.

Discussion

Mung bean is a fast-growing legume and is a good source of dietary protein, calcium, and iron. The yield of this agricultural crop is most commonly reduced by plant diseases. Among these diseases, the harm caused by plant pathogens impacts about 13% of yield losses per annum worldwide51. Among the number of pathogens, over and above 80% of plant diseases and momentous damages to the human diet are due to Fungi. Among different constraints; the most distressing disease of mung bean is leaf spot disease which is caused by innumerable mycological pathogens including Alternaria, Phoma, Drechslera, etc. Different techniques are in use for the prevention and/or control of plant diseases52. The biological control method is considered to be the most effective way to regulate fungi. Many plant species and various plant extracts e.g., eucalyptus, neem, garlic, black pepper, ginger, and many weeds have been analyzed for their antifungal potential with the intent of ascertaining environmentally harmless and cost-effective alternatives for the control of diseases32,33,53,54. Besides the plant extracts; various microorganisms particularly a number of fungal and bacterial species are known to have biocontrol activity55,56,57. Most of the antagonistic species of fungi and bacteria are well known to have effective biocontrol potential against various plant diseases33, especially in fruits and crop plants. Presently, the antifungal potential of metabolites extracts of 5 Penicillium species was tested against P. herbarum to evaluate their biological control potential. It was obvious from the findings that Penicillium species had the innate capability to induce antagonistic effects on the fungal pathogen. The relative intensity of this effect however varied with the species involved, as well as the particular concentrations of the extract employed. The metabolites extract of all Penicillium species significantly reduced the fungal biomass of the target pathogen. Penicillium species are well recognized to secrete a wide range of bioactive metabolites including siderophore, indole acetic acid (IAA), hydrocyanic acid (HCN), lipase, protease, and β-1,3 glucanase that not only facilitate iron uptake in plants but also mediate disease suppression58. In accordance with our present study, Alam and coworkers59 scrutinized the effect of Penicillium sp. EU0013 on Fusarium wilt disease. In dual culture experiments, EU0013 significantly inhibited the growth of Fusarium wilt pathogens on tomato (Solanum lycopersicumL.) and cabbage (Brassica oleraceaL.). In a parallel study, Sreevidya and Gopalakrishnan45 reported the production of citrinin, a secondary metabolite, by Penicillium citrinum VFI-51 which proved to be responsible for regulating the Botrytis gray mold disease in chickpea.

It was observed that the effectiveness of the extracts was found to be associated with the resistance or susceptibility offered by different species of Penicillium. Presently, the percentage inhibition in biomass production was different for all Penicillium species. Inhibition in biomass production indicated that antifungal compounds may be produced by Penicillium species. However, variation in inhibition in biomass production showed differences in the efficiency of each Penicillium species against the pathogen. In a contemporary study, Mamat et al.60 evaluated 7 strains of Penicillium oxalicum against Colletotrichum gloeosporioides. Their findings indicated that among the seven endophytic potential strains, P. oxalicum T3.3 demonstrated the most potent antagonistic activity towards C. gloeosporioides by producing the large inhibition zone against the pathogen tested. Several other workers also reported P. oxalicum to produce an inhibition zone against a wide range of pathogenic fungi during a dual culture test58,61,62.

Currently, it was evident from the antifungal bioassays that all the employed concentrations of metabolite extract suppressed the fungal growth but the highest concentration of metabolites of all Penicillium species suppressed biomass production up to 90 to 95%. In a similar kind of research Trichoderma species viz., Trichoderma viride, Trichoderma aureoviride, Trichoderma reesei, Trichoderma koningii, and Trichoderma harzianum showed a good potency as an antifungal agent against Alternaria citri. Among all culture filtrates T. harzianum was found to be highly effective in subduing the growth of test fungal species up to 93%63.

In the present study, although a significant reduction in biomass production of the pathogen was observed by all concentrations of all metabolites types, however, P. janczewskii proved the most toxic for the fungal growth as it induced more than 50% inhibition in fungal biomass production with the least KI values. These findings are in good agreement with the previously published results that showed the extracts of Trichoderma isolates had good activity against the plant pathogenic fungus Alternaria alternata64.

