Root rot of anthracnose mother plants of garden strawberry: morphological and cultural characteristics of the pathogen and the search for effective fungicides

On the territory of the Krasnodar region, anthracnose root rot, the causative agent Colletotrichum аcutatum Simmonds, is one of the most harmful diseases in the mother plants of garden strawberry. The death of affected plants ranges from 33 to 100 %. Purpose of the research was to study the morphological and cultural characteristics of the causative agent of anthracnose root rot in the mother plant of garden strawberries in the region and to assess the effectiveness of fungicides in controlling the pathogen. The studies were carried out at FGBNU SKFNTSSVV in 2018-2020 using generally accepted techniques. The object of the research is pure cultures of C. acutatum. The morphological and cultural characteristics of C. acutatum were studied on three culture media. A high diversity of the identified morphotypes was shown in terms of the size of conidia and conidiophores, the shape and color of apressoria, and the method of formation of acervules. The influence of seven fungicides of various chemical classes on the development of C. аcutatum spores has been assessed. Under in vitro conditions at the indicated concentrations, the preparations Luna Tranquility, SC (0,15 %), Sercadis Plus, SC (0,1 and 0,15 %), Strobi, WG (0,05 %), Horus, WG (0,04 %) and Skor, EC (0,04 and 0,05 %) completely prevented the formation of fungal spores.


Introduction
Colletotrichum аcutatum Simmonds is one of the most pathogenic species of Colletotrichum Corda, affecting a number of agricultural crops in tropical, subtropical and temperate regions, however, it has the greatest economic importance for the main host plant -garden strawberry (Fragaria ananassa Duch) [1][2][3][4]. As established by foreign and domestic scientists, the disease is due to the fact that after penetration into the plant, the pathogen stops its development until a certain period, and the symptoms of infection can appear for several years [2,5,6]. It was with these asymptomatic plants that the rapid spread of anthracnose took place. Due to its negativity, C. acutatum is included in the quarantine list of the countries of the European Community, its import and distribution with plant products is prohibited [7,8]. In the Krasnodar region, the disease poses a serious danger: the death of plants in uterine plantings is 33-50 %, in years with abundant precipitation -up to 70-100 % [6]. However, on the territory of the Russian Federation, there is no use of fungicides against the causative agent of anthracnose main rot. In this regard, it is relevant to study the morphological and cultural functions of the pathogen of the anthracnose root rot in the region, as well as the development of an effective pathogen; at the first stage, the primary screening of fungicides is carried out. It is carried out by the in vitro method using pure cultures. The objective of the research was to study the morphological and cultural characteristics of the causative agent of anthracnose main rot of strawberries in the uterine plantations of the Krasnodar Territory and to evaluate the effectiveness of fungicides in vitro in controlling the pathogen.

Materials and research methods
The studies were carried out in 2018-2020. at the Federal State Budgetary Scientific Institution "North Caucasian Federal Scientific Center for Horticulture, Viticulture, Winemaking" (Federal State Budgetary Scientific Institution «North Caucasian Federal Scientific Center of Horticulture, Viticulture, Wine-making») in the laboratory for the protection and toxicological monitoring of perennial agrocenoses. The object of the study was pure cultures of the fungus C. аcutatum -the causative agent of strawberry root rot. Isolation of isolates from the affected tissue of the strawberry horns was carried out according to the generally accepted methods with laying in a humid chamber and on potato sucrose agar (PSA). The description of the cultural characteristics of the isolates was carried out on the 10th day of the growth of colonies. The study of the morphological features of the pathogen was carried out for 35 days on potato agar (PA), potato sucrose agar (PSA), potato glucose agar (PGA) [2,[8][9][10][11][12][13]. The effect of chemical fungicides on the development of pathogen spores was carried out by applying a drop of spore suspension to the dry residue of the preparation with the studied concentration. The repetition is five times. The sprouted conidia were counted every other day.

Results and its discussion.
We studied 110 samples of the garden strawberry roots from the mother plants of the Krasnodar region and isolated 53 isolates. It was found that the obtained isolates differ in cultural characteristics, which made it possible to divide them into three morphotypes (Table  1). In isolates of morphotype III, the colony diameter is 13,9-16,9 % larger than in isolates of morphotypes I and II. In all isolates of II and III morphotypes, the colonies had an elevated profile with an aerial mycelium height of 2,0 mm; in isolates of morphotype I, the height was 3,0 mm. The most variable features were the height of the center of the colony, as well as the width of the edge of the colony. The growth rate in isolates of morphotype III was the highest -12,1-12,7 mm / day, in isolates of morphotypes I and II -average and amounted to 10,4-10,7 mm / day. The color of the reverseum of the isolates varied from salmon to dark brown with a violet tint.
The main traits for identifying C. acutatum are the size of the conidia, the presence of setae, and the amount of acervula production. In the Krasnodar population of the pathogen, differences between the isolates in terms of morphological characteristics were recorded. Thus, morphotypes differ in the size of conidia: in isolates assigned to morphotype I, the size of conidia is mean ± SD (range of values within) = 15,0 ± 2,1 x 5,0 ± 0,3 μm, in isolates of morphotype II -mean ± SD = 12,5 ± 2,3 x 4,2 ± 0,5, in isolates of morphotype III -mean ± SD = 13,9 ± 3,2 x 4,8 ± 0,8 μm. Isolates of morphotypes I and II were distinguished by short conidiophores, 4-20 x 2,5-5 and 2,5-7,5 µm in length, respectively. In isolates of morphotype III, conidiophores were of two types: short -3-20,5 μm and long -28,3-63,5 μm. Also, differences between the morphotypes were revealed in the way of the formation of acervules in culture. In isolates of I and III morphotypes on all nutrient media, substrate acervules were formed on the 3-4th day. In isolates of morphotype II on PGA and PSA, deep and surface acervules were formed on days 1-3, while on PA, substrate acervules were formed on the 4th day. The onset of conidiogenesis in the isolated isolates on all substrates was recorded on the 3rd day. The formation of apressoria in isolates of morphotype I on PA and PSA was noted on the 23rd day, on the PGA -on the 31st day. In isolates of morphotype II, apressoria were formed on PA on the 17th day, on the PA and PGA on the 20th day. In isolates of morphotype III, the formation of appressoria was established on the PA on the 31st day, and on the PSA and PGA on the 21st day. The color of the apressoria in the isolates varied from light brown to brown, and their shape varied from ellipsoidal to round with smooth edges.
Based on the results obtained, it can be noted that C. acutatum in the mother plant of garden strawberries from Krasnodar region is characterized by high diversity: the selected morphotypes differ in the size of conidia and conidiophores, in the shape and color of apressoria, in the way of formation of acervules in culture, but do not differ in the type of conidia and mycelium.

Conclusions
Thus, for the causative agent of anthracnose root rot C. acutatum, 53 isolates were obtained in the mother plant of garden strawberries in the Krasnodar region and three morphotypes were isolated, which differ in the size of conidia and conidiophores, the shape and color of the apressoria, the way of formation of acervules in culture, which indicates a high diversity of the fungus in region.
As a result of the primary screening of fungicides for pathogen control, their optimal concentrations were determined depending on the stage of fungal development. Luna Tranquility, SC, registered for use on strawberries, should be used in concentrations of 0.08-0.1 % before infection penetrates the plant, and after infection in a concentration of 0.015 %. In the case of registration for the use of the remaining tested fungicides in the mother plant of garden strawberries, it is advisable to apply them according to the following regulations: