Characteristics of Antagonistic Activity of Two Trichoderma Species New to Kazakhstan Against Soil Pathogens

: The high efficiency of representatives of the Trichoderma genus in suppressing a wide range of plant pathogens contributes to the conduct of large-scale research on the search for local strains. The purpose of this study is to characterize the antagonistic activity of two Trichoderma species new to Kazakhstan ( T. pararogersonii and T. rossicum ) isolated from the rhizosphere of Picea schrenkiana Fisch. et C.A. Mey. and Malus sieversii (Ledeb.) M. Roem. against soil phytopathogens. Identification was carried out based on analysis of its region sequences and determination of antagonistic activity was performed by the dual culture method. In the course of our study, we obtained descriptions of colonies of T. pararogersonii and T. rossicum based on isolated pure cultures, morphological data of both species, the sequence of nucleotides, information about inhibitory activity against some phytopathogens and the effect on the growth of some legumes. T. pararogersonii isolate is characterized by a weak level of inhibitory activity. When simultaneously seeded with most test objects, the radius of the colonies of the latter is the same or greater than the radius of the colonies of the Trichoderma . When using the paper disk method, a significant zone of suppression of the growth of phytopathogenic fungi by T. pararogersonii extract was noted in the variant with Alternaria spoon day 4 (23.55±0.72 mm) and the most insignificant one was noted with Purpureocillium lilacinum (Thom) Luangsa-ard, Houbraken, Hywel-Jones & Samson. T. rossicum was characterized by a higher level of inhibitory activity. It was found that with simultaneous seeding of Trichoderma and test objects on Petri dishes, the rapid growth of T. rossicum was observed in all variants of the experiment, except for the variant with Aspergillus niger Tiegh, where suppression of Trichoderma sporulation was also observed.


Introduction
The genus Trichoderma Pers.(Hypocrea Fr.) is widely distributed in various climatic zones of the world and is of great importance (Jaklitsch and Voglmayer, 2015;Chen and Zhuang, 2017).The popularity of fungi of the genus Trichoderma is due not only to the ability to produce several hundred secondary metabolites, some of which are antimicrobial.It is also able to induce plant resistance to pests and pathogens (Lorito et al., 2010), increase the efficiency of the use of nutrients (especially nitrogen), stimulate plant growth, and impart resistance to abiotic stresses (Mendoza-Mendoza et al., 2018).
Individual strains of the genus Trichoderma can control various phytopathogens that cause the oppression and death of many agricultural plants, such as Botrytis cinerea, Sclerotium cepivorum, Rhizoctonia solani, and others (Alvarado-Marchena and Rivera-Méndez, 2016;You et al., 2016;Abbas et al., 2017).
Currently, significant factual material has been accumulated in various countries on physiological, morphological, biochemical, and genetic studies of species of the genus Trichoderma, as well as on the technology of obtaining biological products and their use (Nicot et al., 2016).A small number of isolates with a high antagonistic ability against a wide range of plant pathogens are used as bioagents (Medeiros et al., 2017).The mechanisms of action of Trichoderma include parasitism, competition for nutrients, and antibiotic synthesis (Sood et al., 2020).Depending on the strain, the use of Trichoderma can promote plant growth, as well as induce resistance (Naher et al., 2014;Kumar and Ashraf, 2017).
Trichoderma species and strains as agents of biological plant protection are a promising and safe alternative to chemical means of combating phytopathogenic fungi since they not only effectively restrain their development and spread but also stimulate plant growth and have a beneficial effect on the microbiological community in the soil (Kumar and Ashraf, 2017;Hassan et al., 2019).
The long-term and successful use of representatives of the genus Trichoderma in agricultural production, as well as their high efficiency in suppressing a wide range of plant pathogens, contribute to the conduct of large-scale studies on the search for strains in Kazakhstan.
From the rhizosphere of wormwood (Artemisia glabella) and shrubby Ajania (Ajania fruticulosa (Ledeb.)Poljak.)cultivated in Kazakhstan on an industrial scale as raw materials for the pharmaceutical industry in the treatment of oncological diseases, four strains of fungi of the genus Trichoderma were obtained, differing in colony density, texture, color, and growth rate.The isolates were identified as Trichoderma viride Pers.(T.lignorum (Tode) Harz.), based on macroscopic and microscopic morphological features (Rakhimova et al., 2012).When studying the antibiotic activity of the isolated strains, it was found that strain 90-2 of T. viride exhibited antagonistic properties against fungi of the genus Fusarium, and strain 9(2) showed antagonistic activity against fungi of the genus Alternaria (Rakhimova et al., 2012).Antagonist strains (Trichoderma viride 22, Trichoderma album 23, Trichoderma asperellum 175, Trichoderma asperellum 1m) have been identified concerning pathogens affecting legumes (chickpeas, peas, beans) and fodder (alfalfa) crops growing in the Almaty region (Bekmakhanova et al., 2015).
The purpose of this study is to characterize the antagonistic activity of two Trichoderma species new to Kazakhstan (T.pararogersonii and T. rossicum) against soil phytopathogens.