Real-time PCR is the most efficient molecular tool in determining the role of genes in disease development65. For real-time PCR, reference genes with stable expression under stimuli or stress play a vital role in comparison and conclusion. The most widely used reference genes are β-tubulin66, GAPDH67, and actin65. Presently, the GAPDH gene was used as a housekeeping gene and the narrow range of cycle threshold (Ct) was observed under all tested conditions. Results of the present study revealed that the higher the concentration of the metabolite more is the transcript for the STE12 gene in P. herbarum. It has been reported that MAPK homologs also regulate the conidia formation and under stress, organisms try to form more spores which could be the possible reason for lower Knockdown values in high metabolites conditions68. In another study by Park and colleagues65, it was observed that under oxidative stress the expression of the STE12 gene is unregulated in rice blast fungus Magnaporthe oryzae. The possibility of new breakthroughs in the control of pathogens involves a better understanding of the virulence mechanisms deployed by E. rostratum as pathogen aggressiveness is controlled by the interactions of several genes that react to signals that appear during host–pathogen interactions24,65.

The pathogenicity of the P. herbarum was carried out through the appressorium which is a specialized cell that has a high ability for an invasion via conidia or hyphae. In fungi, the presence of external stimuli including pheromones, starvation, hyper-osmolarity, and stress condition activates the Mitogen-activated protein kinase (MAPK) pathway for survival69. The MAPK pathway is a major signaling system that controls a variety of biological functions in fungi such as cell cycle, growth, differentiation of cells, virulence, and increase in their survival70. MKPs play a role in mycelial growth and pathogenicity in filamentous fungi, activated through many transcription factors71. Ste12 is a fungal transcription factor responsible for the regulation of other genes and also induces mycelial adaptations during infection as depicted in Fig. 13. In response to the pheromones, phosphorylated Fus3 (Mitogen-activated protein kinase FUS3) activates the transcription factor Ste12 to increase the interaction with DNA to transcribed Fus1 (Mitogen-activated protein kinase FUS1) which will adapt the fungi to enhance its mating ability30. While in the environment of deprived nutrients, MAPK-pathway permits adjustment by activating Ste12 through Kss1 (Mitogen-activated protein kinase gene Kss1) to increase the transcription of Flo11 which has a crucial role in filamentation to increase access to food72. Treatment with secondary metabolites of Penicillium as biocontrol reduced the expression of mRNA of Ste12 which will interrupt the MAPK cascade to attain adaptive responses during infection of P. herbarum.

Thus, the bioassays in the present study conclude the utility of Penicillium metabolites that possessed the strongest antifungal potential against P. herbarum as these metabolites are the precious benediction of nature for disease management against the most devastating pathogen by acting as defense materials against it.

Materials and methods

Phoma herbarum, isolated and identified as a leaf spot pathogen of mungbean in Pakistan73 was obtained from the Fungal Biotechnology research lab, Department of Plant Pathology, Faculty of Agricultural Sciences, University of the Punjab, Lahore, Pakistan. The biological control potential of the metabolites of five Penicillium species was assessed to control Phoma herbarum.

Selection of fungus as a biocontrol agent

Five Penicillium strains (Penicillium oxalicum FCBP-1075, Penicillium crustosum FCBP-1159, Penicillium janczewskii FCBP-1179, Penicillium digitatum FCBP-1160, and Penicillium verrucosum FCBP-1162 A-3) were acquired from the First Fungal Culture Bank of Pakistan, Department of Plant Pathology, Faculty of Agricultural Sciences, University of the Punjab, Lahore on freshly prepared MEA Petri plates. These cultures were utilized in subsequent metabolites extract preparation.

Penicillium metabolites extraction

To prepare a stock solution of fungal metabolites extract, 2% malt extract was prepared and a disc of about 5 mm from freshly revived culture plates was inoculated in each flask containing 100 mL broth medium. The same method was repeated for other strains of Penicillium and left to grow at 25 ± 2 °C for about two weeks. After 15 days, with the completion of fungal mycelium, the metabolites were filtered through 2–threefold of sterilized Whatman filter paper 4 under aseptic conditions and preserved at 4 °C for subsequent use as a biocontrol agent74.

