Comprehensive Characterization and Screening of Different Trichoderma Isolates as Plant Growth Promoters: Insight to Metal Solubilization, Enzymatic Activity, and Antagonistic Effect

doi:10.21203/rs.3.rs-1286108/v1

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Comprehensive Characterization and Screening of Different Trichoderma Isolates as Plant Growth Promoters: Insight to Metal Solubilization, Enzymatic Activity, and Antagonistic Effect

https://doi.org/10.21203/rs.3.rs-1286108/v1

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The present study aimed to isolate a Trichoderma species with dual action as a plant growth promoter in conjunction with antagonistic activity against the plant pathogen Colletotrichum lagenarium. Using internal transcribed spacer (ITS) sequences and inter simple sequence repeat markers (ISSR), eight strains of Trichoderma were identified, and their diversity was studied. In addition, some plant growth promoters such as indole acetic acid (IAA) and element solubilization were evaluated. Moreover, the activity of amylase, pectinase, cellulase, and chitinase was studied in the isolated species. Results showed that Trichoderma viride (T27) and Trichoderma atrobrunneum (T40) produced the highest IAA (80.36, 61.39 µg/mL), respectively and K solubilization efficiency (KE) (12.2 and 14.4, respectively). In contrast, Trichoderma afroharzianum (T31) and Trichoderma atrobrunneum (T40) had the highest zinc (Zn) solubilizer (7.1 and 7.2 cm), respectively. Strains T. viride (T27) and T. atrobrunneum (T41) showed the highest antagonistic activity against anthracnose fungi; therefore, they were examined under scanning electron microscopy. The SDS-PAGE protein pattern and its antigenicity analysis ranged between 40 and 180 kDa. The results of SDS-PAGE indicated that with chitin, both T27 and T40 presented different bands in their protein profile compared to the control (without chitin). The results suggest that T. viride (T27) and T. atrobrunneum (T40) could be used as eco-friendly bio-fungicides with a high potential plant growth promoter production.

Trichoderma

Antifungal

Plant hormones

Chitinase

SDS-PAGE

Filamentous fungi have diverse habitats, including forests, deserts, and agricultural lands [1]. Trichoderma belongs to the family Hypocreaceae present in different soil types, where they are plant growth promoters, opportunistic, and parasites of other fungi [2]. Therefore, several Trichoderma species can form beneficial relationships with several host plants, helping them control diseases and promoting plant growth [3]. In addition, various Trichoderma sp. have the antifungal activity against different phytopathogenic fungi by several biocontrol mechanisms, such as competition for both space and nutrients, mycoparasitism through lytic enzyme production, and antibiosis by the production of bioactive secondary metabolites [4–6]. In addition, they produce various volatile organic compounds (VOCs), controlling disease development and promoting plant growth [6, 7]. Recently, it was reported that VOCs from Trichoderma sp. belong to different chemical classes such as terpenes, ketones, esters, aldehydes, and alcohols [8–10]. Due to their high volatility at room temperature, these compounds can quickly diffuse through the soil [11]. In addition, some Trichoderma sp. can be applied as a biological control agent for plant pathogens due to their potential for enzymatic and antagonistic actions [12, 13].

For soil and agricultural production, different elements such as Fe, Mn, Cu, and Zn are necessary for several physiological processes of the plant. However, they are not found in their active form in the soil, which results in the unavailability of these elements to the plant. Deficiencies of these elements significantly affect the yield and quality of agricultural products [14]. Thus, new methods are essential to increase the bioavailability and uptake of these essential nutrients. For example, the availability of micronutrients at the plant root surface can be mainly facilitated by the biological activities of several microorganisms [15]. These microorganisms can be used as stimulants and enhancers for plant growth. In that context, different fungal species have been used to solubilize different macro-and micro-nutrients such as phosphorus (P), potassium (K), Zn, manganese, copper, and iron [16, 17]. Finally, microorganisms solubilize minerals by several processes, depending on their source and the strain's capacity to solubilize them.

