Screening for resistance to potato wart and late blight
Six potato breeds from Lithuania were tested for resistance to the causal agents of potato wart and late blight: ‘VB Venta’, ‘Goda’, ‘VB Liepa’, ‘VB Meda’, ‘VB Rasa’ and ‘VB Aista’ (Table 1). There are six Lithuanian potato cultivars on the official list of the European Union. The ‘Polish pink’ variety was used as a positive control, and the ‘Glazurna’ variety was used as a negative control for wart sensitivity11. Potato seed material obtained from the Lithuanian Research Centre for Agriculture and Forestry and the Ukrainian Science-Research Plant Quarantine Station Institute of Plant Protection National Academy of Agrarian Science was our breeding stock. These breeds were developed and grown in a state scientific institution.
The variety Nezabudka was used as a positive control for late blight susceptibility, and Glazurna was used as a negative control. In 2020 and 2021, the experiments were conducted in a pest and disease quarantine laboratory at the Ukrainian Science Research Plant Quarantine Station Institute of Plant Protection National Academy of Agrarian Sciences of Ukraine (v. Boiany Chernivtsi district, Chernivtsi region) and in field trials (settlement Berehomet, Vyzhnytsia district, Chernivtsi region).
The experiments were performed in accordance with the methodological recommendations of the station staff12 in accordance with the requirements in the protocol for Synchytrium endobioticum10,27. Under laboratory conditions, potato samples (5 tubers each) were infected in triplicate, with winter zoospores of a common pathotype of the wart pathogen isolated in triplicate from the village of Beregomet, Vyzhnytsia district, Chernivtsi region12. To test wart resistance in the field, 10 potato tubers were used in triplicate. Field research was performed directly at the wart site, with notification and permission from the quarantine service.
Potato samples were planted in special containers with contaminated soil-perlite substrate, which contained 40–50 winter zoosporangia in 1 g of substrate for infecting potato samples with the disease and determining resistance. The seeds were placed in a Climo chamber at a temperature of 18 0 °C, a relative humidity (RH) of 70–80%, and a 12/12 day/night regime.
Once a week, they were watered and loosened; after 60 days, a preliminary examination was performed after infection, and after 75 days, a preliminary examination of the potato lesions was performed. Preliminary accounting includes the inspection of the surface of infected potato stems, and the main accounting includes removing infected plants from their container, examining their tubers and stolons, and determining the infection score.
The tubers of the potato samples were also infected with summer zoospores of the wart pathogen. To this end, paper rings were attached to the top of the potato tuber around the sprout top of the potato tuber part using a mixture of paraffin and Vaseline (1:1). Distilled water was poured into the rings of the potato samples for infection, and clean wart growths with summer zoospores from fresh wart growths of the causative agent with a size (volume) of 0.5 cm3 (which contained up to 50 summer zoosporangia of the pathogen) were added to the water. The samples were incubated with the causative agent of potato wart was from 24 to 48 h with a thermostat at a temperature of 11–13 °C. The potato samples were subsequently placed in a climatic chamber for infection by the causative agent Synchytrium endobioticum. The inoculated samples were placed in a climate chamber with increased humidity, a temperature of 18 °C, and a relative humidity (RH) of 70–80% (with no lighting) for twenty-one days and up to 28 days before the appearance of disease symptoms. The reaction to the infection of the samples was detected on the 28th day after infection with the pathogen, and the disease symptoms of the potato samples were evaluated on a five-point scale, in accordance with the requirements of the EPPO standard.
The degree of resistance was defined on a 5-point scale: 1 – high resistance, early necrosis, soruces absence (R1); 2 – resistant, late necrosis, and simple soruces (R1); 3 – weakly resistant, very late necrosis, and five soruces (R2); 4 – weakly susceptible, dense soruces formation with potato sprouts (S1); and 5 – strongly susceptible, dense soruces, and a wart node (S2)1,28. The experiments were performed in triplicate. The reliability of the differences between the samples was assessed using dispersion analysis, with further evaluation of the least significant difference (LSD) using the Statistica 5 software package.
When research on the selection of wart-resistant potato cultivars under field conditions was conducted in 2020, during the period of infection with the wart pathogen (May-June), the average daily temperature ranged from 12.9 to 19.40 °C, and the amount of precipitation in May was 131 mm (176% of the normal level), and 84 mm in June (112% of the normal level). In 2021, the average daily temperature was 14.20 °C in May and 19.40 °C in June. The amount of precipitation in May was 84 mm (112% of normal), whereas that in June was 83 mm (89% of the norm) (Table 2). The influences of temperature and precipitation were favourable for the manifestation of the disease.
