Modified Method for the Evaluation of Resistance to Fusarium Blight in White Mustard ( Sinapis

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Introduction
White mustard (Sinapis alba L.), black mustard (Brassica nigra (L.) W.D.J.Koch) and false flax (Camelina sativa (L.) Crantz) are valuable crops worldwide [1].White mustard is mainly grown as a green manure crop, improving soil quality and reducing phytopathogenic micromycetes [2].Black mustard seeds are widely used as a food seasoning, and the seed extract of the crop has anti-proliferative (inhibits cell growth) activity against human lung cancer cells [3].False flax oil is a dietary food product that effectively reduces the level of low-density lipids in human blood [4].
Plants of white mustard, black mustard and spring flax are susceptible to various diseases during cultivation.One of the harmful diseases of these crops is Fusarium blight, caused by fungi of the genus Fusarium Link.Infection at early developmental stages (2-4 true leaves and stem extension stages) results in rapid plant death [5][6][7].Penetration of the pathogen's infective matter inside the stem at later stages of development (flowering and green pod stages) leads to occlusion of the vascular bundles, causing premature drying of the plants, reduction in their productivity (by 50-60 %) and seed germination [8][9][10].Visual examination of plants reveals a change in the color of the whole or part of the stem: from green to yellow or reddish brown.The intensity of the coloration depends on the response of the variety, disease development and environmental conditions [11].
One of the ways to protect plants from diseases is to develop resistant varieties of various species of mustard and false flax.A necessary condition for this is the evaluation of disease affection of breeding material of crops.In the Department of Rapeseed and Mustard Breeding of V.S. Pustovoit All-Russian Research Institute of Oil Crops (VNIIMK), the field evaluation of breeding material of oil crops of the cabbage family for resistance to Fusarium blight is carried out annually, during which resistant samples are identified, which will be used in the future for the development of new varieties [12].
Evaluation of resistance of crop varieties to diseases in laboratory conditions helps to intensify and improve the breeding process in the direction of development of resistant genotypes, as it allows to determine the level of their resistance in a short time under controlled conditions.Earlier, at VNIIMK, V.F.Zaichuk et al. (1990) proposed a method for evaluation of Fusarium blight infestation of rapeseed and brown mustard seedlings in laboratory conditions.The Fusarium blight pathogen was isolated from mustard or rapeseed plant residues on potato dextrose agar (PDA) in Petri dishes at air temperature 25-27 °C for 5-6 days.The nutrient substrate was then prepared: rapeseed seeds were boiled in water for 5 minutes, the water was drained and the seeds were placed in Petri dishes (25-30 g of seeds in each Petri dish), then sterilized in an autoclave for 20 minutes at 1.2 atm pressure.Mycelium of the fungus was sown on cooled rapeseed seeds and cultivated for 10 days.Then rapeseed and mustard seedlings at the age of 6-7 days were placed in Petri dishes for fungal colonies, 10-15 pieces of each variety sample.Two samples were placed in each Petri dish.Affection was evaluated after 1, 2 and 3 days, taking into account only the number of affected seedlings.Seedlings were considered susceptible if their tissues were loosened and resistant if the turgor and green color of the seedling tissues were maintained.However, in these studies, the number of affected plants was 71.8% for rapeseed and 88.7% for mustard after just one day.This method is therefore extensive and time-consuming and does not allow rapid evaluation of oil crops in the cabbage family for resistance to Fusarium blight.
In this context, the aim of our study was to modify the laboratory method for the rapid and reliable evaluation of the resistance to Fusarium blight of breeding material of white mustard, black mustard and spring false flax.

Materials and methods
The research was carried out in 2022 under laboratory conditions at VNIIMK on samples of white mustard, black mustard and spring false flax.Three variety samples of each crop, resistant and susceptible to Fusarium blight, were studied in the field under a natural infection background.
Phytopathological analysis showed that in the vast majority of cases the fungus Fusarium oxysporum Schlecht.emend.Synd was isolated from the affected plants of the studied crops, so the research was carried out using this species.
The pathogen was grown on Czapek's solid medium in Petri dishes for 6 days.After sterilization, seeds of white mustard, black mustard and spring false flax were placed in the moist medium of sterile Petri dishes for germination.The 5-day-old apparently healthy seedlings were spread with their roots on the surface of the pathogen mycelium in order to infect them with the exposure duration: 1, 3, 5 hours, as well as 1 and 2 days.
At the end of the exposure period defined in the experiment, the seedlings were transferred to the moist medium of sterile Petri dishes in order to observe their response to infection by the fungus.Observations were made for three days, including the first day of the experiment.A total of 50 seedlings of each breeding sample were studied in each experimental variant (5-6 pieces per 1 Petri dish with pathogen mycelium).
The prevalence and development of the disease on seedlings in each sample were calculated using international formulas.
The prevalence of Fusarium blight of plants was calculated according to formula (1): (1 Where P -is the disease prevalence in the sample, %; n -is the number of affected seedlings in the sample, pcs; N -is the total number of counted seedlings in the sample, pcs.
To calculate the development of the disease according to the formula, we developed a scale of Fusarium blight affection of white mustard, black mustard and spring false flax seedlings (Figure 1): ( Where R -is the disease development, %; Σ(a х b) -is the sum of the products of the number of affected seedlings (а) by their respective affection point (b); N -is the total number of counted seedlings (healthy and affected) in the sample; k -is the highest affection point.

