SCREENING NEW TRICHODERMA ISOLATES FOR ANTAGONISTIC ACTIVITY AGAINST SEVERAL PHYTOPATHOGENIC FUNGI, INCLUDING FUSARIUM SPP

Aims. To obtain and characterize new isolates of Trichoderma antagonistic to phytopathogenic fungi, including Fusarium spp., and 2) to determine their suitability for mass production under different cultivation conditions. Methods. Microbiological, cultural-morphological, statistical. Results. From plants affected by phytopathogenic fungi: cucumber (Cucumis sativus L.), tomato (Solanum lycopersicum L.), white cabbage (Brassica oleracea L.), winter wheat (Triticum aestivum L.), spring barley (Hordeum vulgare L.) in the Forest-Steppe of Ukraine (Kyiv region) 11 new Trichoderma isolates were obtained. Preliminary, morphological determination allocated fi ve of them to T. viride (isolates СК, 165, 27, 49, 35), two of them to T. koningii (21, 64) and four of them to T. longibrachiatum (161, 162, 163, 164). All isolates showed moderate to high antagonistic activity towards 8 phytopathogenic fungal species (Fusarium oxysporum, Fusarium solani, Alternaria cucumerina, Colletotrichum phomoides, Botrytis cinerea, Trichothecium roseum, Penicillium sp., Cladosporium fulvum). In a dual culture experiment they showed generally similar or often higher activity to the above-mentioned fungi than the 8 control strains used in our study, belonging to T. viride (5 strains), T. koningii (2 strains) and T. harzianum (1 strain), which have been maintained since long time in our laboratory. The most active new isolate CK, (presumably) T. viride, showed comparable high activity towards all phytopathogenic fungi as compared to our most active control strain of T. viride, no. 23. The latter is the basis of a biocide Trichodermin, produced by biolaboratories of Ukraine, including the Institute of Plant Protection, NAAS, Kyiv. Chlamydospore production of all isolates and strains studied in submerged culture varied from 106 to 3 · 107 spores/ml, were T. viride isolates and strains were on the higher end. Isolates of ‘T. longibrachiatum’ did not produce chlamydospores in submerged culture. Upon superfi cial cultivation on barley grain, the strains and isolates of T. viride were also characterized by the highest production of spores (6 · 109–9 · 109 spores/g) as compared to those of T. koningii, T. harzianum (5.5 · 109–6.8 · 109 spores/g) and T. longibrachiatum (1.3 · 108–6.8 · 108 spores/g). In an in-vivo experiment under laboratory conditions the most promising antagonistic isolate CK was used to inoculate wheat seed and tested for protection against Fusarium root rot (inoculum a mixture of F. avenaceum, F. culmorum, F. gibbosum, F. oxysporum, in 4·104 spores/g), where it gave an 83 % reduction in root rot as compared to the non-inoculated control. Conclusions. Five new isolates preliminarily (on the basis of morphological characteristics only) allocated to T. viride and four to T. longibrachiatum demonstrated in vitro the highest and widest antagonistic activity against the phytopathogenic fungal species Fusarium oxysporum, Fusarium solani, Alternaria cucumerina, Colletotrichum phomoides, Botrytis cinerea, Trichothecium roseum, Penicillium sp., Cladosporium fulvum, as compared to new isolates, preliminarily allocated to – T. harzianum and T. кoningii. New isolate CK (allocated to T. viride) showed a promising and similar high antagonistic activity as compared to our T. viride 23 strain, which is already successfully used in the biocide Trichodermin. Since this isolate CK also produced a high number of chlamydospores in submerged culture (3 · 107 spores/ml) and conidia (8 · 109 spores/g) when surface cultured on barley grain respectively, it is a potential new candidate for a biocide. When this CK isolate was studied in a small laboratory pot experiment, to control Fusarium root rot in wheat by preventive seed inoculation, it caused an 83 % reduction in this Fusarium root rot. Its usefulness under fi eld conditions and its effect on growth of plants will be investigated in future research.


INTRODUCTION
Trichoderma fungal species are worldwide and in all climate zones an essential part of the microfl ora in all types of soils, both in terrestrial and marine ecosystems (Klein, Eveleigh, 1998). They occur commonly in the rhizosphere, but can also colonize (as endophytes) aboveground plant parts.
