© The Authors, published by EDP Sciences, 2020

1 Introduction

Fusarium is one of the most harmful and widespread diseases of agricultural plants in the world caused by soil phytopathogenic fungi, including representatives of the genus Fusarium. Infection can occur at any stage of crop growth and seriously reduce yield and degrade fruit quality, especially in protected ground [1]. It is economically feasible to use biological control methods that are environmentally friendly in the integrated fight against fusarium [2].

The biological protection of plants from pathogens of Fusarium infections is becoming increasingly important in the production of greenhouse cucumber (Cucumis sativus). Fusarium wilt of a cucumber (tracheomycosis) according to many authors, the disease causes death on average up to 10-15 %, and in some years up to 65 % of cucumber plants. It is possible to successfully reduce the rate of wilting of plants caused by fungi of the genus Fusarium, possibly using antagonistic microorganisms such as Bacillus, Enterobacter, and Pseudomonas, which are the main root colonizers and can stimulate plant protection [3].

Many researchers have shown that in the biological control Fusarium successfully use fungi of the genus Trichoderma spp. [4] five main mechanisms of combating phytopathogens, including mycoparasitism through the secretion of hydrolytic enzymes, competition for nutrition, antibiosis in the production of secondary metabolites, stimulating plant growth and stimulating systemic resistance to diseases in plants [5].

To date, more than 340 species of Trichoderma have been described [6, 7], which have potential biological activity against phytopathogenic fungi. The most commonly used species are T. asperellum, T. atroviride, T. harzianum, and T. polysporum [8].

Thus, based on the foregoing, the relevance of the search for effective biological control agents for fungi of the genus Fusarium, the causative agent of fusarium wilt of cucumber, from the genus Trichoderma, is shown.

The aim of this study was to assess the effect of Trichoderma atrobrunneum exemetabolites VKPM F-1434 and other related strains of microorganisms against fungi of the Fusarium genus, followed by assessment of their effect on the growth and development of cucumber seeds in vitro and in vivo.

2 Materials and methods

2.1 Object of research

For the experiment, we used live cultures of Trichoderma fungi: Trichoderma atrobrunneum VKPM F-1434, Trichoderma harzianum 5/14, Trichoderma Lixii T4/14; phytopathogenic fungi of the genus Fusarium: Fusarium oxysporum B/14, Fusarium oxysporum MOS509, Fusarium fujikuroi IMI 58289 from the academic collection of the department of biotechnology of the Oryol state agrarian University, for a long time stored in the refrigerator at a temperature of +4°. All represented fungi of the genus Trichoderma before 2015 belonged to the same species Trichoderma harzianum [9]. Cucumber seeds of the “German F1”variety were used as objects of research. The cucumber variety “ German F1” is a hybrid universal variety that is suitable for growing in greenhouses and farms.

2.2 Method of research

Direct confrontational analysis of Trichoderma atrobrunneum VKPM F-1434 against Fusarium oxysporum was tested by double culture method on PDA (Zhang, et al 2017) [10]; testing of the antagonistic activity of fungi of the genus Trichoderma was performed using the method (You, et al., 2016) [11]. Spore suspensions of antagonists for research were prepared according to the methods (Regalado et al., 2000) [12]. Determination of enzymatic activity was performed using express tests according to the method (Cruz-Quiroz, et al., 2017) [13]. Germination energy and laboratory germination were determined according to the requirements Russian Standard 12038-84. The prevalence of Fusarium infection was taken into account in the phase of active fruiting on the 40th day of vegetation by a combined method (Chumakova, 1974).

Statistical processing of results was performed using the Microsoft Office 2010 (Excel) package. All experiments were carried out in five-fold repetition.

3 Results

3.1 Determination of antagonistic activity of fungi of the genus Trichoderma

To assess the degree of manifestation of antagonistic activity and mechanisms of action on phytopathogens, the influence of Trichoderma atrobrunneum antagonists strain VKPM F-1434, Trichoderma Lixii isolate T4/14, Trichoderma harzianum isolate 5/14, on strains of phytopathogenic fungi Fusarium oxysporum isolate B/14, Fusarium oxysporum isolate MOS509, Fusarium fujikuroi isolate IMI58289 under in vitro conditions by double culture method.

The results of the research showed that in the control all phytopathogenic microorganisms intensively grew along the Petri dish and occupied almost the entire area of the Petri dish, while they formed a well-developed air mycelium with a bright pigment (Tabl. 1).

It was noted that all antagonists in double culture are able to inhibit the growth of phytopathogens, while the fungus T. atrobrunneum strain VKPM F-1434 on the 10th day of cultivation with phytopathogenic microorganisms showed the highest degree of inhibition (100 %); T. Lixii isolate T4/14 against F. fujikuroi isolate IMI 58289-100 %; T. harzianum isolate 5/14 against F. fujikuroi isolate IMI 58289 – 84 %. T. lixie isolate T4/14 and T. harzianum isolate 5/14 had a very low level of inhibition of F. oxysporum isolate B/14, F. oxysporum isolate MOS509 (<60 %).

3.2 Determination of the activity of enzymes of fungi of the genus Trichoderma associated with mycoparasitism

Inhibition of growth of pathogens by fungi is their generic feature and is due to the ability of mycoparasite to hydrolyze the cell walls of phytopathogenic fungi and use them as a substrate due to the produced enzymes and secreted various compounds [14]. The results of determining the enzymatic activity of the studied antagonist strains are presented in the table 2.

