White Rust Albugo candida (Pers.) Kuntze, 1797 in Horseradish Crops (Armoracia rusticana G. Gaertn., B. Mey. & Scherb., 1800) in the South of the Far East and Measures to Control it

Horseradish is a valuable agricultural crop with gastronomic and medical significance. It contains mono- and polysaccharides, protein compounds and organic acids, minerals and starch, as well as a rich vitamin complex. There is enough oil, nitrogenous and resinous compounds in the plant, a lot of B vitamins. In folk medicine, it is believed that horseradish facilitates the removal of sputum from the respiratory tract, cleanses the body of parasites, helps to fight excessive puffiness, helps to stabilize high blood pressure in hypertension (Agneta et al., 2013, Nguen et al., 2013). Nutritionists recommend using horseradish as a seasoning for favorite dishes for those who want to get rid of extra pounds, because the vegetable is rich in omega-3 and omega-6 fatty acids. In addition, due to the significant fiber content, horseradish is able to control the satiety feeling. In gastronomy it’s root is classified as spicy-flavored crop and it is recommended to serve horseradish with marinades, as well as fatty fish dishes to give them more piquancy (Rivelli et al., 2017). In addition, horseradish leaves are used for preserving tomatoes, cucumbers, beans and therefore it is important to preserve their marketable appearance.
       
At the moment, several varieties of horseradish are known in Russia: “Suzdalian”, “Rizhskij”, “Atlant”, “Valkovsky”, “Tolpukhovsky”, “Bankovskij”. These varieties are suitable for mechanized cultivation. Unfortunately, horseradish diseases are still insufficiently studied, while crop losses from them can be quite significant. This determined the goals of our research.
       
The purpose of our study was to determine the composition of pathogenic fungi on horseradish Armoracia rusticana G. Gaertn., B. Mey. & Scherb. and to develop measures to combat them.

The research was carried out on horseradish varieties “Atlant”, in the plantings of the potato and vegetable department of FSBSI ”Far East Federal Research Center of agrobiotechnology n.a. A.K. Chaika” at 2019 - 2020. In the years preceding the research no diseases were detected in horse radish.
 
Vegetation experiments
 
Preparation of the field for experiments began in the fall after harvesting the predecessor (buckwheat). It consisted in plowing to a depth of 18-20 cm. Spring preparation included harrowing, cultivation and application of mineral fertilizers in a continuous manner at the rate of 300 kg (in physical weight). The day before planting, ridges were cut with a width of 70 cm along the axes of the furrows. Horseradish was planted on April 7 2019, manually, according to the 70x30 cm scheme. The planting density was 47,600 roots/ha. Care for plants began after the appearance of mass horseradish seedlings. In total, three inter-row treatments and two hoeing were carried out during the growing season. The pesticides selection was determined by the assortment of specialized stores for the garden. An indicator of the drug effectiveness in the field was the phytopathogenic load reducing value, symptoms development stopping.
       
The placement of plots in the field experiment was randomized, small-scale. 15 plants treated with a manual sprayer were taken into account. The flow rate of the working fluid was observed in the recommended doses.
       
The phytotoxic effect of the agent was evaluated on a six-point scale, where 0 - no damage; 1- minor damage with up to 5% leaf damage; 2 - mild damage with up to 10% leaf damage; 3 - moderate damage with up to 25% leaf damage, 4 - severe damage with up to 50% leaf damage; 5 - very severe damage with more than 50% leaf damage (Dolzhenko et al., 2018).
       
Disease development account was carried out on a five-point scale: the lowest score - 0 (no damage), 1 - from 1 to 20% of the plant is affected, 2 - 21-40% is affected, 3 - 41-60% is affected, 4 - 61-80% is affected, 5 (the highest) - 81-100% is affected.
       
Based on the results of field experiments, the biological effectiveness of agents was calculated using the standard method (Vasilchenko et al., 2019).
       
Studies of phytopathogens were carried out in the natural context of infection.
 
Seeding on the pure culture
 
The affected plant organs were laid on wet chambers before the mycelium appearance. Subsequently, pieces of mycelium were transferred to the Saburo nutrient medium by a microbiological loop in a sterile box. Transplanting to a pure culture was performed with a Drigalski spatula from a pathogen spores suspension, in the titer 10¹⁰ colony-forming units per milliliter.
       
To prepare the suspension, 5 ml of the pathogen culture was removed from the nutrient medium in a sterile box, placed in plastic tubes “LITOPLAST-Med” with a volume of 50 ml, after which it was centrifuged with 10 ml of distilled water and 5 ml of Twin-80 at 4000 r/min for five minutes. The fraction with spores was selected from a test tube using a single-channel pipet dispenser to a sterile container, the suspension titer was determined in the Gorjaev’s chamber and then they were transfered it to a clean culture in Petri dishes. The differentiator was nystatin (250 thousand units).
       