Fungal growth assay

To perform fungal growth assay in MEA broth, 7 concentrations viz., 0%, 10%, 20%, 30%, 40%, 50%, and 60% of each Penicillium strain were prepared in 60 mL of 2% broth medium containing 1.2gm malt extract per treatment flask. For fungal growth assays, these seven concentrations 0%, … 60% were prepared by adding 0, 6, 12, 18, 24, 30, 36 mL stock solutions (metabolite filtrate) to each flask containing 60 mL, 54 mL, 48 mL, 42 mL, 36 mL, 30 mL, 24 mL of broth, respectively and the final volume was raised up to 60 mL. 0% concentration was selected as the control treatment to compare the results of antifungal activity. The metabolite extract concentrations were divided to make three replicates of each concentration and subsequently simmered into an autoclave at 60 °C for about 20 min with zero pressure74. After simmering, 1 disc of 0.2 mm size from pure fungal culture plates of the pathogen was inoculated in every treatment separately and incubated at 25 ± 3 °C for 8–10 days until the growth in the control treatment reached its maximum. After 10 days, the fungal biomass was collected from all the replicates on the pre-weighed filter papers, oven-dried at 55–60 °C and dry biomass was obtained. The dry weight of biomass was used to assess the efficacy of each concentration of metabolites of all Penicillium strains. Percentage decreases or increases in fungal biomass due to various concentrations of employed extracts were determined by the following formula:

$$ {\text{Biomass inhibition }}\left( \% \right) \, = \,\frac{{{\text{Biomass in control }} - {\text{ Biomass in treatment}}}}{{\,\,{\text{Biomass in control}}}}\,\, \times \,\,100. $$

Percentage inhibition constants were evaluated by a decrease in fungal biomass with respect to various concentrations of metabolite extracts of different Penicillium strains using regression analysis.

Effect of Penicillium metabolites on the expression of STE12 gene

The effect of different concentrations of selected Penicillium metabolite (with maximum antifungal potential) on the transcript level of StSTE12 was studied. The partial Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) coding gene was selected as a housekeeping gene for comparison. The detail of the primers used in the study is presented in Table 3.

Table 3 Detail of genes and primers used for gene expression studies.

Primers were designed based on GenBank database information. Therefore, their specificity was checked by PCR amplification reactions using the fungal genomic DNA of the pathogen and the selected primer pair. The annealing temperatures of selected primers were also optimized.

Quantitative gene expression analysis

RNA from the fungus mycelia grown under selected treatments was isolated using a commercially available RNA isolation GeneAll® biotechnology kit and immediately treated with DNAase enzyme to avoid its degradation. To perform a Real-Time Polymerase Chain Reaction, isolated RNA was converted to complementary DNA (cDNA). The reaction was carried out at 55 °C for 1 h and stopped by incubating the mixture first at 85 °C for 5 min then on ice for 5 min. The synthesized cDNA was stored at − 20 °C until further used. Concentrations of the cDNA were measured using NanoDrop®. The cDNA was then diluted to make the concentration 200 ng/µl to ensure an equal amount of cDNA in the Real-Time PCR analysis. qPCR was conducted using two primers StSTE12 experimental primer and GAPDH as control primer because its expression remains the same throughout the experiment (Supplementary Fig. 1).

Real-time PCR

SYBR® Green Master Mix was used for quantitative gene expression studies in a 20 µL reaction mixture containing 2 µL of cDNA, 0.7 µL each of forward and reverse primer (10 µM), and 10 µL SYBR® green master mix. The PCR reaction was carried out as; one cycle at 95 °C for 10 min followed by 40 cycles each of denaturation at 95 °C for 15 s, annealing at 60 °C for 30 s, and elongation at 72 °C for 30 s.

In-silico studies

The Kyoto Encyclopedia of Genes and Genome (KEGG) database (https://www.genome.jp/kegg/) was used to predict the possible role of the Ste12 transcription factor in the MAPK signaling pathway. It’s an open-source database that includes 16 major databases providing a wide range of systematic genome information50.