Introduction

Root rot of cocoyam (Xanthosoma sagittifolium) caused by Pythium myriotylum is a serious disease in sub-tropical countries including Cameroon (Gomez-Alpizar et al. 2011). Infection occurs in the high humidity conditions during the rainy season. The first symptoms are yellowing and wilting of leaves, root rot, drying, and the infection often results in the death of the whole plant (Adiobo et al. 2007). In the absence of control measures, yield loss could reach up to 80–100%. The most practical methods employed by farmers are the use of resistant cultivars and chemical fungicide. Since the pathogen is soil borne, application of fungicide is difficult in humid conditions, expensive, and their repeated application could result in soil contamination, fungicide resistance, and could be harmful to non-target organisms (Tambong and Hofte 2001).

Hence, alternatives to reduce the use of chemical fungicides, aimed to enhance the productivity with less negative environment and ecological consequences, are needed. Biological control by using beneficial soil, belonging microorganisms, bacteria, and fungi species, are promising methods (Woo et al. 2014). Several species of Trichoderma have been successfully used for biological control of soil borne pathogens (Vinale et al. 2008; Lorito et al. 2010; Tchameni et al. 2017). Mechanisms used by fungi as biological control agents against plant pathogens include competition for nutrients, induced systemic resistance, mycoparasitism, production of antibiotics, and extracellular lytic enzymes. During antagonism, Trichoderma produces volatile and non-volatile organic compounds such as polyphenols, flavonoids, terpenes, pyrones, sesquiterpenes, ketones, thioesters, cyclohexanes, and alcohols (Reino et al. 2008; Vinale et al. 2014). Many studies have shown that the production of these secondary metabolites was associated with inhibition of mycelial growth and spore germination of many plant pathogens (Zhang et al. 2014; Vos et al. 2015; Nieto-Jacobo et al. 2017). In addition, the antagonistic effect of Trichoderma is correlated with the production of extracellular lytic enzymes such as cellulases, proteases, chitinases, and xylanases (Harman 2006). However, successful antagonism often results from synergistic effects from different mechanisms responsible for an antimicrobial interaction.

This work was aimed to evaluate the antimicrobial activity of organic extracts of 4 strains of Trichoderma and the involvement of their proteases, cellulases and xylanases in the inhibition of Pythium myriotylum.

Materials and methods

Pathogen

Pythium myriotylum was isolated from the root of cocoyam showing the typical symptom of root rot disease. Infected roots were rinsed in distilled water and disinfected with sodium hypochlorite (1%) for 5 min. Roots were cut (3–5 mm) and inoculated on a potato dextrose agar (PDA) medium supplemented with penicillin (250 mg/mL) and ampicillin (250 mg/mL) and incubated at 28 °C. Plates were monitored daily, and the suspect pathogen mycelia were transferred in the fresh medium. Pythium myriotylum was identified using the protocol described by Agrios (2005). The pathogen was maintained in PDA medium and stored at 4 °C.

Trichoderma strains

Four strains of Trichoderma were used in this study. These strains came from core culture of the mycotheque of the Laboratory of Biochemistry, University of Douala. These isolates had been previously characterized using microscopic and molecular techniques. The obtained nucleotide sequences were submitted to the NCBI (National Center Biotechnology Information) GenBank data (www.ncbi.nlm.gov/BLAST) for annotation. Sequences are available under accession numbers: JN157757, KX009499, KP115287, and KF040478 for Trichoderma erinaceum (IT-58), Trichoderma gamsii (IT-62), Trichoderma afroharzianum (P8), and Trichoderma harzianum (P11), respectively.

In vitro antagonism assay

Five-millimeter plug of each strain of Trichoderma taken from 2-day-old culture was inoculated at one side of Petri dish containing PDA medium. A 3–5-mmmycelial plug from 5-day-old PDA culture of P. myriotylum was inoculated on the perpendicular opposite side. Plates inoculated only with pathogen served as control. All the plates were incubated at 30 °C for 7 days. The growth of pathogen in both the test and control plates was recorded. The inhibition of mycelial growth of the pathogen was evaluated according the formula: I (%) = ((Do − Dt)/Do)) × 100, where Do is the diameter of growth of the pathogen and Dt is the diameter of growth of the pathogen in paired culture. Each treatment consisted of three replicates, and the experiment was repeated twice.