Materials and Methods
Soil samples were collected in the rhizosphere of various woody plants on the territory of two ridges of the Northern Tien Shan: Dzungar and Kungei Alatau in Kazakhstan.Soil samples were taken during the growing season of 2020-2021 in a soil horizon of 5-20 cm after removing the top layer of litter in dark coniferous and small-leaved forests on mountain forest soils.867 soil samples were selected and examined, while representatives of the genus Trichoderma were found in 95 samples.Two species turned out to be new to the territory of Kazakhstan: Trichoderma pararogersonii (Jaklitsch, 2009) 1).
The geographical location of each sample collection site was recorded using GPS (Germin).Isolation and subsequent identification of soil fungi were carried out according to Khabirova et al., (2022), using the literature on fungi of the genus Trichoderma (Jaklitsch, 2009;2011).The names of fungi species and authors are given following the Index Fungorum database (Fungorum 2022).For molecular genetic identification of fungi samples, 3-7 daily strains of fungi were used.The mycelium was frozen at -20°C.Then it was ground with a pestle in a 1.5 mL Eppendorf test tube to a powdery state.Deoxyribonucleic Acid (DNA) was isolated from the resulting mass using the plant/fungi DNA isolation kit from Norgen Biotek Corp. (Ontario, Canada) according to the manufacturer's protocol.The DNA concentration in the samples was determined using a Qubit tm dsDNA HS assay kit (life technologies, Oregon, USA) fluorimeter on a double-stranded Deoxyribonucleic Acid High Sensitivity (dsDNA HS) scale.The following universal primers of the Internal Transcribed Spacer (ITS) region of fungi were used in the work: ITS1 (5-TCCGTAGGTGAACCTGCGG-3) and (5-TCCTCCGCTTATTGATATGC-3).The reaction mixture for amplification consisted of: 12.5 µL Q5 ® Hot Start High-Fidelity 2× Master Mix, 1.25 µL Forward Primer (10 µm), 1.25 µL Reverse Primer (10 µm), 1.5 µL DNA, and 8.5 water.The total volume of the Polymerase Chain Reaction (PCR) mixture was 25 µL.
PCR was performed on an Eppendorf ProS amplifier (Hamburg, Germany) at the following amplification mode: 94°C for 30 sec; 55°C for 1 min; 72°C for 40 sec in a total of 30 cycles; and 72°C for 10 min.The amplification results were viewed in 1.2% agarose gel.The PCR products were purified with CleanSweep™ PCR Purification reagent (Applied Biosystems, USA).The sequencing reaction was performed using a BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, USA) according to the manufacturer's instructions (BigDye ® Terminator v 3.1 Cycle Sequencing Kit Protocol, Applied Biosystems, USA), followed by fragment separation on an automatic genetic analyzer 3500 DNA Analyzer (Applied Biosystems, USA).The sequencing results were processed in the SeqA software (Applied Biosystems, USA).The obtained nucleotide sequences of the ITS region of fungal DNA were compared with the GeneBank database data (GenBank, 2022), using the BLAST software.The phylogenetic analysis was performed using MEGA6 software.The ClustalW algorithm was used to align nucleotide sequences and the Neighbor-Joining (NJ) method was used to build phylogenetic trees (Simonsen et al., 2008).
Antagonistic properties of isolates against the phytopathogenic fungi Alternaria sp., Aspergillus niger, and Fusarium sp.(strain 320), stored in the collection of the Institute of Botany and Phytointroduction, were determined by the method of dual cultures (Asaturova et al., 2012) at the optimal temperature for the growth of microorganisms 25-26°C on Potato Dextrose Agar (PDA).The size of fungal colonies was measured on days 4-6 using the linear method (mm) from the back of the dish and the percentage of inhibition was calculated (Asaturova et al., 2012).
The inhibitory activity was also determined by the paper disk method.A disk of filter paper moistened with an extract from the Trichoderma mycelium was applied to the test culture of phytopathogenic fungi evenly distributed over the surface of the solid nutrient medium in a petri dish.After incubation, the growth suppression zone of the test culture was determined on the fifth day.
Evaluation of the effect of strains of the genus Trichoderma on the growth of roots and seedlings of legumes was carried out on peas (varieties Nikitka and Zhigalov 112) and beans (varieties Saxa without fiber and Bemol).In the experimental version, seeds (100 seeds in three repetitions) were germinated and treated with Trichoderma spores by powdering until full saturation.The seeds that had not been treated with spores were used in the control variant.The seeds were sown in the Magic bed nutrient soil for seedlings (Buiskie udobreniya, Russia) in mini cassettes for seedlings with a cell size of 55×55×40.On the 7 th -8 th day after germination, the length of stems and roots was measured and the growth rate was calculated (Asaturova et al., 2012).
Statistical analysis of the results (determination of the arithmetic mean and its error) (Zverev and Zefirov, 2013) was carried out using the Excel 2010 software package.

