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

Background: Two Trichoderma species ( T. atroviride and T. citrinovirid e) were evaluated for their potential to have Trichoderma -based biological control agent and biofertilizer properties. Materials and Methods: Eight Trichoderma spp. were identified by molecular methods. The mycoparasitic activities against different phytopathogenic fungi and their capacity to produce extracellular lytic enzymes were investigated. Furthermore, indole-3-acetic acid production and phosphate solubilization capabilities of Trichoderma spp. were evaluated together with the effect of some physical parameters and different carbon:nitrogen sources on mycelial growth and conidium production. Results and Discussion: All strains exhibited lytic enzymes and indole-3 acetic acid production as well as phosphate solubility characteristics. Trichoderma citrinoviride demostrated more mycoparasitic activity against Fusarium oxysporium than T. atroviride whereas T. atroviride was found more effective against Rhizoctonia solani . In par-ticular, at lower temperatures, conidium production of T. atroviride strains were significantly higher than T. citrinoviride strains. Both strains grew well on all carbon sources tested. The effect of organic nitrogen sources on growth were notably higher than inorganic nitrogen sources. Conclusion: The results provided valuable insight in both the highest mycelial growth and conidia production conditions of these cultures for further similar studies related to development of Trichoderma -based new commercial biological control and biofertilizer formulations in different agro-climatic regions.


Introduction
Trichoderma spp. have been studied extensively with respect to many characteristics and are known as successful habitats of the soil ecosystem competing against other fungi [1][2][3][4]. Biocontrol process may be performed as indirectly (i.e.: by competition for nutrients and space, chancing the environmental conditions, or promoting plant growth and plant defense mechanisms and antibiosis) or direct mechanisms, as in mycoparasitism [1]. Trichoderma spp. demonstrate considerable mycoparasitic activity due to the production of lytic enzymes such as chitinases, β-1,3glucanases and various proteases [5,6]. The production of several antibiotics by Trichoderma spp. helps to improve the plant immune system thereby preventing possible plant infections. Compared to bacterial biological control agents (BCAs), fungal based BCAs exhibit a broader spectrum of activity in terms of disease management and therefore they have gained wide acceptance in the recent years [7][8][9]. Trichoderma spp. have an important potential as plant growth promoting fungi [6], comprising of almost 50% of the fungal BCAs market, mostly as plant growth enhancers [7]. Trichoderma spp. have the ability to solubilise valuable nutrients such as phosphate compounds by secreting different organic acids. They also produce different hydrolytic enzymes, contributing to the enrichment of the soil composition. Trichoderma spp. are also effective bioremediation agents due to their ability to degrade xenobiotics, removing potential hazards from the soil. Besides their plant growth enhancement properties, Trichoderma spp. are important candidates for use as BCAs [4].
Some fungal genera such as Trichoderma, Aspergillus, Fusarium, Penicillium are most frequently reported plant growth-promoting (PG-P) fungi and owing to their multiple beneficial effects on plant quantity and quality have attracted considerable interest as bio-fertilisers [10].
In this study, eight Trichoderma spp. isolates were evaluated in respect of their potential as BCAs and biofertilizer. The mycoparasitic activity, lytic enzyme productions indole-3-acetic acid (IAA) production and phosphate solubilization (P-S) capabilities of the species were tested together with the effects of temperature in light/dark, different carbon and nitrogen sources on mycelial growth and conidia production (M-G and C-P).
For the isolation of Trichoderma spp., the soil dilution plating method was used [2]. The diluted samples were directly plated onto Rose Bengal Chloramphenicol agar as a selective medium. After 6 days of incubation at 28°C, fungal colonies were transferred to Malt Extract Agar (MEA) slants and they were stored +4°C for further investigations.
Determining the optimal medium for Trichoderma spp.
Five different growth media were used for evaluation of M-G and C-P at 28°C for 6 days. Tested growth media were as follows; Malt Yeast Glucose agar (MYGA) medium, MEA, Czapek Dox Agar (CDA), Modified CDA and Mendel's Medium [7,12]. Since optimum pH values for the growth of the isolates were found as between pH 5.0 and 6.0 previously [13], it was kept constant at pH 5.50 in further experiments performed in this study.

DNA extraction, PCR amplifications and sequencing
For DNA extraction, fungal isolates were cultivated on MYGA at 28°C for 6 days. DNA extraction was carried out as described previously by Chen et al. [14]. The ITS region of the nuclear rDNA gene cluster was amplified from DNA using primers ITS1 (TCCGTAGGTGAACCTGCGG) and ITS4 (TCCTCCGCTTATTGATATGC) [15]. PCR amplifications and sequencing were performed by REFGEN (Turkey). The sequences were aligned in Molecular Evolutionary Genetics Analysis (MEGA) v.6 using Clustal W. The Phylogenetic trees were established with Maximum Likelihood method in MEGA v.6. For determining each clade, a bootstrap analysis was performed with 1000 replication.

Screening of Trichoderma spp. as a BCAs in vitro
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].
This medium (50 mL), in 250 mL flask, was inoculated with 1.0 × 10 8 (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.

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].

Screening of Trichoderma spp. as biofertilizers in vitro
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 HClO 4 , 250 mL of distilled water and 7.5 mL of 0.5 M FeCl 3 · 6H 2 O) [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 × 10 8 spores per mL. The medium contained L −1 : glucose, 10 g, Ca 3 (PO 4 ) 2 , 5 g, MgCl 2 · 6H 2 O, 5 g, MgSO 4 · 7H 2 O, 0.25 g, KCl, 0.2 g and (NH 4 ) 2 SO 4 , 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].

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.

Statistical analysis
The experiments were carried out in triplicate. The results were statistically analyzed and Tukey test was used to compare the differences and to determine the standard deviations and standard errors of the means and to test the significance of treatment at p ≤ 0.05.

Results and discussion
Determination of the optimal medium for Trichoderma spp.
Among the tested media, MYGA was determined as a optimal medium for the cultivation of Trichoderma spp. after observational comparison (data not shown) [7]. Further studies were carried out by using MYGA.

Identification of Trichoderma strains by molecular methods
The ITS rDNA region of Trichoderma spp. was sequenced and the fungal isolates were identified as T. atroviride (n = 3), T. citrinoviride (n = 5). The GenBank accession numbers are shown on Table 1 and the phylogenetic trees were constructed with MEGA v.6 as seen on Figure 1.

Screening of Trichoderma spp. as a BCAs in vitro
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.

Screening of Trichoderma spp. in respect of biofertilizer properties in vitro
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.

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

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.
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).
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).

Conclusion
The results provided valuable data for achieving highest M-G and C-P of Trichoderma species in terms of their portential as for the preparation of new commercial biological control and biofertilizer formulations under different agro-climatic regions since they exposed growth in a wide temperature range. In particular, to the best of our knowledge it was the first study concerning the T. citrinoviride in respect of BCAs and biofertilizer properties.
According to the results obtained, it was clearly seen that T. citrinoviride strains might have potential for use as efficient BCAs and PG-P fungi.