Recent investigations have shown that Trichoderma spp. can sense, penetrate and kill other fungi and this has led to the development of a large number of successful biopesticides with Trichoderma spp. as basis (Benitez T et al, 2004;Harman GE et al, 2005;Schuster, Schmoll, 2010;Kubicek CP et al, 2011;Błaszczyk et al, 2014;Sawant IS, 2014;Sood et al, 2020).
Trichoderma spp. produce a wide spectrum of (volatile) antibiotics killing a number of fungi and nematodes. Their metabolites, including enzymes are able to stimulate plant growth, to induce resistance in plants against pathogens and induce abiotic stress tolerance and are used in industrial processes (Mastouri F et al, 2010;Lorito et al, 2010;Contreras-Cornejo HA et al, 2011;Sawant IS, 2014;López-Bucio J et al, 2015;Fiorentino N et al, 2018). Some Trichderma spp are noxious, because they are pathogenic to humans (Sandoval-Denis SA et al, 2014) and cultivated mushroom (Goltapeh EM, Danesh YR, 2006).
In addition to high antagonistic potential, the fungi of this genus are notable for good growth rate and the possibility of cultivating on non-expensive substrates, thus, they are widely used in the elaborations of biological preparations in industrial conditions. The cultivation of Trichoderma fungi is done both superfi cially using loose substrates (Cavalcante RS et al, 2008, Motta FL et al, 2012, and by the submerged technology in liquid culture media (Jakubíková L et al, 2006;Šimkovič M et al, 2008;Al-Taweil HI et al, 2009;Kobori NN et al, 2015;Ferdous Akter M et al, 2018).
Biofungicides, based on Trichoderma spp. were presented worldwide in 2014 by over 250 products and comprised about 60 % of the international market of biofungicides. The most common species in most products, based on Trichoderma, is T. harzianum (83 %), followed by T. viride, and and T. koningii (Woo et al, 2014). T. harzianum and T. viride are mainly used to treat soil for about 87 different crops against 70 soil pathogens and 18 foliar pathogens, predominantly fungi such as Verticillium spp., Fusarium spp., Rhizoctonia solani, Sclerotinia sclerotiorum, Alternaria radicina, Pythium spp, and Botrytis cinerea (Topolovec-Pintarić, S., 2019).
The aims of our study were 1) to obtain and characterize new isolates of Trichoderma antagonistic to phytopathogenic fungi, including Fusarium spp., and 2) to determine their suitability for mass production under different cultivation conditions.

MATERIALS AND METHODS
The studies were conducted in the Laboratory of Microbiological Methods of Plant Protection at the Institute of Plant Protection, NAAS, Kyiv in 2015-2019.
Trichoderma fungi were isolated from plants affected by phytopathogenic fungi: cucumber (Cucumis sativus L.), tomato (Solanum lycopersicum L.), white cabbage (Brassica oleracea L.), winter wheat (Triticum aestivum L.), spring barley (Hordeum vulgare L.) in the Forest-Steppe of Ukraine (Kyiv region) (Table  1). Isolates were obtained by transferring germinated conidia, which appeared on the surface of parts of plants affected by phytopathogenic fungi, to wort agar medium.
Preliminary allocation of the new isolates to a Trichoderma species was done on the basis of morphological features only, following the keys of Gams W and Bissett J, (2002).
The antagonistic activity of 11 new isolates and 8 collection strains of Trichoderma (kept for 30 years in the Laboratory of Microbiological Methods of Plant Protection at the Institute of Plant Protection, NAAS) (see Table 2) was determined by the method of blocks the dual culture assay, using different species of phytopathogenic fungi, isolated by us from diseased plant material and maintained as agar slants in our culture collection (Table 2) -Fusarium oxysporum (isolated from the roots of cucumbers), Fusarium solani (isolated from a tomato stalk), Alternaria cucumerina (isolated from the leaves of cucumbers), Colletotrichum phomoides (isolated from the fruit of the tomato), Botrytis cinerea (isolated from the fruit of the cucumber), Trichothecium roseum (isolated from soybean seeds, Glycine max (L) Merr), Penicillium sp. (isolated from soybean seeds), and Cladosporium fulvum (isolated from cucumber leaves).