The agents were diluted in 10%, 30% and 70% of the recommended dose by the manufacturer. In addition, the experiment variants used the recommended dose, as well as studied control without treatment. Each version was prepared in three repetitions. There were 5 iterations of the experiment for each version.
       
The fungicides were applied to the nutrient medium with a pasteur pipet a day after the pathogen was seeded.
       
The fungicidal effect of the agents was evaluated by the rate of colonies growth and sporulation activity. The activity of sporulation was estimated on a ten-point scale, where 1 -sporulation absents; 3 - sporulation takes up to 10% of the nutrient medium surface; 5 - sporulation takes from 10 to 25% of the nutrient medium surface; 7 - sporulation takes from 25 to 50% of the surface environment; 8 - sporulation takes from 50 to 75% of the nutrient medium surface; 9 - sporulation is more than 75%.
       
The conidia of each sample were counted in 10 fields of microscope view, the area of 10 mm2 on a rank scale: 1 (very rare) - 1-50, 2 (rare) - 51-150, 3 (moderately encountered) - 151-200, 4 (frequent) - 201 - 250, 5 (very frequent) - more than 250. Next, the index of conidia occurrence (CI) were calculated.
   
Where
FCS - the percentage of occurrence of samples with very few conidia; RC - percentage of occurrence of samples with rare conidia, MC - the percentage of samples with moderate frequency of conidia, FC - percentage of occurrence of samples with frequent conidia, VFC - percentage of occurrence of samples with very frequent conidia.
       
The aggressiveness index of field populations was determined by the formula:
   
Where
AI - aggression index D - development DI - development index CI – conidia formation index (Vasilchenko et al., 2019).

Microscopy was performed using Levenhuk D740T, 5,1 MP. Photofixation of lifetime injuries was performed by Sony SAL1855. Processing of the microscopy results were carried out using programs Outfi, PluriIQ, CellProfiler.
 
Investigational agents
 
In an experiment to study the fungicidal effectiveness of agents in relation to horseradish diseases, 10 agents were used (given from an Internet source http://www.pesticidy.ru/):
1. Acrobat WG (BASF). Active ingredient - dimethomorph +mancozeb (40 g/10l).  Disrupts spore formation, acting on the cellular level. Inhibits the spread of infection.
2. Bravo SC (Syngenta). Active ingredient - chlorothalonil, consumption rate (500 g/l). A broad spectrum of activity against the late blight, the downy mildew, early blight. High efficiency against a number of barley and wheat leaves and ear diseases.
3. Zummer SC (Cheminova). Active ingredient - fluazinam (500 g/l). Increases yield and product quality. Provides ong-lasting leaves and stems protection. It is a tool for resistance preventing.
4. Consento SC (Bayer). Active ingredient - propamocarb hydrochloride+phenamidone (375 + 75 g/l). Provides late blight, early blight, of peronosporosis control. It is used in all vegetation phases . Antisporous properties.
5. Infinito SC (Bayer). Active ingredient - hydrochloride propamocarb+fluopicolide (6,25 + 6,25 g/l). Provides protection from late blight on leaves and stems, anti-spore properties.
6. Topaz EW (Syngenta). Active ingredient - peconazole (100 g/l). Systemic fungicide for the protection of pome fruits, stone fruits, berries, vegetables, ornamental crops and vines from powdery mildew and other diseases.
7. Ordan SP (Avgust). Active ingredient - copper oxychloride + cymoxanil (689 g/kg + 42 g/kg). Fungicide for vegetable crops and potatoes protection from fungal and bacterial diseases. High fungicidal activity against peronosporosis.
8. Thiovit Jet WG (Syngenta). Active ingredient-sulfur (800 g / kg). Fungicide and acaricide for vegetable, fruit, lower crops and vineyards protection from real powdery mildew, some other diseases and mites.
9. Rajok EW (Avgust). Active ingredient - diphenoconazole (250 g/l). Systemic fungicide for apple, pear, sugar and feed beets, potatoes and tomatoes protection from a complex of diseases. Antisporous properties.
10. Proton Extra WG (Technoexport). Active ingredient- copper chloroxide + oxadixyl (670 + 130 g/kg). A new fungicide of protective and healing action. Designed to protect vegetable crops from late blight, early blight and downy mildew, mildew.
11. Ridomil Gold MZ WG (Syngenta). Active ingredient - mancozeb+mefenoxam (640+40 g/kg). Fungicide of systemic and contact action, effective against pathogens of potato and tomato late blight and early blight, cucumbers and onions peronosporosis, mildew and grape anthracnose.
      