Assay of proteases

The culture medium (25 mL) used for protease production was a mineral medium containing the following (g/L): MgSO4 0.52, KCl 0.52, KH2PO4 1.52, FeSO4 × 7H2O 0.01, ZnSO4 × 7H2O 0.01, and casein 5. The culture medium was first inoculated with a 0.5-cm mycelial plug and incubated at 25 ± 2 °C for 7 days. The culture was then centrifuged (5000 rpm, 10 min, 4 °C), and the supernatant (crude enzyme) was collected for analysis. A proteolytic activity assay, using casein as the substrate, was performed according to the method described by Ramakrishna and Pandit (1998) with some modifications. Enzyme activity was determined by incubating 250 μL of the culture supernatant with 500 μL 1% (w/v) casein sodium salt in 50 mM buffer pH 7.0 for 2 h at 30 °C. The reaction was stopped by the addition of 375 μL 20% (w/v) trichloroacetic acid. The tubes were placed in an ice bath for 30 min and then centrifuged at 5000 rpm for 15 min at 4 °C, and the absorbance of the supernatant was measured at 280 nm versus an appropriate blank. The specific protease activity was defined as the amount of enzyme that hydrolyzes casein to produce equivalent absorbance to 1 μmol of tyrosine/min/mg of protein, with tyrosine as standard. The soluble protein content in supernatant was estimated by the method described by Bradford (1976).

Assay of cellulases

The cellulase activity was assayed based on the method described by Gautam et al. (2011). To initiate the cellulase assay, 25 mL of culture broth (3 g NaNO3, 0.1 g KH2PO4, 0.5 g MgSO4 × 7H2O, 0.5 g KCl, 1% CMC) was first inoculated with a 0.5-cm mycelial plug and incubated at 25 ± 2 °C for 7 days. The culture was then centrifuged (5000 rpm, 10 min, 4 °C), and the supernatant (crude enzyme) was collected for analysis. The supernatant (0.5 mL) was mixed with 1.5 mL reaction mixture (0.5 mL of 1% CMC, 1 mL of 0.1 M sodium citrate buffer at pH 4.8) and incubated for 60 min at 50 ± 2 °C. The reducing sugar produced was determined using dinitrosalicylic acid (DNS) (Miller 1959) where 3 mL of DNS reagent was added to the reaction mixture, heated at 90 °C for 10 min, followed by the addition of 1 mL of 40% potassium sodium tartrate. After cooling, the absorbance was read at 540 nm. The amount of glucose released was calculated from the glucose standard curve (0–35 μmol/L). The specific cellulase activity was defined as the amount of enzyme needed to release 1 μmol of glucose per min per mg of protein under assay conditions described.

Assay of xylanases

Five-millimeter mycelial plug (7 days) of each strain of Trichoderma was incubated into 250-mL Erlenmeyer flasks containing 50 mL of isolation media (devoid of agar) during 7 days under agitation at 180 rpm. The culture was filtered (Whatman no. 1), the filtrate was then centrifuged (5000 rpm, 10 min, 4 °C), and the supernatant (crude enzyme) was collected for analysis. Xylanase activity was assayed in 2% corncob powder, 0.4% NH4NO3, 0.05% K2HPO4, 0.05% MgSO4 × 7H2O, 0.05% KCl, and 0.001% FeSO4 × 7H2O. The xylanase activity was measured using 3,5-dinitrosalicylic acid (DNS). At 50 °C and pH 5.0, activity of xylanase was defined as the amount of enzyme required to release 1 μmol xylose per minute per mg of protein. Xylose was used as standard for xylanase analysis.

Liquid culture and extraction of organic compounds

Two 5-mm-diameter plugs of each Trichoderma strain, obtained from actively growing margins on PDA cultures, were inoculated into 1-L conical flasks containing 250 mL of sterile potato dextrose broth (PDB). Cultures were incubated for 14 days at 30 °C and 120 rpm. The cultures were filtered under vacuum through filter paper (Whatman No. 4; Brentford, UK), and the filtrates stored at 4 °C for 24 h. The organic metabolites were extracted exhaustively with ethyl acetate (3 times), and the combined organic fraction was dried (Na2SO4) and evaporated under reduced pressure at 40 °C. The red-brown residue recovered was stored at 4 °C until further analysis.

Evaluation of total phenol and total flavonoid

Total Polyphenol Content

The total polyphenol content was determined by applying the Folin-Ciocalteu method (Cudalbeanu et al. 2018) to a 96-well plate analysis. Twenty-five microliters of Folin-Ciocalteu reagent was added to 10 μL of extract. After 5 min of incubation, 25 μL of a 20% aqueous sodium carbonate solution and then ultrapure water was added until the final volume reached 200 μL. Blanks were also prepared for each sample by replacing the Folin-Ciocalteu reagent with ultrapure water. A freshly prepared gallic acid solution was used as a standard reference, and the results are given in equivalents of gallic acid per 1 g of sample. After 30 min, the absorbance values of the samples at 760 nm were recorded using a multiwell plate reader (Tecan Pro 200).