Results
When black mustard seedlings were infected with Fusarium blight pathogen under laboratory conditions in the variants with exposure to mycelium for 1 and 3 hours, it was possible to carry out recording after 1 day, because on the day of the experiment there was no difference between the variants, and with exposure for 5 hours, recording was carried out immediately after the end of exposure, because the difference in the affection between resistant and susceptible samples was clearly visible (Table 1).The number of affected black mustard seedlings in 1 and 2 variants after 1 day in susceptible samples was high (73.0-82.0%), and in resistant samples it was 6.0-18.0% less, amounting to 55.0-76.0%.Disease development in these variants in susceptible samples was high, ranging from 64.2 to 73.0 %, while in resistant samples it was average, amounting to 21.2-34.6%.
The largest difference between resistant and susceptible samples on the prevalence of Fusarium blight was observed in the 3rd variant at exposure of 5 hours and recording on the day of the experiment: in susceptible samples it was 93.0 %, in resistant samples -71.0 %.Disease development in this variant in susceptible samples was high (70.3%), and in resistant samples -average (42.5 %), as in other variants.
Two days after infection, black mustard seedlings of both resistance groups showed symptoms of disease and 4 points of affection degree, i.e. both prevalence and development of disease were 100%.

E3S Web of Conferences 463, 01019 (2023) EESTE2023
https://doi.org/10.1051/e3sconf/202346301019 The evaluation of white mustard and spring false flax seedlings affected by Fusarium blight, carried out after 1 day, showed that the prevalence of the disease in susceptible samples was 100% in all variants in both crops, and in resistant samples -50.0-78.0 and 48.0-76.0%,respectively, with the maximum value at exposure of 5 hours (Table 2).The highest development of the disease was observed when seedlings were exposed to the pathogen mycelia for 5 hours.In susceptible samples it was 76.5 % in white mustard and 72.4 % in false flax; in resistant samples it was 37.5 and 35.4 %, respectively.
Two days after infection of white mustard and spring false flax seedlings with the fungus F. oxysporum, the prevalence of Fusarium blight in resistant samples increased up to 100 % in all experimental variants in both crops.At the same time, the development of the disease on mustard and false flax seedlings increased: in susceptible samples up to 94.5-100 and 92.6-100 %; in resistant samples -up to 70.4-97.3and 80.0-98.2%, respectively (Table 3).In the variants where seedlings of white mustard, black mustard and spring false flax were on the surface of the pathogen mycelium for 1 and 2 days, it was found that the prevalence and development of Fusarium blight was 100% when recorded immediately after the end of the exposure period of the experiment (

Discussion
Data from laboratory studies on the resistance of white mustard, black mustard and spring false flax to Fusarium blight are consistent with field data, i.e. resistant samples under field conditions showed significantly lower disease affection than susceptible samples.For black mustard, the largest difference in Fusarium blight prevalence, combined with a significant difference in disease development between samples of different resistance groups, was observed at the 5 h exposure time and recording on the day of the experiment.Disease prevalence in resistant samples was 22.0% lower and disease development was 39.0% lower compared to susceptible samples.In this experimental exposure, prevalence in resistant samples of black mustard averaged 71.0% and disease development 43.0%.On this basis, we propose to accept these indicators as a criterion in the evaluation of disease affection of seedlings.Thus, we propose that samples with a disease prevalence of 71.0% or less and a disease development of 43.0% or less be considered resistant to Fusarium blight under laboratory conditions.
In white mustard and spring false flax, the optimum combination of difference in disease prevalence and development between samples of different resistance groups was observed with the 5 hour exposure and recording after 1 day.Although the difference in the prevalence of Fusarium blight between susceptible and resistant samples of both crops was not so high in this variant (22.0 and 24.0 %, respectively), the difference in disease development between them was significant (39.0 and 37.0 %, respectively).In this experimental exposure, the disease prevalence in resistant samples averaged in white mustard -78.0 %, in spring false flax -76.0 %; disease development -37.5 and 35.4 %, respectively.We consider it possible to propose these indicators as a criterion for evaluating samples of these crops for resistance to Fusarium blight.We propose to consider samples of white mustard with disease prevalence of 78.0% and less, disease development of 38.0% and less, and samples of spring false flax with disease prevalence of 76.0% and less, disease development of 36.0%and less, as resistant to Fusarium blight.

Conclusion
Evaluation of breeding material of white mustard, black mustard and spring false flax for resistance to Fusarium blight under laboratory conditions, which is rapid and reliable, should be carried out by exposing seedlings to mycelia of the pathogen for 5 hours and E3S Web of Conferences 463, 01019 (2023) EESTE2023 https://doi.org/10.1051/e3sconf/202346301019recording the affection of seedlings of black mustard on the day of infection and of white mustard and spring false flax 1 day after the end of the exposure time of the experiment.

Fig. 1 .
Fig. 1.Visual scale of white mustard seedlings affected by Fusarium blight: (a) 0 points; (b) 1 point; (c) 2 points; (d) 3 points; (e) 4 points. 0 points -seedlings are healthy, root hairs are developed. 1 point -dark spots on stem and root or darkening of 1/5 of the seedling root, root hairs developed. 2 points -darkening of 1/3 of the seedling root, root hairs are developed. 3 points -darkening of half of the seedling root, thinning of the root is not observed, root hairs are practically absent.

Table 2 .
Affection of white mustard and spring false flax seedlings by the fungus Fusarium oxysporum Schlecht.emend.Synd.et Hans (recording after 1 day).

Table 3 .
Affection of white mustard and spring false flax seedlings by the fungus Fusarium oxysporum Schlecht.emend.Synd.et Hans (recording after 2 days).

Table 4 .
Affection of white mustard, black mustard and false flax seedlings by the fungus Fusarium oxysporum Schlecht.emend.Synd.et Hans under prolonged exposure conditions.