Evaluation ofTrichoderma atroviride and Trichoderma citrinoviride growth profiles and their potentials as biocontrol agent and biofertilizer

Analysis of mycoparasitic activity against some phytopathogenic fungi in vitro

Three phytopathogenic fungi (Verticillium sp., R. solani and F. oxysporium) were obtained from the culture collection of Ege University Department of Plant Protection. The mycoparasitic activities of Trichoderma spp. were tested by dual culture techniques against phytopathogenic fungi after co-cultivation on PDA at 28°C for 10 days. Antagonism was also carried out according to the classification proposed by Bell et al. [2], [16], [17].

Determination of lytic enzyme activities

β-1.3 Glucanase activity (EC 3.2.1.39), chitinase activity (EC 3.2.1.14) and protease activity (EC3.4.21.4) were determined [18], [19] by using modified Trichoderma Liquid Enzyme (TLE) [20]. The components of the medium used for lytic enzyme production were as follows: 0.1% bactopeptone, 1.43% corn steep solids (CSS), KH₂PO₄ 0.2%, 0.03% MgSO₄·7H₂O, 0.03% glucose, and 0.1% (v/v) trace elements solution containing Fe²⁺, Zn²⁺, Mn²⁺, Cu²⁺ [20].

This medium (50 mL), in 250 mL flask, was inoculated with 1.0×10⁸ (microscopic count) spores of Trichoderma spp., and incubated on rotary shaker (180 rpm) at 27°C and 30°C, respectively, for 4 and 7 days. Supernatant was separated and stocked at −20°C until enzyme activity measurement. All experiments were performed in duplicate.

Analysis of β-1, 3-glucanase activity

β-1, 3-glucanase activity (EC 3.2.1.39) was measured using 25% (w/v) suspension of laminarin that dissolved in 50 mM acetate buffer (pH 5.0) at 40°C for 30 min. Reducing sugars liberated from the substrate were determined with dinitrosalicylic acid (DNS) method described by Miller [18]. One unit of β-1, 3-glucanase activity was defined as the amount of enzyme that released 1 μmol reducing sugar in 1 min under assay conditions.

Determination of chitinase activity

Chitin (0.5%) obtained from crab shells (Sigma C7170) was used as substrate in modified TLE medium for chitinase activity. Chitinase activity was measured in terms of the amount of N-acetyl-D-glucosamine sugars released from chitin. Reducing sugars liberated from the substrate were determined with DNS method described by Miller [18]. One unit (U) of chitinase activity was defined as the amount of enzyme necessary to produce 1 μmol of N-acetylglucosamine (NAG) in 1 min [18].

Analysis of protease activity

Protease activity (EC3.4.21.4) was measured using azocasein (Sigma, A2765) as the substrate as described by Chun et al. [19]. One unit (U) protease activity was defined as the amount necessary to increase the absorbance at the rate of 0.01 at 440 nm in 1 min [19].

Analysis of IAA production by Trichoderma spp.

Flasks (250 mL) containing 50 mL of half-strength Tryptic Soy Broth (TSB, Sigma-Aldrich) supplemented with 200 μg/mL L-tryptophan (Fluka, 22092) were inoculated with three agar plugs of 6 mm from 6 days old Trichoderma spp. and the flasks were incubated at 27°C at 150 rpm [21]. After 7 days of incubation, the cultures were filtered. IAA (mg/L) was measured by mixing 1 mL of the filtrate with 2 mL of Salkowski reagent (150 mL of HClO₄, 250 mL of distilled water and 7.5 mL of 0.5 M FeCl₃·6H₂O) [21]. Reaction mixtures were incubated at room temperature for 20 min and the absorbances were measured at 535 nm [21].

Analysis of P-S capacity of Trichoderma spp.

P-S was determined using the method previously described by Murphy and Riley [22]. The 250 mL flasks containing 50 mL National Botanical Research Institute’s Phosphate growth medium (NBRIP) were inoculated with 1.0×10⁸ spores per mL.

The medium contained L⁻¹: glucose, 10 g, Ca₃(PO₄)₂, 5 g, MgCl₂·6H₂O, 5 g, MgSO₄·7H₂O, 0.25 g, KCl, 0.2 g and (NH₄)₂SO₄, 0.1 g [23]. All flasks were incubated for 10 days at 27°C, 150 rpm. After incubation, cultures were filtrated and P-S (mg/L) was measured by mixing 40 mL of the filtrate with 8 mL of ascorbic acid solution. Reaction mixtures were allowed to stay at room temperature for 10 min and the absorbances were measured at 700 nm.

Effect of temperature, carbon and nitrogen sources on M-G and C-P

Highest M-G and C-P at different conditions were evaluated, including different incubation temperature in light and dark conditions, the use of different carbon and nitrogen sources [12], [24], [25], [26].