Furthermore, certain Trichoderma species can directly boost plant development by producing plant growth-promoting chemicals and plant growth regulators such as auxin, gibberellin, cytokinin, and ethylene. [18]. In addition, they may indirectly enhance plant growth via the production of siderophores and antagonistic substances, such as antibiotics and cell wall-degrading enzymes [19, 20]. Recently, it was reported that some species could work as a biostimulant by enhancing the activity of soil enzymes and increasing nutrient uptake [21, 22]. Recent studies confirmed that Trichoderma spp. can protect the plant against pathogens through many mechanisms such as mycoparasitism [23], competition for nutrients and locations, antibiosis, enzymes and volatile compounds [24, 25].

A variety of enzymes, including amylase and chitinases, are produced by Trichoderma species to exploit a wide range of carbon (C) and nitrogen (N) sources. These enzymes mainly help break down large polymers into primary sugars but also help in the mycoparasitism of Trichoderma against other fungal pathogens [26]. Previously, it was reported that some Trichoderma species could produce many amylase enzymes, including T. harzianum [27, 28]. From an industrial aspect, enzymatic hydrolysis of lignocellulose to produce fermentable sugars is crucial in biorefinery (Gupta, Kubicek et al. 2016, Guo, Chang et al. The produced sugars can be converted to yield lactic acid [29], bioethanol [30], or biohydrogen [31]. However, the big challenge is the high cost of enzymatic treatment [32–34]. Chitinase is a recently discussed cell wall-degrading enzyme that catalyzes chitin's degradation by breaking down β-1,4 linkages [26]. Due to its ability to produce many chitinases, Trichoderma has played an essential role in controlling plant diseases via different mechanisms.

Regarding food security and reducing fungal pathogens, using bio-pesticide production is essential to use promising alternatives to chemical pesticides. One of these recently discussed mechanisms involves inhibiting the growth of phytopathogen hyphae by coiling then penetrating the hyphae using some enzymes, i.e. chitinase, for fungal cell wall degrading [35–37]. Anthracnose, caused by Colletotrichum species, is one of the recent widely discussed devastating plant diseases, with 100% disease incidence on plant leaves and 30-40% yield loss [38]. Amongst, Colletotrichum lagenarium is a hemibiotrophic fungus that causes anthracnose disease and significantly limits the growth and development of cucumber plants worldwide [39]. Therefore, it was selected in the antagonistic experiments as a plant pathogen in the present study.

To have the most beneficial influence on plant production, it is critical to select a Trichoderma species with a high potential to stimulate plant development through many mechanisms of action such as secretion of plant growth promotors, metal solubilization, and antagonistic activity. Therefore, the present study aimed to isolate and screen different Trichoderma sp. for the possibility of plant growth promotion. Their ability to produce indole acetic acid (IAA) and solubilize several nutrients (P, K, and Zn) were evaluated. Moreover, the enzymatic activity of Trichoderma strains, including chitinase, pectinase, amylase, and cellulase production, was also measured. Moreover, the antagonistic activity by which the selected Trichoderma strain can overcome the growth of C. lagenarium was studied.

2.1 Trichoderma isolation and identification

Eight Trichoderma isolates were collected and purified from rhizosphere soil samples from seven geographical locations in Egypt (Fig. 1), as described in the previous study [33]. Identification of eight selected isolates (T1, T3, T5, T8, T27, T28, T31, T40) was carried out by amplification of 5.8S ITS regions using a pair of general primers, ITS4 and ITS5 (Eurofins), as described previously [33, 40]. The homology identification was assessed using the nucleotide BLAST to compare the strains with the available sequences at the GenBank database. In addition, a set of six ISSR-PCR primers (ISSR-1, ISSR-3, ISSR-5, UBC-807, UBC-812, and UBC-826) were designed by Macrogen (Seoul, Korea) for their ability to produce polymorphic patterns among the eight Trichoderma isolates. PCR was carried out in 20-µL volumes containing 10 ng of extracted DNA and 0.5 µM of each primer (10 pmol). To produce a similarity matrix, the amplified PCR products of the ISSR bands were scored as 1 (present) or 0 (absent) for the eight Trichoderma strains. ISSR agarose gel electrophoresis was analyzed using Numerical Taxonomy System Biostatistics (NTSYSpc ver. 2.2), and Jaccard’s similarity coefficients were analyzed. A phylogenetic tree was drawn by grouping the Jaccard’s coefficients using the Sequential Agglomerative Hierarchical Non-overlapping clustering software [41–43].