Total Flavonoid Content

The total flavonoid content was quantified through a 96-well plate analysis using aluminum chloride. One hundred microliters of a 2% aqueous aluminum chloride solution was added to 100 μL of sample. After 15 min of incubation, the absorbance values at 415 nm were determined using the Tecan Pro 200 multiwell plate reader. Quercetin was used as a reference standard, and the results are given in equivalents of quercetin per 1 g of sample.

VOC analysis by GC-MS

For the identification of volatile compounds (VOCs) from Trichoderma spp., the concentrated extracts were dissolved in 100 μL of methanol, passed through a 0.45-μm disposable PTFE filter, and used for GC-MS analysis. The GC-MS analyses were performed on a Varian 4000 electron impact mass spectrometer using a Varian CP-8400 injector. A 30 m × 0.25-mm factor four capillary column with a particle size of 0.25 μm (Varian) was used. The injection temperature was set at 250 °C. The helium gas flow rate through the column was 1 mL/min, ions were generated at an electron impact (EI) of 70 kV, the ion source temperature was set at 200 °C, and the mass range was m/z 50–1000. The column temperature was maintained isothermally at 70 °C for 2 min and then raised to 300 °C at a rate of 10 °C/min. Authentic analytical standards were employed for retention time and retention index comparison with the VOCs detected. VOCs were identified against a library of mass spectra (National Institute of Standard and Technology v2.1 mass spectral database).

Antimicrobial assay of organic extract

The organic crude extracts were tested in vitro against P. myriotylum to evaluate their antimicrobial properties. Pathogen plugs (5-mm diameter) from growing edges of colonies were placed in the center of Petri dishes containing PDA. Ten microliters of crude extracts, at concentrations varying from 10 to 80 μg/μL, was applied on the top of each plug. Controls were obtained by applying 10 μL of DMSO. The solvent was evaporated in a laminar flow cabinet, and the plates were incubated at 30 °C for 5 days. The pathogen growth was measured daily as colony diameter. Each treatment consisted of three replicates and the experiment was repeated twice. The growth inhibition (GI) was determined on the third day using the following equation: GI = ((Do − Dt)/Do) × 100 where Do is the diameter of mycelial growth of pathogen and Dt is the diameter of mycelial growth of pathogen during antagonism.

Statistical analysis

Statistical analyses of the data were performed using STATGRAPHICS Computer Software, version 5.0. The differences among treatments for all the studied characteristics mentioned above were analyzed for statistical significance using variance analysis (ANOVA). Treatments were compared by using Duncan’s multiple range test at the p < 0.05 significance level.

Results

In vitro antagonism of Trichoderma

Four strains of Trichoderma were tested for their ability to inhibit mycelial growth of P. myriotylum in vitro by the paired culture technique. The results showed that all the strains used inhibit the mycelial growth of P. myriotylum (Table 1). The degree of inhibition was 48.68%, 50.67%, 57.0%, and 63.33%, respectively, for T. erinaceum (IT-58), T. gamsii (IT-62), T. afroharzianum (P8), and T. harzianum (P11). According to these results, statistical analysis revealed that there is significant difference (p ≤ 0.05) between the inhibitions percentage.

Table 1 Inhibitory effects of Trichoderma against P. myriotylum in dual culture
Full size table

Effects of organic crude extract of Trichoderma spp. on mycelial growth of P. myriotylum

All the organic extracts from the strains of Trichoderma used significantly inhibited the mycelial growth of P. myriotylum (Table 2). The total inhibition growth was evaluated at four different concentrations for extracts from T. erinaceum (IT-58), T. gamsii (IT-62), T. afroharzianum (P8), and T. harzianum (P11), respectively. Negative and significant correlation was obtained between the antagonistic activity and the concentration of inhibition of mycelial growth of crude organic extract. The organic extract from T. harzianum (P11) which had the highest antagonistic activity also had the minimum inhibitory concentration among all the organic extracts.