The strains of phytopathogenic fungi (Table 2) were grown in Erlenmeyer fl asks on an orbital shaker (PSU-20i, Biosan, Latvia) in liquid wort medium for 72 h at 200 rpm and 25 °C in the dark. Inoculum for fl asks was obtained by rinsing with sterile water spore-mycelial mass from the surface of agar in test tubes. One ml of liquid culture of each phytopathogenic strain was added to a Petri dish and mixed with 15 ml of wort agar. Trichoderma isolates and strains under investigation were grown in Petri dishes using wort agar for 5 days at 25 °C. Agar blocks (5 mm diam.) were cut out with a cork borer from the margins of 7-day-old colonies and placed into the Petri dishes with the just seeded phytopathogenic fungi, 3 blocks per Petri dish, in equal distance from each other. Incubation was for 5 days at 25 °C in an incubator (TS-80, Czech Republic).
The antagonistic activity was determined in the dual culture assay on day 5 of the experiment by measuring the growth inhibition zones of test objects using the following scale: inhibition zone 0 mm, inactive; 1-14 mm poor activity; 15-25 mm moderate activity; >26 mm strong activity. The dual culture assay was done in three repeats.
The technological suitability for mass production of the new Trichoderma isolates was determined by submerged and surface cultivation using chlamydospore formation and biomass production as indices.
Submerged cultivation was done in 300 ml Erlenmeyer fl asks with 50 ml of medium on a rotary shaker (200 rpm) at 25-26 °С for 72 h in the medium, using corn extract (side product of starch and syrup production, PrJSC 'Dneprovsky Starch and Syrup Integrated Works', Dniprovske, Dnipropetrovskyi region) and beet molasses (side product of sugar beet production, PrJCT 'Salyvonkivskyi Sugar Plant', Hrebinky, Kyiv region): corn extract -1 %; molasses -3 %; NH 4 NO 3 -0.5 %; KH 2 PO 4 anhydrous -0.5 %; MgSO 4 ·7H 2 O -0.2 %. (Tkalenko AN, Goral SV, 2013). Flasks with medium were sterilized in an autoclave at 0.1 MPa for 30 minutes. Inoculation of fl asks was performed with 7-day cultures grown in test tubes. The inoculum concentration was 5 · 10 6 spores/ml. Surface cultivation was done in 1 L Erlenmeyer fl asks with barley seeds. The fl asks were fi lled with substrate to 1/3 volume, moistened with water (70-80 % by weight) and sterilized in an autoclave at 0.1 MPa for 30 minutes. Inoculation of fl asks was performed with 7-day cultures grown in test tubes. The inoculum concentration was 1 · 10 6 spores/g. (Bondarenko et al, 1985) Cultivation was performed for 10 days till massive formation of air-distributed conidia at 25-26 °С occurred.
To determine the biomass of Trichoderma fungi, 10 ml of liquid culture was fi ltered through a paper fi lter (pore size 4-7 μм), washed with distilled water and dried in an drying oven at 110 °С for 1 hour. The biomass was determined by weighing using a precision scale (Axis, Poland). The productivity of fungal spore formation was determined via direct calculation of spores in a Goryaev's chamber (haemacytometer with 90 nL capacity instead of the usual 100 nL) with a microscope (Zeiss Primo Star, ×400).
The calculation of optimal inoculum doses of a liquid biopreparation based on isolate T. viride СК (3.5 × × 10 7 chlamydospores/ml) was done using the pretreatment of winter wheat seeds of Myronivska 808  СК, 165, 27, 49, 35; T. koningii, isolates 21, 64; T. longibrachiatum, isolates 161, 162, 163, 164. It was determined that the 11 new Trichoderma isolates differed in their antagonistic activity against the eight phytopathogenic fungi tested (Table 2). In general, the solates, morphologically allocated to T. viride and T. longibrachiatum showed a higher activity and broader spectrum of action than the isolates allocated to T. harzianum and T. koningii.
Seven out of ten investigated isolates and strains of T. viride (23, СК, Тр-1, 75, 27, 49, 35) demonstrated high antagonistic activity regarding all fungal spp. tested, both in producing antibiotic substances and in hyperparasitic activity with the build-up of phytopathogen colonies and abundant spore formation (Table 2). Isolate T. viride CK showed the highest activity apart from the control strain for this species, T. viride 23 (Fig. 1). Isolate T. viride 165 demonstrated much lower antagonistic activity regarding two Fusarium species (2-2.5 times smaller diameters of the growth inhibition zones of phytopathogens compared with isolate T. viride CK and strain T. viride 23), and this isolate did not inhibit the growth of Trichothecium roseum and Cladosporium fulvum.