2.2 Evaluation of plant growth promotion parameters

2.2.1 Auxin production (IAA) assay

The production of IAA by Trichoderma species was tested as one of the plant growth promoters using the Salkowski test [44]. Briefly, the quantitative estimation of IAA was carried out using the inoculation of one pure disc (5 mm diameter) of the active Trichoderma culture in 250 mL Erlenmeyer flasks containing 50 mL of PDB medium. After incubation at 28°C ±1°C with shaking at 150 rpm for 10 days, cultures were centrifuged at 5000× g for 15 min, and the supernatant was collected. One millilitre of the filtrate was mixed with 2 mL of Salkowski reagent at room temperature for 30–45 minutes before measuring the quantification by a spectrophotometer at 530 nm [45]. The concentration of IAA was determined based on a constructed standard curve using different concentrations of IAA.

2.2.2 Metal solubilization

To test the efficacy of Trichoderma species for zinc-solubilizing, the isolates were cultivated on Tris-minimal agar medium supplemented with D-glucose amended with Zn carbonate (1.728 g/L) as an insoluble Zn source at a final concentration of 0.1% zinc. The inoculated plates were incubated at 28°C for 7 days in the dark, and the growth zone around the cultured disc was observed [46]. The eight isolates were tested for K solubilization activity using the Aleksandrov Agar medium, which shows growth zone formation as an indicator of K solubilizers [47]. In addition, isolates were cultivated on Petri dishes containing 15 mL of NBRIP agar to study the capability of strains for solubilizing insoluble phosphate [46, 48]. The K solubilization efficiency (KE) was measured using the formula:

$$\text{K}\text{E} \left(\text{\%}\right)=(\text{S}\text{o}\text{l}\text{u}\text{b}\text{i}\text{l}\text{i}\text{z}\text{a}\text{t}\text{i}\text{o}\text{n} \text{h}\text{a}\text{l}\text{o} \text{z}\text{o}\text{n}\text{e} \text{d}\text{i}\text{a}\text{m}\text{e}\text{t}\text{e}\text{r}+\text{D}\text{i}\text{a}\text{m}\text{e}\text{t}\text{e}\text{r} \text{o}\text{f} \text{t}\text{h}\text{e} \text{c}\text{o}\text{l}\text{o}\text{n}\text{y})/ \text{D}\text{i}\text{a}\text{m}\text{e}\text{t}\text{e}\text{r} \text{o}\text{f} \text{c}\text{o}\text{l}\text{o}\text{n}\text{y}$$

[49, 50]. The phosphate solubilization index (PSI) was calculated using PSI = fungal colony diameter + halo zone diameter / fungal colony diameter [51]. The effective isolates were stored at -80°C for future use.

2.3 In vitro antagonistic activity

2.3.1 Morphology examination

Trichoderma isolates were evaluated against C. lagenarium to test their antifungal capability in a dual-culture assay that involved placing two 7-day-old cultures (5 mm diameter) of the biological agent (Trichoderma) and the pathogen (C. lagenarium) in the same petri dish containing PDA medium. Both fungi were placed simultaneously along a diametrical axis, 0.5 cm from the edge of the Petri dish, then incubated for 7–9 days in the dark at 28°C [52]. The pathogen's percentage inhibition of radial growth (PIRG) was calculated [53]. To examine the hyphal interaction between the selected isolates and C. lagenarium, SEM (JEOL JSM 5200) was used after 6 days of incubation according to the methodology [54].