Results
The antagonistic activity was determined for two species new to the territory of Kazakhstan: Trichoderma pararogersonii (Jaklitsch, 2009), Voglmayr and T. rossicum Bissett, C.P. Kubicek et Szakács.

Cultural and Morphological Features of Trichoderma Pararogersonii
On the PDA medium, colonies were well-growing, densely fluffy, patchy, and brownish-green, with welldefined concentric zones (Fig. 2).The substrate and aerial mycelium was well-developed.The aerial hyphae were especially numerous along the border of the colonies.The reverse was not stained.
The degree of homology with the nearest strain KY750455.1:53-596Trichoderma pararogersonii isolate CTCCSJ-F-KZ40688 was 100.00%, which allows us to attribute the studied sample to this species (Fig. 4).The obtained isolate of Trichoderma pararogersonii was characterized by a weak level of inhibitory activity.In the conditions of simultaneous seeding with most test objects, the radius of the colonies of the latter was the same or greater than the radius of the colonies of the Trichoderma (Table 1, Figs. 5-6).The rapid growth of Trichoderma pararogersonii and suppression of the growth of the test object was observed only in the variant of the experiment with Penicillium expansum with simultaneous seeding with the test object (Table 1).When using the paper disk method, a significant zone of suppression of the growth of phytopathogenic fungi with Trichoderma pararogersonii extract (strain 249v) was noted in the variant with Alternaria sp. on day 4 and the most insignificant one was noted in the variant with Purpureocillium lilacinum (Table 2). Test Test    When studying the effect of Trichoderma pararogersonii strain 249 v on the growth of legume seedlings, it was found that in all studied varieties of peas and beans, the length of the ground and underground parts on the eighth day in the experimental variants was slightly bigger than in the control variant (Table 3).
Strain 249 v of Trichoderma pararogersonii obtained by us had a positive effect on the growth rate of peas and beans.