Some of our laboratory strains showed rather disappointing results concerning antagonism of some phytopathogenic fungi: high activity of the T. viride T-3 strain was found only against Trichothecium roseum, as for Alternaria cucumerina, Colletotrichum phomoides, Botrytis cinerea, Penicillium sp., Cladosporium fulvum, it had moderate activity against F. oxysporum and no activity against Fusarium solani. T. viride ТМ demonstrated poor antagonistic activity against Alternaria cucumerina, Colletotrichum phomoides, Botrytis cinerea, Cladosporium fulvum, and did not inhibit the growth of F. oxysporum, F. solani, Penicillium sp.
Both laboratory strains of T. harzianum demonstrated high antagonistic activity, inhibiting the growth of all the investigated species of phytopathogenic fungi.
The isolates of T. longibrachiatum showed also a good antagonistic activity, except against both Fusarium spp. tested.
The isolates and control strains of T. koningii were characterized by low to moderate antagonistic activity only against all fungal spp. tested.
Under submerged cultivation conditions in liquid c orn extract/molasse medium, all 19 investigated isolates and strains produced almost a similar amount of biomass over a period of 72 hours cultivation at 25 °C, namely 10.2-14.7 g/l (Table 3).
All studied strains and isolates of T. viride (Fig.2), T. harzianum and T. koningii produced chlamydospores in the submerged culture.
Spore formation productivity of the new isolate T. viride СК was on the level of the biocide Trichodermin control strain T. viride 23, namely 3 · 10 7 chlamydospores/ml after 72 hours growth in corn extract/molasse medium at 25 °C. Other isolates and strains of T. viride produced a smaller amount of chlamydospores, 1.0 · 10 7 -1.5 · 10 7 /ml. The productivity of T. harzianum strains was 8 · 10 6 chlamydospores/ ml. A considerably smaller number of chlamydospores was produced by the T. koningii control strain and the two new isolates -0.7·10 6 -1.4·10 6 chlamydospores/ml.
The new isolates of T. longibrachiatum did not produce chlamydospores in the submerged culture (Fig. 3). Under surface cultivation on barley seeds, T. viride isolates and strains were characterized by higher productivity compared to strains of the other three species (T. koningii, T. harzianum, T. longibrachiatum of aerial spores (conidia) -6.2 · 10 9 -9.2 · 10 9 spores/g, and only two strains of this species (ТМ and Т-3) produced a smaller number of conidia, viz. 3.8 · 10 9 -4.3 · 10 9 spores/g. The productivity of T. koningii and T. harzianum control strains was on the level of 5.5 · 10 9 -6.8 · 10 9 spores/g, and the productivity of conidia formation in the four new T. longibrachiatum isolates was the lowest, namely 1.3 · 10 8 -6.8 · 10 8 spores/g. T. longibrachiatum isolates were notable for later formation of spores (by 7-10 days) as compared to T. viride strains and isolates.
The treatment of seeds with a preparation with T. viride isolate CK in a 1 % concentration decreased the infection of plants with root rots by 83 % as compared to the control. After twenty-fold decrease in the preparation concentration the biological effi cacy decreased twice (40 %), see Table 4.

DISCUSSION
Mass production of biological control preparations requires the availability of initial strains with high antagonistic activity and specifi c, suitable growth characteristics: high productivity, synchronic growth (biomass) and spore formation. Due to observed loss of their initial antagonistic traits, physiological features, change in morphological characteristics, once active strains may show lower or zero activity after long-term storage and subculturing on artifi cial media. Therefore, existing industrial strains of microorganisms require periodic check-up by the main indices -sporulation rate, technological specifi cities, activity rate.
This study was aimed at estimating the antagonistic activity and suitability for cultivation of 19 Trichoderma spp. isolates and strains -11 new isolates and 8 collection strains, the latter have been maintained by us for a long time and used to produce biopreparation Trichodermin.