2.3.2 Enzymatic activities

Chitinase, cellulase, pectinase, and amylase enzymes were determined in the present study. For cellulase activity, carboxymethylcellulose (CMC) medium was inoculated with each strain and incubated at 28 ºC and 150 rpm for 7 days, then centrifuged at 10,000 rpm for 10 min at 4 ºC to obtain the crude enzyme (the supernatant). The enzyme activity was estimated as described by [55]. Briefly, 300 µL of culture supernatant was added to 900 µL of 1.0% CMC in 0.05 M sodium acetate buffer (pH 7.0). Then, the mixture was incubated at 50°C for 25 min, and 1800 µL of dinitrosalicylic acid reagent was added to the reaction mixture. The mixture was boiled for 5 min before measuring the absorbance at 540 nm after cooling for 5 min, then the concentration of reducing sugars was determined and calculated as glucose [55].

For pectinase activity, Trichoderma isolates were tested using pectinase agar medium (g/100 mL) and incubated at 28°C for 7 days, flooded with 50 mM K iodide-iodine solution. The growth zone around the colonies indicated the ability of an isolate to produce pectinase [56]. Moreover, amylase production was determined by the dinitrosalicylic acid (DNS) method [55]. Then, the optical density at 575 nm was measured using a spectrophotometer. Finally, the quantity of reducing sugars was calculated using a standard glucose curve.

Colloidal chitin was prepared from the commercial chitin (Sigma C7170-100G) to test the chitinase activity and supplemented in the chitinase assay medium (CAM) as a sole C source. One pure disc of Trichoderma was tested for chitinase potential after incubation at 28°C ±2°C until the formation of colored zones after 7 days of incubation [57]. In addition, SDS-PAGE protein with/without chitin was used to test the gene expression of chitinase in the studied isolates. The extracellular proteins were extracted from the supernatants of different strains grown in a fermentation medium supplemented with chitin. This test compared the original strains (without chitin) with the treated cultures (with chitin). Electrophoresis was conducted according to the protocol by Laemmli [58], and the gel was stained with Coomassie blue R250 to detect protein bands [59].

2.4 Statistical analysis

All experiments were conducted in triplicates, and results are presented as the mean ± standard deviation (SD). The results were analyzed using one-way ANOVA followed by Tukey’s post-test using SPSS (v20, IBM).

3.1 Trichoderma isolates

Table 1 shows the species names and accession numbers of the best NCBI hits for the eight isolated species. All strains exhibited varying percentages of the maximum identity, with “best hits” of 99–100% similarity. Among the eight isolates, T3, T5, and T8 strains were identified as T. harizanum, T1 strain was T. asperelloides, T27 was identified as T. viride, T31 and T40 were T. afroharzianum and T. atrobrunneum, respectively (Table 1). Phylogenetic trees showed the distances of the eight isolates with the closely similar sequences on the GenBank database (Fig. 2). Genetic analysis using ISSR markers revealed polymorphic and reproducible patterns among different Trichoderma strains (Fig. S1). A total of six ISSR primers amplified 116 amplicons and were scored in size range of 300–1500 bp among the eight Trichoderma strains. The total amplicons from PCR amplification with the primers UBC 807 and UBC 826 were 19 and 10, respectively, while the maximum number of bands was 25 with primers ISSR1 and ISSR3 and ISSR5. In addition, the Unweighted Pair-Group Methods with Arithmetic Average method was used to estimate the polymorphic loci and indicated diversity among the strains (Fig. S1).

Table 1

Phylogenetic details with accession numbers of the eight *Trichoderma* isolates
Isolates	Best match (Accession number)	PCR product (bp)	Query covers (%)	Percent identity (%)	Accession number
T1	Trichoderma asperelloides (MF114226.1)	583	98	99.65	MH908491
T3	Trichoderma harzianum (MH151203.1)	572	100	100	MH908493
T5	Trichoderma harzianum (KC576719.1)	590	99	99.83	MH908495
T8	Trichoderma harzianum (MH151203.1)	593	100	99.33	MH908498
T27	Trichoderma viride (MH333256.1)	596	96	99.13	MH908510
T28	Trichoderma sp. (MK870994.1)	576	100	100	MH908511
T31	Trichoderma afroharzianum (MF116235.1)	578	100	100	MH908513
T40	Trichoderma atrobrunneum (MT000972.1)	582	100	100	MH908517

