Antimycotic activity of bacterial strains against the pathogen of grape necrotic leaf spotting Alternaria sp

Species of the genera Bacillus Cohn and Pseudomonas Migula are successfully used as biocontrol agents for many mycopathogens, including Alternaria sp. To assess the bacteria antagonistic potential against Alternaria sp, we used the method of counter cultures on various nutrient media – universal and specialized. Of the 24 bacterial strains of the genus Bacillus, 18 strains were developed antagonistic activity to Alternaria sp. after 10 days of co-cultivation on the Tylona-3 medium. The maximum sterile zone (8.0 mm) was formed by bacterial strains (R-9, 5B-1, 01 cor f Bacillus sp.). 3 strains of 9 bacteria of the genus Pseudomonas, developed antagonistic activity to Alternaria sp. on Kinga B medium 14-3 Pseudomonas sp., Oif 2-1 Pseudomonas sp., 14-4 Pseudomonas sp. The maximum antibiotic activity on potato-sucrose agar was established in strains 3-3 Bacillus sp., and K 1-1 Bacillus sp. (sterile zone 7.5 mm). All the tested bacteria most often caused one sign of antagonism – antibiosis.


Introduction
Alternaria Nees species are mainly saprotrophic fungi. Nevertheless, some species have acquired the ability to cause diseases of a wide range of host plants [1]. Alternaria fungi are pathogens of aboveground plant organs, their main harmfulness lies in the suppression of the photosynthetic apparatus of plants, the pathogenesis is quite slow, but with a wide distribution. The infection leads to the formation of necrotic lesions on the leaves in the form of spots, which are a reaction of the hypersensitivity of the plant tissues in response to pathogen invasion. The process of plant infection with Alternaria fungi includes the germination of conidia, penetration and colonization of the plant surface. Conidia usually produce several germ tubes. According to the results of studies by a number of authors, the penetration of the pathogen into plant tissues occurs either directly through the epidermis or microcracks in it, or through the stomata or lenticels [2][3][4]. The site of pathogen penetration is surrounded by necrotic and often chlorotic halos. Such tissue damage is caused by the diffusion of secondary fungal metabolites -toxins.
Alternaria spp. It belongs to the kingdom Fungi, subclass Eumycotera, phylum Fungi Imperfecti, class Hyphomycetes, order Moniliales, family Dematiaceae, genus Alternaria [1,[5][6]. All known teleomorphs of Alternaria belong to the genus Lewia Barr & Simmons [7]. It is believed that the overwintering stock of the fungus is a seed infection, or infected plant remains, where the infection accumulates and stored for a long time in the form of mycelium and conidiospores. A seed-borne infection is dangerous because almost immediately the disease attacks the sprouts or seedlings of agricultural crops, leading to great damage.
Data on grape Alternaria blight are extremely limited. The authors of the published works provide signs of grape infection with Alternaria blight with reference to the only source F. Cavara (1888) [8]. The described signs of the disease are always grayish spots with a brownish coating, localized along the leaf veins. The plaque is a mycelium and bundles of conidiophores with conidia. D.P. Lawrence and other (2013) with reference to P. Joly (1964) states that the species Alternaria vitis is not a specialized pathogen of grape, but is actually a cosmopolitan species of Alternaria tenuissima [9].
When studying the Alternaria blight of grapes in the Krasnodar Krai, the species A. tenuissima was noted as the causative agent, while the signs of the disease were radically different from those listed above. The disease is described as leaf spotting, which begins with the appearance of dark dots first on the underside, which increase and become visible on the upper side in the form of small rounded spots, and as they grow, they cover almost the entire leaf surface [10].
There are more and more reports about the spread and harmful manifestation of Alternaria blight in various crops [11][12][13][14][15][16], in this connection, the study of the features of pathogenesis and the development of measures to combat these diseases is becoming more relevant. The world is actively searching for effective producers of biological products -strains of antagonistic bacteria. The global biotechnological trend is the species and strains from the genera Bacillus and Pseudomonas. Thus, according to the results of Aldib et al. P. brassicacearum and P. jessenii strains effectively suppressed the potato pathogen Alternaria solani [17]. In tests of antibiotic activity against A. alternata, six bacteria were tested (Bacillus subtilis F9-2, F9-8, and F9-12, Pseudomonas arsenicoxydans F9-7, P. koreensis F9-9, and P. moraviensis F9-11), the growth of fungal mycelium was inhibited by an average of 27% [18]. Chinese researchers (Dai et.al., 2021) isolated a strain Bacillus velezensis C16 with high antifungal activity. Antagonistic mechanisms (LPs) and volatile organic compounds (VOCs) produced by the C16 strain against A. solani were studied. The results showed that VOCs and unstable lipopeptides showed significant antagonistic activity against A. solani, with a decrease in colony diameter and significant inhibition of conidia germination [19]. In another study, an endophytic strain of B. velezensis SEB1 isolated from Vigna mungo (L.) Hepper was tested for the effectiveness of biocontrol of the pathogenic potato isolate A. alternata. SEB1 was accepted to be effective for inhibiting pathogen growth in double culture in vitro. SEB1 secretes thermally stable antimycotic metabolites [20].

