EAT. Chudinov, V.A. Platonov, A.V. Alexandrova, S.N. Elansky
It has recently been shown that the ascomycete fungus Ilyonectria crassa is capable of infecting potato tubers. This work is the first to analyze the biological characteristics and resistance to some fungicides of the I. crassa strain isolated from potatoes. The sequences of species-specific regions of the “potato” strain coincided with those obtained earlier for fungi isolated from the roots of daffodil, ginseng, aspen and beech, lily bulbs and tulip leaves. Apparently, many wild and garden plants can be reserves of I. crassa. The investigated strain infected tomato and potato slices, but did not infect the whole tomato fruit and intact potato tuber. This shows that I. crassa is a wound parasite. Evaluation of resistance to fludioxonil, difenoconazole and azoxystrobin on a nutrient medium showed high efficacy of these drugs.
The EC50 index (the concentration of the fungicide, which slows down by 2 times the rate of radial growth of the colony relative to the fungicidal control) was equal to 0.4; 7.4 and 4 mg / l, respectively. The possibility of the development of the disease caused by I. crassa should be taken into account when phytopathological assessment of potato tubers and the development of plant protection measures.
The development of phytopathogenic microorganisms leads to high losses at all stages of growing and storing potatoes. When planning protective measures, as a rule, well-known pathogens are taken into account, such as species of the genera Alternaria, Fusarium, Phoma, Helminthosporium, Colletotrichum, Phytophthora, etc. However, in recent years, more and more reports have appeared about the appearance of new phytopathogenic microorganisms on potatoes. Their biology is poorly studied, the effectiveness of fungicides used on potatoes in relation to them is unknown, diagnostic methods have not been developed. With mass development, they are capable of causing significant damage to the potato crop. One of these microorganisms is the ascomycete fungus Ilyonectria crassa (Wollenw.) A. Cabral & Crous, first discovered by the authors on potato tubers (Chudinova et al., 2019).
This work presents the results of the analysis of the I. crassa strain isolated from potato tubers. The morphology of colonies and mycelial structures of I. crassa, nucleotide sequences of species-specific DNA regions, virulence to potatoes and tomatoes, and resistance to some popular fungicides were studied.
Materials and methods
We used the I. crassa 18KSuPT2 strain isolated in 2018 from the infected potato tuber grown in the Kostroma region. The tuber was affected by a dry rot type with a cavity covered with light brown mycelium. Using a sterile dissecting needle, the fungal mycelium was transferred into a Petri dish with an agar medium (beer wort 10%, agar 1.5%, penicillin 1000 U / ml). The plates were incubated in the dark at 24 ° C.
A Leica DM2500 light microscope with an ICC50 HD digital camera and a Leica M80 binocular microscope with an IC80HD digital camera (Leica Microsystems, Germany) were used to photograph, evaluate the size and morphology of spores and spore organs.
To isolate DNA, the fungal mycelium was grown in liquid pea medium, then frozen in liquid nitrogen, homogenized, incubated in CTAB buffer, purified with chloroform, and washed twice with 2% alcohol.
The DNA extraction method is described in detail in the article by Kutuzova et al. (2017).
To determine the species by molecular methods and compare with other known I. crassa strains, PCR was carried out with primers allowing amplification of species-specific DNA regions: ITS1-5,8S-ITS2 (primers ITS5 / ITS4, White et al., 1990), gene regions b -tubulin (Bt2a / Bt2b, Glass, Donaldson, 1995) and translation elongation factor 1α (tef1α) (primers EF1-728F / EF1-986R, Carbone and Kohn, 1999). Amplicons of the desired length were extracted from the gel using the Evrogen CleanUp kit. The amplified regions were sequenced using the BigDye® Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, CA, USA) on an Applied Biosystems 3730 xl automated sequencer (Applied Biosystems, CA, USA). The resulting nucleotide sequences were used to search for a match in the US National Center for Biotechnology Information (NCBI) GenBank database. Phylogenetic analysis was performed using the MEGA 6 program (Tamura et al., 2013).