Based on the cluster analysis of ISSR data, Trichoderma species were divided into two main groups. The first one included T5 and T8, and the second group was divided into sub-groups which provided the other strains. Interestingly, T. viride T27 isolated from southern Gaza in North Sinai joined with T. atrobrunneum T40, isolated from Saint Catherine in South Sinai. T. asperelloides T1 isolated from Menoufia showed a high genetic similarity with T. harzianum T3 isolated from the same province in one cluster. However, ITS analysis for T1 and T3 revealed two different species, T. asperelloides and T. harzianum. Besides, T. harzianum T5 and T8 were isolated from different locations (Kafr El-Sheikh and Minya, respectively). However, they identified as T. harzianum and were clustered in one group with a 69% coefficient (Fig. S1). The overall mean value of the similarity coefficient was 0.76 based on ISSR analysis of the ISSR markers.

3.2 Metal solubilization

Zn is an essential micronutrient element required for plant growth and development, involved in cellular metabolism. Zn solubilization was significantly different among the studied Trichoderma strains (Fig. 3A, B). Overall, strains T40, T31, and T27 showed the highest growth zone diameters with ZSI, Fig. 3A (F = 53.4, df = 7, 23, P < 0.05). While strain T28 showed the lowest growth zone diameter, with a ZSI of 31.11%. K is one of the most vital macronutrients involved in plant cell metabolic functions, helping in protein synthesis and photosynthesis. It is considered the third most significant macronutrient for plants after N and P. Potassium-solubilizing microorganisms, especially fungi, play a unique role in the K cycle, ensuring K availability. K solubilization was evaluated for the isolates based on the clear zone around the colonies of Trichoderma spp. on Aleksandrov agar. Trichoderma spp. can produce different acids that help K solubilization in the medium, leading to changes in medium color (Fig. 3C). As shown in Fig. 3A, all the screened Trichoderma strains showed a potential for K solubilization with varied ability. T40 showed the highest K solubilization ability, followed by T27, which had significantly higher K solubilization ability than the other strains. These findings indicated the ability of all tested strains to solubilize K to its ionic form.

P is the second most essential element for plant growth after N, and its availability via fungal solubilizing activity plays a critical role in plant productivity. The eight Trichoderma strains isolated from different geographical locations in Egypt were tested under insoluble P stress (Fig. 3A, D). A clear zone was measured as an indicator of P solubility around the inoculated Trichoderma disk on solid NBRIP media. All species of Trichoderma showed the potential for solubilizing phosphorus, with halos greater than 15 mm. Interestingly, different halo diameter zones, from 16.67 mm in Trichoderma harzianum (T5) to 64.67 mm in Trichoderma viride (T27), were observed, indicating different abilities to solubilize phosphate. Among the various tested strains, T. viride produced the largest halo diameter in the NBRIP plate assay, forming a visible growth zone (Fig. 3D).

3.3 IAA assay

IAA production was significantly different between the studies Trichoderma isolates (Fig. 4). Overall, strain T27 showed the highest significant concentration of IAA production (80.4 µg/mL) (F = 88.2, df = 7, 23, P < 0.05). in addition, strains T40, T28, and T1 produced a significant amount of IAA (61.4, 56.8, and 54.2 µg/mL, respectively). Conversely, T3, T5, and T8 showed the lowest IAA production (27.6, 19.3, and 14.9 µg/mL, respectively).

3.4 Enzymatic activities of Trichoderma species

Pectinase catalyzes the degradation of pectin by two different mechanisms, de-esterification and de-polymerization, resulting in a growth zone around the inoculated fungal colony. The current study revealed that all tested Trichoderma strains except isolate T8 have pectinolytic activity at different rates (Fig. 5A). The highest activity was achieved by strains T27, T5, and T1, respectively, and the activity was significantly higher than that of other strains. However, moderate activity was achieved by strain T3, with the lowest activity recorded in T40. Amylase hydrolyzes α-(1-4)-glucosidic linkages in the starch, producing maltodextrins with different molecular weights. Amylase activity was measured based on the color produced after adding the dinitrosalicylic acid (DNS) reagent. Results revealed that all tested Trichoderma strains could produce extracellular amylase at different rates. The highest activity was detected in the T1 isolate (45.78 U/mL), while T8 showed the lowest activity (8.36 U/mL) (Fig. 5B). The ability of different Trichoderma spp. to produce cellulase was also assessed by carboxymethylcellulose (CMC) as a substrate. Results showed that all tested Trichoderma strains have cellulase activity at different rates (Fig. 5C). The highest activity was achieved by strains T1, T3, T8, and T5 (87.28, 84.05, and 83.23 U/mL, respectively). However, moderate activity was recorded for strain T31 (61.79 U/mL), while the lowest activity was recorded for isolate T40 (24.05 U/mL) (Fig. 7C).