The growth of each strain was measured by colony diameter [17]. The conidia production of Trichoderma spp. was graded as (−), 1(+), 2(+), 3(+), 4(+), 5(+).

Analysis of different temperatures in light and dark conditions

The effect of different temperatures on M-G and C-P under light (130 lux) [27] and dark conditions were investigated using 12 different temperatures (4, 10, 15, 20, 24, 27, 30, 33, 35, 40, 43 and 45°C). An agar plug of 6 mm, from 6 days old Trichoderma spp. was centrally inoculated on MYGA and the plates were incubated at temperatures listed above.

Effect of different carbon sources on growth

Sixteen different carbon sources (glucose, fructose, galactose, maltose, saccharose, lactose, raffinose, arabinose, cellulose, chitin, glycerol, mannitol, sorbitol, xylitol, trehalose and xylose) were used to determine their effect on growth. Each carbon source at a final concentration of 1% (w/v) was added into MCMA (carbon source, 10.0 g/L; yeast extract, 7.0 g/L; NaNO₃, 2 g/L; KCl, 0.5 g/L; MgSO₄, 0.5 g/L; FeSO₄, 0.01; K₂HPO₄, 1.0 g/L; agar, 20.0 g/L) [12] and all plates were incubated at 28°C for 3 days.

Effect of different nitrogen sources on growth

Eight different nitrogen sources (yeast extract, corn steep solid, soybean flour, peptone, urea, sodium nitrate, ammonium sulfate and diammonium hydrogen phosphate) were used to test their effect on growth. Each nitrogen source at a final concentration of 0.7% (w/v) was added into MCMA (nitrogen source, 7.0 g/L; glucose, 10.0 g/L; NaNO₃, 2 g/L; KCl, 0.5 g/L; MgSO₄, 0.5 g/L; FeSO₄, 0.01; K₂HPO₄, 1.0 g/L; agar, 20 g/L) [12]. All plates were incubated at 28°C for 3 days.

Analysis of mycoparasitic activity of Trichoderma spp. against some phytopathogenic fungi in vitro

Trichoderma spp. vary considerably in their mycoparazitic activity against phytopathogenic fungi [8], [28]. For instance, it has been reported that T. atroviride caused significant decrease in growth rate of Leptosphaeria spp. [3], and R. solani [8], [28].

In this study, all of the Trichoderma spp. overgrew on the mycelia of Verticillium sp. Interestingly, T. citrinoviride demostrated strong effect against F. oxysporium and T. atroviride was very effective against R. solani. This is particularly important to improve the antagonistic effects of BCAs on phytopathogenic fungi and particularly important for the biocontrol of phytopathogenic fungi such as F. oxysporium and R. solani [1].

Mycoparasitism also involves some morphological changes [9]. As seen on Figure 2, significant antagonist action leading to morphological changes in vitro was achieved by T. citrinoviride (EGE-K-72 and EGE-K-128) against F. oxysporium.

Figure 2:

Dual growth Trichoderma spp. and three phytopathogenic fungi (Verticillium sp, Rhizoctonia solani and Fusarium oxysporium) at 27°C for 10 days.

According to Bell’s classification each number represents a different growth pattern as follows: (1) Trichoderma completely overgrew the pathogen and covered the entire medium surface, (2) Trichoderma overgrew at least two-thirds of the medium surface, (3) Trichoderma and the pathogen each colonized approximately one-half of the medium surface and neither organism dominated the other, (4) the pathogen colonized at least two-thirds of the medium surface and withstood encroachment by Trichoderma and (5) the pathogen completely dominated Trichoderma, overgrew it, and occupied the entire medium surface.

Lytic enzyme activities

Chitinase, protease and β-1,3-glucanase are known as mycoparasitism-related enzymes (Table 2), since they are degrading the cell wall and inhibiting the growth of phytopathogenic fungi. Furthermore, β-1,3-glucanase inhibits spore germination of phytopathogen together with chitinases [1]. In the present study, all Trichoderma spp. produced lytic enzymes such as chitinase, protease and β-1,3-glucanase. The chitinase, 1,3-glucanase and protease activities for T. citrinoviride and T. atroviride species were determined as 0.17–0.21 and 0.14–0.17 U/mL, 0.12–0.21 and 0.07–0.25 at U/mL and, 0.64–2.29 and 1.76–1.22 U/mL, respectively (Table 2).

Analysis of IAA production by Trichoderma spp.

Several Trichoderma species can produce PG-P substances such as the auxin phytohormone IAA. Its production has been suggested to promote root growth, and resulting in increased root mass [29].

IAA production levels in this study were determined to be a minimum of 5.65±0.05 and 11.56±1.92 mg/L and a maximum of 8.81±0.03 for T. atroviride and 57.01±3.11 mg/L for T. citrinoviride (Table 2).