Determination of virulence was carried out on whole green fruits of large-fruited tomato (variety Dubrava) and potato tubers (variety Gala). In addition, to simulate damage to damaged fruits and tubers, we used slices of the same fruits and tubers. Slices of tubers were placed in humid chambers, which were Petri dishes with wet filter paper at the bottom. A slide was placed on the paper, on which, in turn, slices of tubers or fruits were placed. Whole tubers and fruits were also placed in containers with wet filter paper at the bottom. In the center of the slice (or on the intact surface of the tuber or fruit), a piece of agar (5 × 5 mm) with fungal hyphae was placed after 5 days of growing on wort agar.
The assessment of the resistance of fungal strains to fungicides was carried out in laboratory conditions on agar nutrient medium. We studied the susceptibility to fungicidal drugs Maxim, KS (active ingredient fludioxonil, 25 g / l), Quadris, KS (azoxystrobin 250 g / l), Scor, EC (difenoconazole 250 g / l) (State catalog ..., 2020). Evaluation was carried out in Petri dishes on wort-agar medium with the addition of the studied drugs at concentrations of the active substance 0.1; one; 1 ppm (mg / L) (for fludioxonil and difenoconazole), 10; ten; 1 ppm (for azoxystrobin) and in media without fungicide (control). The fungicide was added to the melted and cooled to 10 ° C medium, after which the medium was poured into Petri dishes. An agar block with fungal mycelium was placed in the center of a Petri dish and cultured at a temperature of 100 ° C in the dark. After 60 days of incubation, the diameters of the colonies were measured in two mutually perpendicular directions; the measurement results for each colony were averaged. The experiments were performed in triplicate. Based on the results of the analyzes, the EC24 was calculated, equal to the concentration of the fungicide, which halved the rate of radial growth of the colony relative to the fungicidal control.
Results and discussion
On petri dishes with wort agar, the fungus formed colonies with white flocculent mycelium. The medium under the mycelium turned reddish-brown. When the medium dries up, the fungus formed spores of two types on single and aggregated conidiophores in small sporodochia. Macroconidia are elongated, cylindrical, with one to three septa, average length 27.2 µm with a range of values from 23.2 to 32.2 µm, width - up to 4.9 µm (Fig. 1). The average length of microconidia is 14.3 µm with a range of values from 10.3 to 18.1 µm, the width is up to 4.0 µm. All macro- and micromorphological characters fit into the range of variation of the species Ilyonectria crassa (Cabral et al., 2012).
The sequences of species-specific DNA regions (ITS, b-tubulin, TEF 1α) completely coincided with the sequences of the I. crassa strains we studied earlier (Chudinova et al., 2019, Table 1). To study the prevalence of I. crassa in other regions and to analyze the spectrum of affected cultures, analogous DNA sequences in the GenBank database were analyzed (Table 1). The overlap was 86 to 100%. The sequences of all three DNA regions of the “potato” I. crassa strain were identical to the sequences of the strains isolated from the lily bulb and narcissus roots in the Netherlands and from the ginseng root in Canada. We failed to find other I. crassa strains with three analyzed similar sequences in open databases. However, analysis of the deposited ITS and b-tubulin sequences showed the presence of I. crassa on tulip leaves in the UK. Fungi with a similar ITS sequence were identified in the analysis of the mycobiota of aspen roots in Canada and beech roots in Italy, potato tubers in Saudi Arabia (Table 1). The results of this study show that I. crassa has a global distribution and is capable of infecting various plant species.
When determining the pathogenicity on slices of tomato and potato on the 5th day, the diameter of the lesion reached 1.5 cm. The investigated strain did not infect the whole tomato fruit and intact potato tuber. However, the sepals were affected on the tomato. To exclude the possibility of contamination, a fungal isolate from the mycelium developed on a potato tuber slice was isolated into a pure culture. It was completely identical to the parental strain. Apparently, I. crassa is a wound parasite.
Pre-planting treatment of seed tubers with fungicides reduces the development of diseases on plants during the growing season. For the selection of effective fungicides, it is important to assess which of them are effective against I. сrassa. The work studied the widespread active substances of fungicides - fludioxonil, azoxystrobin, difenoconazole. Fludioxonil is included in several mixtures used for dressing seeds and seed tubers before planting. Fludioxonil (Maxim) is also used to treat seed tubers before storage. Difenoconazole and azoxystrobin are also included in a number of preparations used for processing seed material, as well as in preparations intended for processing vegetative plants (State catalog ..., 2020).