3.5 Antagonistic activity and mechanism

The average percentage of inhibition (%) of mycelial growth for C. lagenarium pathogen is presented in Fig. 6. All Trichoderma isolates inhibited the mycelial growth of the pathogens. The percentage of reduction in the growth of the pathogen ranged between 65% and 95%. Among all, T40 showed a significantly higher percentage of inhibition (95%) against C. lagenarium, followed by T31 (79.6%), while T5 showed the lowest significant inhibition percentage (65.0%). The mechanism of action was further studied in the following sections.

3.5.1 Chitinase potential

Due to chitinase production, Trichoderma can break chitin down into N-acetyl glucosamine, changing the pH to alkalinity and changing the bromocresol purple to a purple zone around the inoculated colony. All Trichoderma strains showed chitinolytic potential at different rates after screening on solid medium amended with colloidal chitin (Fig. 6). The highest chitinase ability was detected among different studied isolates for T27, T3, and T28.

3.5.2 Extracellular protein analysis (SDS-PAGE)

The total intracellular protein profile (SDS-PAGE) was extracted after adding the chitin to the media from 5-day-old and submerged cultures of eight different Trichoderma isolates. As shown in Fig. 7A, substantial changes in protein profiles were observed after growing the cultures on media amended with chitin. The changes of protein patterns were observed at the 40–180 kDa size range, which generally corresponds to the predicted sizes of the enzymes involved in chitin metabolism.

3.5.3 Microscopic characteristics

Based on the physiological activity of the studied isolates discussed in the previous sections, two Trichoderma isolates (T27 and T40) were selected to examine their morphology under a microscope. After five days of incubation, a pure disc of T. atrobrunneum was cultured onto PDA at 28°C (Fig. S2). Under a light microscope, the whole conidiophores system appeared to be filiform, with the primary branches arising from below to the top.

SEM allows the study of the morphological mycoparasitic interaction of Trichoderma spp. with the anthracnose fungus Colletotrichum lagenarium. All strains were antagonistically active against Colletotrichum lagenarium, but only the most promising strains (T27 and T40) were selected for visualization by SEM based on the Chitinase activity and protein profile. As shown in Fig. 7B-D, the interaction points between the two fungi could be visualized clearly. The hyphae of the two selected Trichoderma spp. were observed to coil around the hyphal pathogen when confronted with it. The results showed complete colonization of C. lagenarium with the Trichoderma strains T27 and T40. The hypha of Trichoderma spp. was grown by producing spores and coiling the hyphae of the pathogen without penetrating its cell wall. This finding confirms the antagonistic effect via the breakdown of the hyphae of the pathogen by Trichoderma spp. could be due to lytic enzymes produced by Trichoderma spp., particularly chitinase.

Using morphological, molecular, and physiological approaches, the present study identifies and characterises eight Trichoderma isolates. The Trichoderma strains showed a precise variety in their morphological and genetic characteristics for identification. Furthermore, the assessment of the genetic relationship using ISSR markers revealed polymorphic and reproducible patterns among Trichoderma strains. A recent study [60] scored 54 ISSR bands, ranging from 0.1–5 kb among 91 Trichoderma strains with 42.59% polymorphism. A similar study [43] reported that ISSR markers were considered unique amplicons and reliable approaches to identifying two efficient biocontrol agents, T. koningii MTCC 796 and T. virens NBAII Tvs12. Fifteen ISSR primers were amplified to characterize 12 isolates belonging to T. harzianum and T. viride, with a total of 70 amplified bands ranging from 100 to 950 bp in another study [61]. In general, ISSR markers are necessary to study the polymorphic patterns and evaluate the biodiversity of Trichoderma species after isolation from different geographical locations.