Cultural and Morphological Features of Trichoderma Rossicum
The colonies were colorless, thin, usually non-zonal, and rapidly growing (Fig. 7).The aerial hyphae were quite abundant.At 25°C on the PDA, the mycelium covered the surface of the dish for a week or 10 days.Sporulation began on days 2-3, while the surface of the colony became coarse and gradually acquired a yellowish-green hue.The reverse side of the colonies was not colored.
The conidiophores developed slowly and their concentration was observed at the borders of colonies.The size of the phialides was 5-7×3.5-4.5 µm.The conidia were oval or ellipsoid, 4.5-5×2.7-3µm in size, and smooth (Fig. 8).Chlamydospores were not observed.

Cultural and Morphological Features of Trichoderma Rossicum
The degree of homology with the nearest strain MF408977.1:40-579 of Trichoderma rossicum isolate CTCCSJ-W-QT23800 was 100.00%, which made it possible to attribute the studied sample to this species (Fig. 9).
When determining the inhibitory activity of Trichoderma rossicum strain 439a by the method of dual cultures, it was found that with simultaneous seeding of Trichoderma and test objects on petri dishes, rapid growth of T. rossicum was observed in all variants, except for the variant with Aspergillus niger (Table 4,.Aspergillus niger suppressed the sporulation of Trichoderma rossicum strain 439a. When studying the effect of Trichoderma rossicum strain 439a on the growth of legumes, it was found that in all studied varieties of peas and beans, the length of the ground and underground parts on the eighth day was slightly longer than in the control variant (Table 5).
Strain 439a of Trichoderma rossicum obtained by us had a positive effect on the growth rate of peas and beans. Test