Analysis of P-S capacity of Trichoderma spp.

Trichoderma species are potential phosphate solubilizing microorganisms and play an important role in providing phosphorus to plants [22], [23], [30], [31]. Studies related to several Trichoderma-based biofertilizers are available and they are used for controlling plant diseases and promoting plant growth [10], [30]. Therefore, T. atroviride and T. citrinoviride were screened for in-vitro P-solubilization. The average concentrations of phosphate (mg/L) were determined as 6.01 mg/L for T. atroviride and 12.54 mg/L for T. citrinoviride (Table 2). Although this value is higher than that obtained in Gravel et al. [21], there are some reports mentioning much higher amounts of phosphate solubility (up to 404.07 μg·mL⁻¹) values as compared to this study [30], [31]. Since the main aim was not to optimize phosphate solubilisation, further optimization need to be performed to be able to obtain higher values especially considering the incubation period with time course productions.

Analysis of different temperatures in light and dark conditions

Studies indicated that there were significant differences on the fungal M-G and C-P due to physical growth conditions such as incubation temperature together with light and dark conditions and nutritional requirements using different carbon and nitrogen sources [9], [24], [32].

The mycelia growth and conidia production of all T. atroviride and T. citrinoviride species in the present study were affected by different temperatures as well as light and dark conditions. The differences are significant (p<0.05) between T. citrinoviride and T. atroviride species at different temperatures in light/dark conditions.

Optimum growth temperatures were found to be 27°C for T. atroviride and between 27 and 35°C for T. citrinoviride (Figures 3A,B, Figure 4A,B).

Figure 3:

(A) Effect of different temperature (4, 10, 15, 20, 24, 27, 30, 33, 35, 40 and 45°C) on mycelia growth profiling of T. citrinoviride in light (130 lux) for 3 days. (B) Effect of different temperature (4, 10, 15, 20, 24, 27, 30, 33, 35, 40 and 45°C) on mycelia growth profiling of T. citrinoviride in dark condition for 3 days.

The differences are significant (p<0.05) between T. citrinoviride and T. atroviride species at different temperatures in light.

Figure 4:

(A) Effect of different temperature (4, 10, 15, 20, 24, 27, 30, 33, 35, 40 and 45°C) on mycelia growth profiling of T. atroviride in the light (130 lux) condition for 3 days. (B) Effect of different temperature (4, 10, 15, 20, 24, 27, 30, 33, 35, 40 and 45°C) mycelia growth profiling of T. atroviride in the dark condition for 3 days.

The differences are significant (p<0.05) between T. citrinoviride and T. atroviride species at different temperatures in dark.

Although strains of all five T. citrinoviride grew well between at 15°C and 40°C, no growth was observed above 40°C and below 15°C after 3 days of incubation under light conditions (Figure 3A). However, all T. citrinoviride strains were able to grow at 43°C for 3 days under dark condition (Figure 3B). Interestingly, T. citrinoviride EGE-KL-128 grew at 10°C for 3 days under both light and dark conditions (Figure 3A,B).

Strains of T. atroviride grew well between 15°C and 30°C after 3 days of incubation under light conditions (Figure 4A) and they were able to grow at 15°C–33°C for 3 days under dark conditions (Figure 4B). Previous studies by Daryaei et al. [25] have shown that conidia production was enhanced by T. atroviride LU132 colonies with the exposure of dark conditions. All strains grew at 10°C after 1 week in both light and dark conditions except for T. atroviride EGE-K-131 which grew at 10°C for 3 days (Figure 4A,B).

In general, conidia production temperature ranges were determined as 15–30°C and 20–35°C for T. atroviride and T. citrinoviride (Table 3). These results are comparable with the study reported by Daryaei et al. [24] where lower amount of conidia produced at 30°C. They reported that the best conidia production for T. atroviride was produced at 25°C.

None of the strains produced conidia at 4°C, 10°C, 43°C and 45°C for 10 days in the present study. It is also worth mentioning that, at lower temperatures, conidia production of T. atroviride was significantly higher than T. citrinoviride (Table 3). On the other hand, the highest conidia production for T. citrinoviride was achieved between 33°C and 35°C and it declined at 40°C (Table 3).

Overall evaluation of temparature effect on growth suggested that T. atroviride produced higher amount of conidia at lower temperatures as compared to T. citrinoviride. Likewise, the conidia production pattern of T. citrinoviride seemed to increase as the temperature increased. Significant differences were found when growing both species at different temperatures under the effect of light and dark. Thus, the highest conidia production perfomances of both Trichoderma species in the wide temperature range provided that it is possible to take this advantage for the production of BCAs in different climatic conditions around the world.