Materials and methods
The causative agent of necrotic leaf spotting Alternaria sp. was isolated from the affected grape leaves in the vineyard of the Riton variety in the Krasnodar Krai. Bacterial strains for their antimycotic activity determination were taken from the collection of Dr. L.V. Maslienko (FSBSI FSC "VNIIMK", city of Krasnodar, Russia).
From seven-day cultures on potato sucrose agar (PSA) Alternaria sp. and each of thirtythree species of bacteria (Bacillus sp. Cohn, Pseudomonas sp. Migula), medium-sized discs (5 mm 3 ) were cut out and used for inoculation on Petri dishes 9 cm in diameter containing three agar culture media -PSA, Tylona-3 and Kinga B. Disc with Alternaria sp. culture and discs with cultures of bacteria were placed at a distance of 2 cm from opposite edges of the Petri dish (4.5 cm from each other), along the same diameter. Five control dishes with a single microorganism were also prepared. All the dishes were incubated in the dark at room temperature for ten days.
The degree of bacteria antagonistic activity was determined on the 10th day of cultivation by the area occupied by the antagonist and pathogen, as well as by the presence and size of the antibiotic zone; cultures of antagonists and pathogens sown on different Petri dishes (PD) were taken as controls [21].

Results and discussions
Analysis of screening results of 24 strains of Bacillus sp. Cohn on Tylona-3 medium, specialized for them, showed that 18 of them had antimycotic activity against the causative agent of necrotic leaf spotting for the type of antibiosis (Table 1). Strains R-9 Bacillus sp., 5B-1 Bacillus sp. and 01 cor f Bacillus sp. had the greatest antimycotic activity against Alternaria sp. on the Tylona-3 medium, a sterile zone was 8.0 mm when co-cultured (Fig. 1). On the PSA strains of Bacillus sp. together with the causative agent of necrotic leaf spotting showed slightly less antimycotic activity. The maximum sterile zone in the double culture was shown by strains 3-3 and K 1-1, which was 7.5 mm (Table 2). One of the work stages was the screening of bacterial strains of antagonists from the genus Pseudomonas.
The tests were carried out on 2 different media -PSA and Kinga B. The Kinga B medium was selected as a specialized medium for Pseudomonas bacteria (Table 3). Of the nine collection bacteria of the genus Pseudomonas, only three bacterial strains -14-3, 14-4, Oif 2-1 -showed antibiotic activity against the pathogen agent of necrotic leaf spotting ( Figure 2). It was found that on potato-sucrose agar, as well as on Kinga B medium, the maximum sterile zone in the double culture was formed by the strain 14-3 Pseudomonas sp. -9.0 mm. The suppression zone of Alternaria sp. growth on the PSA by strains Oif 2-1 and 14-4 was 6.5 and 5.0 mm, respectively (
It was found that the size of the antibiotic zone was larger on the media specialized for each bacteria genus than on the universal culture medium.