The ability of Trichoderma sp. to sparingly solubilize soluble minerals, including Zn and rock phosphorus, in vitro was demonstrated in another study [17]. The present study confirmed the solubilization of metallic Zn by different isolates, which is considered a high-potential and eco-friendly tool for increasing the bioavailability of native and applied Zn to plants under the current experimental conditions. Zn solubilization by Trichoderma spp. has been reported in previous studies [62, 63]. However, the present study showed the highest Zn solubilization with T. atrobrunneum (T40) and T. viride (T27). The current study also investigated K solubilization, which can increase the availability of K to the plant. This process is the vital key in the K cycle, making it available for the plant. The results indicated that T. atrobrunneum and T. viride (T40 and T27) also had the highest K and P solubilization activities. Similarly, it was reported that Trichoderma strain LX-7 could solubilize K to its ionic form [64].

Interestingly, several fungi, including Aspergillus, Cladosporium, Sclerotinia, and Trichoderma, have been reported to have the ability of K solubilization [65]. The variation in the diameter of the growth zone surrounding Trichoderma inoculum may be due to the variations in the produced quantities of organic acids between different strains, which reduced the pH value and enhanced the solubilization process of both micro-and macronutrients. However, these results are inconsistent with the findings of a previous study [66]. It was concluded that different species of Trichoderma had a significant capability for solubilizing rock phosphate and K [67, 68].

As a universal enzyme produced by both macro and microorganisms, amylase has gained significant interest, and the microbial sources of this enzyme are effective for many applications [69, 70]. However, it has been reported in many previous studies that different species of Trichoderma can produce large quantities of extracellular amylase on soluble starch [71, 72]. The current results revealed that all tested Trichoderma isolates could produce extracellular amylase enzymes at different rates. The highest activity was achieved by T. asperelloides, followed by T. atrobrunneum. In parallel, previous studies have reported the production of amylase by T. pseudokoningii [73], T. harzianum [27, 71], T. viride [74], and T. asperellum [75]. Furthermore, fungal species, including Trichoderma spp., Aspergillus spp., and Penicillium spp., are considered high cellulase producers and have gained significant interest [76]. The current results showed that all tested Trichoderma strains have cellulase activity at different values. The mechanism of cellulase production includes the action of three groups: carboxymethyl cellulases, which release cello-oligosaccharides; cellobiohydrolases, which generate cellobiose; and β-glucosidases, which act on both cello-oligosaccharides and cellobiose, producing different monomers of glucose [77]. These enzymes act synergistically during cellulose hydrolysis [78]. The cellulase production was reported from T. viride [77]. Among all producers, T. reesei was reported to produce a vital cellulase enzyme [79, 80].

Fungi can produce many extracellular enzymes, which facilitate their mission in nature: helping to decompose diverse organic compounds in the surrounding environment. As one of these enzymes, pectinase catalyzes the degradation of pectin-containing compounds by two mechanisms: de-esterification and de-polymerization [81]. Many groups of microorganisms can synthesize pectinases, but it was reported that molds such as T. harzianum and A. niger are preferred because they produce around 90% of the enzyme into the culture medium [82]. In the current study, T. viride (T27), T. harzianum (T5), and T. asperelloides (T1) had the highest activity of enzymatic activity. Trichoderma produces this enzyme to help the breakdown of the middle lamella; it then extracts nutrients from the tissues or penetrates pathogen hyphae [83]. This feature of Trichoderma can be used as a biocontrol agent against Aspergillus and Fusarium moniliforme, as reported previously [84]. Biological control agents are capable of solubilizing various elements through different mechanisms. These mechanisms help these agents achieve their roles under different environmental conditions [85]. It was reported that the antagonistic effect of T. harzianum against Rhizoctonia solani is attributed to the breakdown of the pathogen’s hyphae by the extracellular chitinase enzyme [86]. In the mycoparasitic process, Trichoderma species produces lytic enzymes in its hosts, attaching coils around the host hyphae [87]. Another study reported that Trichoderma harzianum antagonized pathogenic isolated fungi [88]. It was also reported that Trichoderma harzianum could inhibit R. solani, P. ultimum, and A. solani [89].