The growth rate of I. crassa was studied on media (Fig. 2) with different concentrations of active ingredients: fludioxonil (EC50 = 0.4 ppm), azoxystrobin (EC50 = 4 ppm), and difenoconazole (EC50 = 7.4 ppm) (Table 2). These preparations can be considered highly effective against I. crassa, since their EC50 is significantly lower than the recommended concentration of the preparation in the working fluid used for treating tubers. According to the State Catalog ... (2020), the concentration of fludioxonil in the liquid for treating potato tubers is from 500 to 1000 ppm, azoxystrobin (in the liquid for treating the bottom of the furrow) - 3750-9375 ppm, difenoconazole (in the liquid for treating vegetative plants) - 187.5– 625 ppm.
Table 1. Sequence similarity of species-specific sequences of strain 18KSuPT2 and strains of Ilyonectria crassa available in the Genbank database
Strain | Host plant, site of excretion | Sequence numbers deposited with GenBank, percentage of similarities | Link | ||
ITS | β-tubulin | TEF 1α | |||
17KSPT1 and 18KSuPT2 | Potato tuber, Kostroma region | MH818326 | MH822872 | MK281307 | Chudinova et al., 2019, this work |
CBS 158/31 | Narcissus roots, Netherlands | JF735276 100 | JF735394 100 | JF735724 99.3 | Cabral et al., 2012 |
CBS 139/30 | Lily bulb, Netherlands | JF735275 100 | JF735393 99.7 | JF735723 99.3 |
|
NSAC-SH-1 | Ginseng root, Canada | AY295311 99.4 | JF735395 100 | JF735 / 725 99.6 |
|
RHS235138 | Tulip leaf, UK | KJ475469 100 | KJ513266 100 | ND | Denton, Denton, 2014 |
MT294410 | Aspen roots, Canada | MT294410 100 | ND | ND | Ramsfield et al., 2020 |
ER1937 | Beech, Italy | KR019363 99.65 | ND | ND | Tizzani, Haegi, Motta. Direct submission |
KAUF19 | Potato tuber, Saudi Arabia | HE649390 98.3 | ND | ND | Gashgari, Gherbawy, 2013 |
ND = not deposited
Table 2. Resistance of Ilyonectria crassa to fungicides
(active substance) | EC50, ppm | ||||
3 day | 5 day | 7 day | |||
Control | 17±2 | 33±5 | 47±3 | ||
Quadris, KS (fsoxystrobin) | 18±1 | 34±2 | 48±2 | ||
11±1 | 11±1 | 12±1 | |||
11±1 | 11±1 | 12±1 | |||
Maxim, KS (fludioxonil) | 16±1 | 28±2 | 48±2 | ||
7±1 | 13±3 | 19±4 | |||
5±1 | 12±1 | 17±5 | |||
Skor, EC (difenoconazole) | 18±1 | 35±2 | 48±1 | ||
11±1 | 24±3 | 35±4 | |||
11±1 | 13±1 | 17±3 |
In our work, I. crassa strains were isolated from potato tubers in the Kostroma and Moscow (Chudinova et al., 2019) regions. A high proportion of fungal strains with ITS sequences identical to I. crassa was revealed when analyzing the mycobiota of potato tubers in Saudi Arabia (Gashgari and Gherbawy, 2013). Apparently, I. crassa is not as rare on potatoes as it might seem. Our experiments showed that the fungus could infect damaged tomato fruits. It is known from the literature that I. crassa is capable of developing in the soil saprotrophically (Moll et al., 2016), as well as affecting a variety of plants, even taxonomically distant ones such as daffodils, lilies, ginseng, aspen, and beech (Table 1). one). Apparently, many wild and garden plants can be reserves of I. crassa. The above shows that when developing protection measures, it is necessary to take into account the possibility of affecting potato tubers with this fungus. Widespread preparations for the treatment of potato tubers containing fludioxonil, azoxystrobin and difenoconazole have shown high fungicidal efficacy against I. crassa.
This work was supported by the Russian Foundation for Basic Research (Grant No. 20-016-00139).
The article was published in the journal "Plant Protection Bulletin", 2020, 103 (3)