Table 2 Inhibitory effects of organic extract from Trichoderma against P. myriotylum
Full size table

Activity of extracellular lytic enzymes

Activity of cellulase

Trichoderma harzianum (P11) recorded the highest activities of cellulase followed by T. afroharzianum (P8), T. gamsii (IT-62), and T. erinaceum (IT-58) (Fig. 1). Trichoderma harzianum (P11), which recorded the highest inhibition, exhibited the highest cellulase activity; T. erinaceum (IT-58), which recorded least inhibition, produced the lowest cellulase activity.

Fig. 1
figure 1

Activity of cellulases produced by Trichoderma

Full size image

Activity of xylanase

Results presented in Fig. 2 showed that T. erinaceum (IT-58) exhibited the highest xylanase activity followed by T. harzianum (P11), T. afroharzianum (P8), and T. gamsii (IT-62). T. erinaceum (IT-58) which had the lowest antagonistic effect recorded the highest xylanase activity.

Fig. 2
figure 2

Activity of xylanases produced by Trichoderma

Full size image

Activity of protease

The results of protease activity are presented in the Fig. 3. T. harzianum (P11) recorded the highest activity followed by T. afroharzianum (P8), T. gamsii (IT-62), and T. erinaceum (IT-58). T. harzianum (P11) which exhibited the highest antagonistic activity had the highest activity of protease.

Fig. 3
figure 3

Activity of proteases produced by Trichoderma

Full size image

Total phenolic compounds and flavonoid content

The highest total phenolic compounds were produced by T. harzianum (P11) followed by T. afroharzianum (P8), T. gamsii (IT-62), and T. erinaceum (IT-58) (Fig. 4). The flavonoid production was also maximum with T. harzianum (P11) followed by T. afroharzianum (P8), T. erinaceum (IT-58), and T. gamsii (IT-62) (Fig. 5). A significant correlation was observed between inhibitory effect of secondary metabolites and the production of total phenols and flavonoids. T. harzianum (P11) which has the lowest minimal inhibitory concentration exhibited the highest production of phenols and flavonoid compounds.

Fig. 4
figure 4

Polyphenol content in organic extract produced by Trichoderma

Full size image
Fig. 5
figure 5

Flavonoid content in organic extract produced by Trichoderma. IT-58 Trichoderma erinaceum, IT-62 Trichoderma gamsii, P8 Trichoderma afroharzianum, P11 Trichoderma harzianum. According to DUNCAN test, the same letters are not significantly different at p < 0.05. Each treatment was made by three replicates, and the experiment was repeated twice

Full size image

Analysis of volatile organic compounds

Volatile organic compounds were analyzed by GC-MS. Results showed that the quality and the quantity of volatile compounds produced are variable for each strain of Trichoderma (Table 2). GC-MS analysis of extract from T. erinaceum (IT-58) showed the presence of 22 different components representing 98.75%. The major components were 2-(3-acetoxy-4,4,10,13,14-pentamethyl-2,3,4,5,6,7,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl) propanoic acid (34.19%); 15,17,19-nonacosatriynoic acid (14.70%); and 6β-hydroxyfluoxymesterone (11.60%). Results of analysis of T. gamsii (IT-62) revealed the presence of 20 different compounds representing 98.33%. The main components were 1,3-dioxolane-2-(1-hydroxyethyl)-methylate (24.64%) followed by 2,6-Bis(1,1-dimethylethyl)-4-(1-oxopropyl) phenol (12.06%) and 2-myristynoyl pantetheine (10.11%). Chemical analysis of volatile compounds from T. afroharzianum (P8) extract revealed the presence of 23 different compounds (97.8%), with ethyl iso-allocholate (33.76%) and 2-(3-acetoxy-4,4,10,13,14-pentamethyl-2,3,4,5,6,7,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl) propanoic acid (11.84%) as major components. Twenty-two compounds were obtained from GC-MS of T. harzianum (P11) organic extract (98.41%). Among them, 2-(3-acetoxy-4,4,10,13,14-pentamethyl-2,3,4,5,6,7,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl) propanoic acid (20.9%), 1,3-dioxolane-2-(1-hydroxyethyl)-methylate (14.50%), and harzianolide (12.13%) were the main compounds (Table 3).

Table 3 GC-MS analysis of volatile organic extract from Trichoderma
Full size table

Discussion

Trichoderma spp. have been successfully used in agriculture for the control of soil borne disease. In the present study, the four strains of Trichoderma tested were found to be effective in inhibiting the mycelial growth of Pythium myriotylum and the inhibiting percentage was variable for each of the species of Trichoderma used. T. harzianum (P11) performed the best in the antagonistic test. These results suggest that Trichoderma could produce different antimicrobial components. One of the major mechanisms used by Trichoderma as biocontrol agent is mycoparasitism. Several cell wall degrading enzymes such as chitinase, cellulase, xylanase, and glucanase are involved in the process. Many studies have been showed that biocontrol agents including Trichoderma species secrete such enzymes able to degrade cellulose, chitin, and glucan, which are the major constituents of cell wall of fungal and oomycete (Meena et al. 2001: Gruber et al. 2011; Saravanakuma et al. 2018). Viterbo et al. (2002) showed that, during the antagonism, Trichoderma produced chitinase, glucanase, and protease. Marco et al. (2003) demonstrated that T. harzianum produced a novel protease which inhibited the growth of C. perniciosa, the pathogen of witches’ broom disease. In the same way, several studies have been shown that the antagonistic activities of Trichoderma against soil borne pathogens are correlated with the production of extracellular lytic enzymes (Zhang et al. 2014; Kredics et al. 2005; Szekeres et al. 2004; Gajera et al. 2012). De la Cruz-Quiroz et al. (2018) showed that cellulase and chitinase produced by T. harzianum and T. asperellum were involved in the suppression of mycelial growth of Phytophthora capsici and Colletotrichum gloeosporioides. In the present study, the production of these enzymes could be implicated in the in vitro inhibition of P. myriotylum mycelial growth.

During the antagonistic activity, Trichoderma species are known to produce non-volatile and volatile organic metabolites (Lorito et al. 2010; Vinale et al. 2014). These compounds could play a key role during the microbe attack. Many studies have demonstrated that non-volatile secondary metabolites, mainly phenolic compounds and flavonoids, can inhibit the mycelial growth and spore germination of soil borne pathogens (Mazzei et al. 2016; Pakora et al. 2017). The present study showed that the non-volatile organic compounds produced by the four strains of Trichoderma significantly reduced the mycelial growth of P. myriotylum in vitro. This suggests that during the antagonism, these compounds may be involved in the suppression of mycelial growth of P. myriotylum. Similar results were obtained by Pakora et al. (2017) who reported that crude organic extracts from T. viride and T. harzianum inhibited the mycelial growth and spore germination of P. palmivora, P. megakarya, and P capsici, the oomycetes associated to cocoa black pod. Moreover, Leylaie and Zafari (2018) obtained a significant correlation between the anthraquinones (polyphenolic compounds) produced by Trichoderma and the antimicrobial activities against a panel of fungus pathogens. Our results also showed a significant correlation between the antimicrobial effects and the production of total polyphenols and flavonoids. Polyphenol compounds such as flavonoids could act by attaching to the cell wall of pathogens and disorganizing their structure. They could also be able to inhibit the activity of enzymes released by the pathogen (Vinale et al. 2014). The antimicrobial activities of secondary metabolites could be due to synergistic effects of non-volatile and volatile organic compounds. Many species of Trichoderma produce volatile organic compounds that have inhibited soil borne pathogens (Vinale et al. 2008, 2014). Volatile organic compounds confer resistance to Trichoderma against environmental stress factors and could protect its cell walls against the action of hydrolytic enzymes released by pathogens. In addition, in dual culture, the production of volatile compounds increased when Trichoderma was confronted with pathogens (El-Hasan et al. 2018). In the present study, all the strains used produced varying volatile organic compounds. Some of major compounds produced such as harzianolide, 6β-hydroxyfluoxymesterone, and 2,6-Bis (1,1-dimethylethyl)-4-(1-oxopropyl) phenol may have a role in the antimicrobial activity against phytopathogens [9]. In addition, some minor volatile compounds present in our extract, such as gliotoxin, 6-penthylpyrone, and harzianic acid, were previously isolated from many species of Trichoderma and have exhibited antimicrobial activity against soil borne pathogens (Vinale et al. 2014). The antimicrobial activities of our strains of Trichoderma could be due to the major and also the minor VOC produced. One of the mechanisms of action of these VOC could be due to their toxicity caused by loss of osmoregulation in cell membranes of P. myriotylum.

In conclusion, the results of this work revealed that T. erinaceum (IT-58), T. gamsii (IT-62), T. afroharzianum (P8), and T. harzianum (P11) significantly inhibited the in vitro development of P. myriotylum. This could be due to the combined effect of lytic enzymes, volatile and non-volatile organic components produced by Trichoderma in paired cultures. The activity of each enzyme and the nature of secondary metabolites implicated depend on the different Trichoderma species. However, the isolation and the test of secondary metabolites are further required in order to fully understand their roles.