Effect of different carbon sources on growth

One of the most important factor that effect production of Trichoderma-based BCAs is the carbon source and several scientists have tried different substrates as a carbon source to obtain high sporulation [33], [34], [35].

Both species (T. atroviride and T. citrinoviride) grew well on all carbon sources tested in the present study. No significant differences in mycelial growth were observed with respect to different carbon sources used tested (Figure 5A and B). In a similar study, sucrose, mannose, glucose, xylose, and starch were found effective for the growth of T. harzianum as compared to maltose and D-galactose [33] whereas glucose [36] sucrose [35] and cellobiose [37] were found favourable in other studies.

Figure 5:

(A) Effect of different carbon sources on mycelia growth profiling of T. citrinoviride at 28°C for 3 days, (B) Effect of different carbon sources on mycelia growth profiling of T. atroviride at 28°C for 3 days.

The differences are significant (p<0.05) between T. citrinoviride and T. atroviride species with different carbon sources.

It is worth mentioning that no conidia production was observed after 10 days of incubation with some carbon sources tested in this study. These carbon sources are maltose, raffinose, cellulose, chitin, sorbitol for T. atroviride EGE-K-65; saccharose, raffinose, cellulose for T. citrinoviride EGE-K-72; maltose, saccharose, raffinose, cellulose, chitin, xylitol for T. citrinoviride EGE-K-129; and saccharose, lactose, raffinose, cellulose, chitin, xylitol for T. citrinoviride EGE-K-130 (data not shown).

It has been reported that the Carbon:Nitrogen (C:N) ratios of media had significant effects on the spore yield of fungal BCAs to optimize nutritional conditions for mass production [38]. Daryaei et al. [24] reported that different C:N ratio and carbon contents for maximal conidia production of T. atroviride LU132 were 5:1 and 4.2 g/L, respectively. The C:N value for maximal M-G and C-P was found as 2:1 in our experimental conditions, which is quite similar to Daryaei et al. [24]. They reported that the greatest inhibition on R. solani growth was achieved from conidia produced at high concentration of trehalose. Trehalose accumulation was induced by stress conditions which is required for stress tolerance like dehydration of fungal cells under high temperatures [8], [24], [26], [28]. The highest mycelial growth for T. citrinoviride in this study was found not only when mannitol and trehalose were used but also with glucose, galactose glycerol, mannitol and xylose containing media (Figure 5A).

Effect of different nitrogen sources on growth

All Trichoderma spp. demonstrated pronounced growth with CSS (Figure 6A and B). T. citrinoviride strains, with the exception of T. citrinoviride EGE-KL-72, exhibited good mycelium growth with yeast extract and soybean flour (Figure 6A). The highest mycelial growth was achieved by T. citrinoviride EGE-K-129 on pepton (Figure 6A).

Figure 6:

(A) Effect of different nitrogen sources on mycelia growth profiling of T. citrinoviride at 28°C for 3 days. (B) Effect of different nitrogen sources on mycelia growth profiling of T. atroviride at 28°C for 3 days.

The differences are significant (p<0.05) between T. citrinoviride and T. atroviride species with different nitrogen sources.

The results showed that the effect of organic nitrogen sources (corn steep solids>yeast extract>soybean flour) on the growth of T. atroviride and T. citrinoviride species were significantly higher than those observed with inorganic nitrogen sources with the exception of T. citrinoviride EGE-KL-72 (Figure 6A and B). The effect of organic nitrogen sources on growth was notably higher than inorganic nitrogen sources.

T. citrinoviride EGE-K-72 showed very weak growth on sodium nitrate and ammonium sulfate and did not grow in the medium containing diammonium hydrogen phosphate. Among the T. citrinoviride strains, T. citrinoviride EGE-K-129 demostrated better growth with inorganic nitrogen sources as compared to the other species (Figure 6A).

T. atroviride strains revealed similar growth patterns on sodium nitrate and ammonium sulfate and their effect on growth was significantly higher than the one obtained by diammonium hydrogen phosphate. Our results were in agreement with other results obtained by other researchers in which the best growth and sporulation of T. viride and T. harzianum were favored by ammonium forms of nitrogen as compared to nitrite or nitrate forms [35], [39].

As seen on Figure 6A and B, none of the Trichoderma species can grow on urea up to 3 days of cultivation. The minimum mycelial growth was initiated after 4 days of incubation. Towards the end of the incubation period differences between the colony diameters were very high ranging between 10 and 40 mm.

All species produced significant amounts of conidia on all organic nitrogen sources. But, none of the Trichoderma species produce conidia on urea which is in accordance to those reported previously [33]. Trichoderma citrinoviride EGE-K-72 did not produce conidia on inorganic nitrogen sources during 10 days. Exceptionally, T. citrinoviride EGE-K-129 produced conidia on diammonium hydrogen phosphate (data not shown).