In our study, all Trichoderma species are capable of producing lytic enzymes. In T. atrobrunneum strain VKPM F-1434, the degree of manifestation of lipase and chitinase activity is strong, average proteinase. Strains of T. Lixii strain T4 / 14 and T. harzianum strain 5/14 have medium lipase and chitinase activity, weak – proteinase. Which confirms the data of mycoparasitic ability of strains within species [15].

3.3 Determination of the stimulating and fungicidal effect of presowing treatment of cucumber seeds with spore suspensions of the studied antagonists in vitro

Many researchers have shown that microorganisms with antagonistic activity can stimulate the growth and development of various plants, as well as change the soil microbiota, thereby improving the phytosanitary state of the soil [16].

В наших исследованиях особое значение придавалось использованию аборигенных штаммов антагонистов, так как их биологическая активность непосредственно связана with habitat and with the entire soil complex as a whole [17]. Based on the results of the antagonistic, mycoparasitic, and enzymatic activity of the studied antagonist microorganisms, in vitro experiments were performed on their ability to stimulate the germination of cucumber seeds. The results are presented in Figure 1.

The maximum germination energy of cucumber seeds was noted in the variant with T. atrobrunneum strain VKPM F-1434, T. Lixii isolate T4/14. The greatest number of seedlings was observed in the variant with T. atrobrunneum strain VKPM F-1434, T. harzianum strain 5/14, which exceeded the control by 5 %. Thus, the studied antagonists possessed growth-promoting ability (in vitro), which positively affected the germination of cucumber seeds.

Fig. 1 Indicators of germination energy (3rd day), germination of cucumber seeds (7th day) for various processing options, %

3.4 Biocontrol potential of the studied fungi of the genus Trichoderma against fungi of the genus Fusarium in vivo

Trichoderma species produce a huge amount of water-soluble metabolites, including pyrenes, terpenoids, steroids and polyketides, and others [5]. Able to inhibit the growth of plant pathogens in vitro and in vivo. The biocontrol potential of the studied antagonist microorganisms was evaluated in vivo against an artificially created infectious background against cucumber microplants. 50 ml of an aqueous suspension of Fusarium fungi, the titer of which is higher than that of the antagonists (10⁹ conidia/ml), was added to plastic cuvettes with 7-day-old seeds of cucumber treated with spore suspensions of the studied antagonist microorganisms. T. atrobrunneum strain VKPM F-1434 and T. harzianum isolate 5/14 stimulate root growth, while T. Lixii isolate T4/14 is characterized by stimulation of both roots and seedlings.

It was shown that by the end of the experiment, the number of phytopathogenic populations decreased by 75-80 % compared with the initial number of phytopathogenic fungi due to the hyperparasitism of the T. atrobrunneum micromycete strain VKPM F-1434, which no longer affected the susceptibility of cucumber seedlings, but at the same time decreased the number of introduced antagonist by 52 % due to the decrease in substrate (phytopathogens) and the processes of restoring the structure of the microbial pool in the soil. The number of populations of micromycetes of the genus Fusarium decreased by 58-82 % compared with the initial number of phytopathogenic fungi due to hyperparasitism of micromycete T. harzianum isolate 5/14, while the number of introduced antagonist decreased by 64 %. The population of micromycetes of the genus Fusarium decreased by 37-39 %, Fusarium fujikuroi isolate IMI 58289 by 79 % compared to the initial number of phytopathogenic fungi due to the hyperparasitism of micromycete T. Lixii isolate T4/14, which no longer affected the susceptibility of cucumber seedlings, even at the same time, the number of introduced antagonist decreased by 35 % (Fig. 2).

By the end of the experiment, the degree of development of a fusarium infection of 40-day-old cucumber plants was evaluated under the action of the studied antagonist microorganisms in the root zone. The degree of damage to cucumber plants by Fusarium infections when using T. atrobrunneum strain VKPM F-1434 was 0.5 %, which is 95 % lower than the control variant (variant without treatment). Compared with the control, T. harzianum decreases by 55 %, T. Lixii isolate T4 / 14 by 28 %.

Fig. 2 Changes in the number of phytopathogenic micromycetes of the genus Fusarium under the influence of antagonists of the genus T. atrobrunneum strain VKPM F-1434, T. harzianum isolate 5/14, T. Lixii isolate T4/14

4 Conclusion

The results of tests of the biological activity of antagonist strains obtained in laboratory conditions and in the open ground can vary significantly, since microbial antagonism in the soil proceeds taking into account many natural factors, often significantly different from the antagonism of the same microbes on artificial nutrient media [18].

Therefore, the search for antagonist microorganisms should include studies of the interaction of microorganisms in controlled conditions and in a natural environment. Given the widespread prevalence of fungal diseases of cucumber, especially protected soil, it is especially important to select indigenous strains of antagonists that are able to efficiently reduce the number of phytopathogens and at the same time stimulate plant growth and development of an environmentally friendly crop to protect the culture. The Trichoderma atrobrunneum VKPM F-1434 strain meets these requirements.

References

All Tables

All Figures

	Fig. 1 Indicators of germination energy (3rd day), germination of cucumber seeds (7th day) for various processing options, %
In the text

	Fig. 2 Changes in the number of phytopathogenic micromycetes of the genus Fusarium under the influence of antagonists of the genus T. atrobrunneum strain VKPM F-1434, T. harzianum isolate 5/14, T. Lixii isolate T4/14
In the text