Discussion
According to our study and scientific data (Rakhimova et al., 2012;Bekmakhanova et al., 2015), 18 species of the genus Trichoderma have been found in Kazakhstan, two of which are new: T. pararogersonii and T. rossicum.For T. rossicum, the most closely related species is T. stromaticum, known from tropical America and usually forming associations with Theobroma cacao L. The endogenous strains of T. stromaticum penetrating deeply into the vascular system of cocoa stems have been described (Samuels et al., 2012).The habitat of T. rossicum is soil, the known distribution has been found in Russia, Austria, and Peru (Jaklitsch, 2011).T. stromaticum, T. rossicum and the recently discovered species T. barbatum, T. caesareum, T. floccosum, T. ivoriense, T. lanuginosum, and T. vermipilum form a unique section of Trichoderma, clade Stromaticum (Samuels et al., 2012).T. pararogersonii is characteristic of the wood and bark of broad-leaved trees and is widespread in Mediterranean Europe, in particular, in Croatia and Greece (Jaklitsch and Voglmayer, 2015).This species belongs to the section (clade) Viride, which also includes T. viride, T. viridescens, T. asperellum, T. hamatum, T. koningii, T. koningiopsis, etc.In contrast to the Stromaticum clade, where antagonistic activity has been studied only for new species of T. hebeiense, T. sichuanense and T. verticillatum (Chen and Zhuang, 2017), antagonist strains from the viride clade have been studied quite well.
When studying the antagonistic activity of 15 Trichoderma isolates belonging to the species T. harzianum, T. asperellum, T. longibrachiatum, T. viride against Fusarium oxysporum f. sp.capsici, it was found that the value of inhibition of mycelial growth of the pathogen in vitro ranged between 35.7% and 85.8% (Hewedy et al., 2020).In our case, suppression by Trossicum of mycelial growth of Fusarium sp.equaled 72.5%.
Strains of antagonists of Trichoderma viride, T. album, and T. asperellum species have been identified concerning pathogens affecting legumes (chickpeas, peas, beans) and fodder (alfalfa) crops growing in the Almaty region.The strains showed the highest antagonistic activity and growth rate against Fusarium solani, F. sporotrichiella var.poae, and Alternaria compacta, with the diameter of the pathogen growth suppression zone ranging from 28-39 mm.Trichoderma strains were less effective on Fusarium oxysporum, Alternaria alternate, and A. tenuis: The diameter of the pathogen growth suppression zone ranged from 22-30 mm (Bekmakhanova et al., 2015).In our experiments, the zone of suppression of the growth of phytopathogenic fungi (test objects) by extracts of Trichoderma pararogersonii was significantly smaller and ranged from 11.57±0.53-23.20±0.44 mm on the sixth day.
Antibiotic activity against toxin-forming strains of Fusarium sambucinum and F. sporotrichioides was shown by strains MG/6, T-30, and TH-7 of Trichoderma asperellum.The highest antagonistic activity against phytopathogens of the genus Fusarium was noted for strains MG/6 and T-30 (Popova and Sadykova, 2014).Some researchers believe that T. harzianum and T. viride strains were the best antagonists for some plant pathogens with an inhibition percentage of 60-80% (Abd Elahi et al., 2012;Kumar et al., 2012).In our case, the suppression by T. rossicum of mycelial growth of test objects was up to 72.5%.
When studying the effect of Trichoderma asperellum MG-97 spores and Fusarium sporotrichioides Z3-06 metabolites on the physiological parameters of young wheat plants (Triticum aestivum), it was found that T. asperellum increased the laboratory germination of seeds and enhanced plant growth on the 10 th and 30 th days of vegetation.Therefore, the leaf area, the mass of the aboveground part of plants, and the mass of roots increased.In turn, F. sporotrichioides metabolites negatively affected most of the listed indicators.With the combined action of Trichoderma spores and fusarium metabolites, growth rates were close to the control variant, but seed germination and germination energy were suppressed (Golovanova et al., 2020).Strain 439a of Trichoderma rossicum obtained by us had a positive effect on the growing intensity of the ground and underground parts of legume seedlings (peas and beans).However, we did not evaluate the germination of seeds and germination energy.
Thus, the strain of T. rossicum isolated by us actively restrained the growth and development of pathogens and positively affected the growth rate of peas and beans, which makes it possible to use the studied strain as a biological plant protection agent, especially since local strains are more productive and also more resistant to biocontrol since they are well adapted to conditions of the local environment.
T. pararogersonii isolate is characterized by a weak level of inhibitory activity.At the same time, T. rossicum is characterized by a higher level of antagonistic activity.We found that with simultaneous seeding of Trichoderma and test objects on Petri dishes, the rapid growth of T. rossicum was observed in all variants of the experiment, except for the variant with Aspergillus niger Tiegh, where suppression of Trichoderma sporulation was also observed.In addition, T. rossicum had a positive effect on the growth rate of peas and beans.
The level of antagonistic activity of the T. rossicum strain isolated by us makes it possible to use it as a biological plant protection agent, especially since local strains are more productive, as well as more stable under biocontrol since they are well adapted to the conditions of the local environment.

Fig. 4 :
Fig. 4: A phylogenetic tree constructed by comparing the ITS region of the studied sample with the sequences of reference strains placed in the blast international database

Fig. 9 :
Fig. 9: A phylogenetic tree constructed by comparing the ITS region of the studied sample with the sequences of reference strains placed in the Blast International Database

Table 1 :
Determination of the inhibitory activity of Trichoderma pararogersonii isolate during simultaneous seeding with test objects (by the method of dual cultures) Mycelium growth inhibition of the test object, %

Table 2 :
The zone of suppression of the growth of phytopathogenic fungi by extracts of Trichoderma pararogersonii (strain 249v) by the method of paper disks, mm Growth suppression zone, mm -

Table 3 :
Effect of the Trichoderma pararogersonii strain on the growth of roots and seedlings of peas and beans, on day 8, cm

Table 4 :
Determination of the inhibitory activity of Trichoderma rossicum strain 439a during simultaneous seeding with test objects (by the method of dual cultures)Mycelium growth inhibition of the test object, %

Table 5 :
Effect of Trichoderma rossicum strain 439a on the growth of roots and seedlings of peas and beans, on day 8, cm