Submerged cultivation has several advantages, including shorter period of cultivation and the possibility of automation of many operations. Since not all the fungi are capable of spore formation in the submerged culture, the selection of the corresponding strain is of utmost signifi cance (Jakubíková L. et al, 2006). Submerged conidia, produced by some strains of Trichoderma spp., have thinner cell wall compared to air-distributed conidia and lower survival rate while stored (Watanabe S et al, 2006). Chlamydospores are suggested as the most promising propagules to produce preparations using the submerged technology, as they are capable of surviving in soil for a long time, having faster germination in soil and lower sensitivity to soil fungistasis than conidia. Chlamydospore-based formulations have other benefi cial features, including resistance to drying and low temperatures, insensitivity to soil antibiotics, and extended preservation time, which in turn simplifi es processing, storage, and transport of the biological control agent (Beagle-Ristaino JE, Papavizas GC, 1985;Li et al, 2016).
The ability of the investigated Trichoderma strains to produce chlamydospores in the submerged culture (spores of vegetative reproduction), which are the forms in the cycle of fungus development, most resistant to the impact of unfavorable environmental factors, ensuring their survival in soil, is the prerequisite for the elaboration of the submerged technology of liquid preparation production. Therefore, following the results of our initial laboratory assessment, the new T. viride СК isolate was selected for further studies, as it is the most promising candidate for a microbiological preparation, judging by its antagonistic activity and technological traits under submerged cultivation. Growth characteristics of T. viride isolate CK after incubation of 5 days on wort agar at 25 °C are shown in Fig. 4.
The suitability of Trichoderma spp. isolates and strains for mass cultivation was determined by the indices of conidia formation during superfi cial cultivation on grain and the capability of producing chlamydo-  Data presented as mean values ± standard error spores in the submerged culture. Submerged cultivation was conducted using a culture medium, based on cheap organic sources of nutrients -corn extract and molasses, which can completely meet the demands of fungi for necessary nutrients and are economically reasonable, being by-products of food industry. Optimization of the culture medium, selection of the components and determination of their optimal content for the cultivation of the new T. viride isolate СК will be a further direction of our research.
T. viride isolate CK, a promising highly antagonistic isolate in vitro, also showed promising antagonistic activity towards Fusarium spp. (reduced the number of plants affected by root rot by more than 80 %) when tested in a small laboratory based experiment where its effect on the reduction of root rot in wheat, variety Myronivska 808, was investigated.
In our earlier studies, we tested a preparation based on the T. viride CK isolate in the fi eld using cucumber. Pre-treatment of cucumber seeds hybrid Courage F1 at a rate of 2 ml/kg provided complete protection against plant loss during the seedling phase as a result of root rot caused by Fusarium spp. in the greenhouse (Balvas-Hremiakova K, 2019).
We also found that in addition to the protective effect, the T. viride CK isolate also demonstrated its growth-stimulating activity, increasing the germination by 20-25 %, furthermore metabolites, synthesized this isolate, stimulated growth activity of meristem cells -the length of the root and stem of cu-cumber plants was 25-30 % larger during the seedling phase as compared to the control (Balvas-Hremiakova K and Goral S, 2019).

CONCLUSIONS
Five new isolates preliminarily (on the basis of morphological characteristics only) allocated to T. viride and four allocated to T. longibrachiatum, demonstrated in vitro the highest and widest antagonistic activity against the phytopathogenic fungal species Fusarium oxysporum, F. solani, Alternaria cucumerina, Colletotrichum phomoides, Botrytis cinerea, Trichothecium roseum, Penicillium sp., Cladosporium fulvum, as compared to new isolates, preliminarily allocated to T. harzianum and T. кoningii. New isolate CK (allocated to T. viride) showed a promising and similar high antagonistic activity as compared to our T. viride 23 strain, which is already successfully used in the biocide Trichodermin. Since this isolate CK also produced a high number of chlamydospores in submerged culture (1 · 10 7 -3 · 10 7 spores/ml) and conidia (6 · 10 9 -9 · 10 9 spores/g) respectively when surface cultured on barley grain, it is a potential new candidate for a biocide. When this CK isolate was studied in a small laboratory pot experiment, to control Fusarium root rot in wheat, by preventive seed inoculation, it caused an 83 % reduction in this Fusarium root rot. Its usefulness under fi eld conditions and its effect on growth of wheat plants will be investigated in future research.