Organisms that produce chitinases have been discussed as the best biocontrol agents against many phytopathogens [90, 91]. The current study showed that all tested Trichoderma isolates have chitinolytic activity with different rates during their screening on solid medium amended with colloidal chitin. These results are consistent with the previous studies, which reported the production of chitinase by different Trichoderma species, such as T. koningiopsis UFSMQ40 [82], T. asperellum PQ34 [92], T. harzianum GIM 3.44 [93], and T. asperellum [94]. The action of the three chitinolytic enzymes limits the mechanism of complete hydrolysis of chitin. These enzymes include exochitinase (acts on the non-reducing end of the chitin polymer), endochitinase (randomly hydrolyzes chitin polymers), and chitobiose (acts on the chitobiose dimer). The three enzymes consecutively release N-acetyl glucosamine units [95, 96]. Therefore, protein profiling of all tested Trichoderma strains showed high fluctuations in response to chitin in addition to the medium. This indicates that Trichoderma strains adjusted their protein profile and physiological cell culture to adapt to the nutritional conditions. The changes were observed between the 40–180 kDa size range, which generally corresponds to the predicted sizes of the chitin metabolism enzymes. These observations are in agreement with those of previous studies, which reported shifts in proteome in response to several nutrients’ limitations in fungi [97–99]. Thus, Trichoderma species can excrete different lytic enzymes during mycoparasitism, such as chitinase and proteases.

The present results indicated the interaction points between the Trichoderma spp. strains (T27 and T40) and Colletotrichum lagenarium as a plant pathogen. The hyphae of Trichoderma spp. were observed to coil around the pathogens’ hyphae. In addition, these results suggested that Trichoderma spp. may increase the IAA production and nutrient solubility (i.e., P, K, and Zn). Additionally, Trichoderma strains demonstrated sufficient hydrolytic enzymes, including chitinase, pectinase, amylase, and cellulase. Therefore, Trichoderma could be used as a biocontrol agent against the plant pathogen with a high potential to produce plant growth promoters. Therefore, application studies to evaluate Trichoderma strains' impact on plant growth need to be implemented in future work.

Author contributions: Conceptualization, O.A.H., K.S.A. and R.M.E.; Isolation: O.A.H., and M.H.I., Methodology, O.A.H., K.S.A. and R.M.E.; Formal analysis, A.M., and M.G.A.; resources, O.A.H., M.H.I. and K.S.A.; - Original draft O.A.H., R.M.E., M.G.A., and A.M.; Sampling and software, A.M., M.H.I. and M.G.A.; Supervision and editing O.A.H.

All authors have read and approved the manuscript for publication.

Conflict of Interest Statement

None declared.

Acknowledgements

The authors would like to thank Dr Abd El-Fatah Abomohra, Professor at Chengdu University, China, for his valuable and critical suggestions to the manuscript.

Data availability: The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

Ethics approval and consent to participate: Not applicable

Funding Information: No funding information was provided.

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Comprehensive Characterization and Screening of Different Trichoderma Isolates as Plant Growth Promoters: Insight to Metal Solubilization, Enzymatic Activity, and Antagonistic Effect

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Abstract

Figures

1. Introduction

2. Materials And Methods

2.1 Trichoderma isolation and identification

2.2 Evaluation of plant growth promotion parameters

2.2.1 Auxin production (IAA) assay

2.2.2 Metal solubilization

2.3 In vitro antagonistic activity

2.3.1 Morphology examination

2.3.2 Enzymatic activities

2.4 Statistical analysis

3. Results

3.1 Trichoderma isolates

3.2 Metal solubilization

3.3 IAA assay

3.4 Enzymatic activities of Trichoderma species

3.5 Antagonistic activity and mechanism

3.5.1 Chitinase potential

3.5.2 Extracellular protein analysis (SDS-PAGE)

3.5.3 Microscopic characteristics

Discussion

Conclusion

Declarations

References

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