Late blight was tested using the widely accepted laboratory and field trials of artificial infection by separate tubers and leaves from Phytophthora infestans(Mont) de Bary fungal inoculum (race 1.2.3.4.5.6 + 0.7.8.9.10.1 xyz), as created by the Institute for Potato Study, National Academy of Agrarian Sciences (NAAS), which was received in 20204. The targets are selected for infection, uninjured, or without signs of damage caused by other tuber diseases. After being washed in water, the plants are treated with alcohol, and burnt tubers are dipped for 5 min in a suspension of conidia and zoospores of the fungus. The inoculum load is 25–30 conidia in the field of view of the microscope at a magnification of 120 times. The period of use for the suspension is 3 h. Infected tubers (5 pieces of each sample) are placed in polyethylene pots, and the infected pots are placed in a special chamber and covered with filter paper and then glass. The chamber is maintained under optimal conditions for infection and development disease temperature, with high (over 90%) relative air humidity, which is reached by moistening the filter paper in the pots and the one with which they are covered. Excess water drains through the holes in the pots, which prevents not only waterlogging but also the appearance of wet rots. Thirty days after infection, the resistance of each sample tuber to the causative agent of late blight is determined by inspecting the surface and area of the tuber after it is cut in half. The following scale is used:
1–2, very susceptible (more than 75% affected);
3–4, susceptible (50.1–75% affected);
5–6, moderately resistant (affected from 26 to 50%);
7–8, resistant (affected tissue occupies 10–25.9% of the tuber surface or section); and.
9 – highly resistant (affected tissue occupies less than 10% of the surface or section tubers).
Field studies were conducted on a natural infectious background, where the most common race of late blight in Ukraine is 1.2.3.4.5.6 + 0.7.8.9.10.11 xyz. Inoculation of potato plants was performed during the budding-flowering phase. The degree of infection was scored on a 9-point scale: 9 − 8, very high resistance (symptoms of infection absent); 7 − 6, relatively high resistance (infected tissue ranging from 10 to 25% surface and cut from tubers); 5 − 4, moderate resistance (infected from 25 to 50%); 3 − 2, low resistance (infected from 50 to 75%); and 1, very low resistance (infected more than 75%)4.
During the growing season, favourable circumstances occurred for late blight development, such as air temperatures between 10.1 and 21.2 °C in 2020 and between 10.4 and 22.6 °C in 2021 and precipitation totals of 402 mm in 2020 and 373 mm in 2021 (Table 2).
Microsatellite analysis
The total DNA from the young leaves was extracted using the DNeasy Plant Mini Kit (QIAGEN N.V., Hilden, Germany) according to the manufacturer’s instructions. When the genetic diversity of cultivars is evaluated via SSR markers, microsatellite DNA sequences are ideal molecular markers for discriminating among genotypes. The microsatellite 6-carboxyfluorescein (FAM)-labelled primers used in the studies are listed in Table 329,30. The PCR was performed in a Veriti-96 Gradient Thermocycler (Applied Biosystems, USA) in a 20 µL volume by applying 10 µL of KAPATaq Ready Mix (KAPAbiosystems, USA) according to the manufacturer’s instructions. SSR alleles were detected and scored using GeneScan analysis software (Applied Biosystems).
Data analysis
The microsatellite loci were estimated by polymorphic information content (PIC):
$$\:\text{P}\text{I}\text{C}=1-?\left({\text{p}}_{\text{i}}^{2}\right)$$
(1)
where pi is the frequency of the ithallele detected in all individuals of the population31.
Genetic similarity and cluster analyses of genetic distances between individuals were calculated using a factor based on the presence or absence of the relevant alleles. The similarity matrix was calculated using Sokal and Sneath 2:
$$\:{\text{D}}_{\text{i}\text{j}}=\frac{2\left(\text{b}+\text{c}\right)}{\text{a}+2\left(\text{b}+\text{c}\right)}$$
(2)
where i and j are genotype reference indices; a is the number of cases at all loci when allele (1) is present in both genotypes; and b and c are the number of cases in which the genotype has one allele (1) and (0)32. For the statistical evaluation of SSR data, cluster and factor analyses were performed using DARwin 6.0.021 software [updated 26/04/2019]. A dendrogram was generated using the unweighted pair group arithmetic mean (UPGMA) algorithm. Genotype distances were calculated using the UPGMA method with Sokal and Sneath 2 dissimilarity indices.