Statistical processing of the experiment results was performed using the program Past 4.03. The variance and the mean square deviation were calculated.

The first disease manifestations were observed on 06.07.2020 in the production plantings of horseradish belonging to the department of potato growing of FSBSI ”Far East Federal Research Center of agrobiotechnology n.a. A.K. Chaika”. There were chlorotic spots on the upper leaves surface. By 13.06. 2020, the spots had developed to pustules on the leaf underside (Fig 1, Fig 2).  Pustules broke through the epidermis and sporangial sporulation could be observed. In General, the picture corresponded to that described by Gabor et al., (2013) and therefore it was concluded about development of Albugo candida (Pers.) Kuntze,  also called white rust.
 

 

       
The causative agent of white rust belongs to the Albuginaceae family. It is a family of obligate parasites of herbaceous plants, including some cultivated crops such as gilliflower and horseradish. They are characterized by mycelium endophytic development in the intercellular plants spaces. Non-branching conidiophores develop under the host’s epidermis, forming a dense layer that breaks the plant’s cover. Conidia germinate with zoospores and sexual reproduction and oospore formation occurs in host tissues (Rimmer et al., 2007).
       
To confirm the diagnosis, the material was seeded on nutrient media with subsequent removal to a clean culture and microscopy in a light and dark field, as well as microscopy in vivo.
       
Eucarpic mycelium was observed during the in vivo microscopy. Hyphae were coenocytic, aseptate and abundantly branched (Fig 3). They formed short, thin-walled, square-headed conidiophores at right angles to the nutrient medium. After preparing the Ziehl-Neelsen smear, there were noted concave-convex, single-core zoospores characteristic of Albugo candida (Fig 4).
 

 

       
At the moment, the methods of fighting white rust on horseradish are not sufficiently studied. In this regard, we have established an experiment on the fungicidal activity of chemicals against Albugo candida. The main results of the experiment are presented in Table 1.
 

     
On average, for five iterations, the greatest fungicidal activity was demonstrated by the Acrobat WG, Topaz EW and Proton Extra WG agents. In these variants the mycelium was poorly developed, unpolished, conidial sporulation was practically absent and zoospores were not formed. All three agents are different groups of active substances (Fig 5).
 

       
Acrobat WG consists of dimetomorf and mancozeb. It is known that mancozeb itself does not have fungicidal activity, but when dissolved in water it forms ethylene bisisothiocyanate sulfide, which is converted to ethylene bisisothiocyanate under the action of ultraviolet light. Both substances affect the fungi enzyme systems that contain sulphydryl groups. They disrupt about six important biochemical processes in the mitochondria and cytoplasm of fungal cells (Nikitin, 2012; Gawade et al., 2009).  Dimetomorph in this case causes an anti-spore-forming effect. Changes the natural morphogenesis of the fungi cell wall, disrupting their normal development cycle (Zinchenko, 2012; Valaramathi, 2018).
       
Penconazole, which is included in Topaz EW, inhibits sterols biosynthesis (Belov, 2003) and stops the growth tube into the leaf tissue penetration (Golyshn, 1993; Andreeva, Zinchenko, 2002; Srivastava et al., 2015). It acts in very low concentrations on the pathogen during its introduction into the leaf tissue and gaustoria formation. Oxadixyl is quickly absorbed and penetrates deeply into the plants leaves, affects the pathogens RNA, disrupting the reproduction processes.
       
All three actors are recommended for peronosporosis fungi management. The maximum effectiveness was observed for the Ridomil Gold MZ WG, which is confirmed by the studies of Stone et al., (1987). According to their data, metalaxil was active against the 7^th race of  Albugo candida  on  Brassica campestris cv. Torch. When plants were sprayed for 4 days after inoculation, it reduced leaf infection by 95% and seed treatment with metalaxil at a dose of 5.0 g/kg controlled leaf infection until the sixth leaf stage in the growth chamber.              
Moderate activity was showed by Bravo SC, Zummer SC and Rajok EW actors. Thus, the use of Zummer SC inhibited the zoospores formation, which is consistent with the references data (Ganiev, Nedorezkov, 2005). However, this pesticide had no effect on the mycelium growth, as in the case of Bravo SC.  Adding the latter to the milieu partially stopped the conidia and spores germination. Rajok EW inhibited the sporulation productivity, as shown by Supranovich R.V. and Matveychik M.A. (1995), but did not inhibit the fungi development.
       
The lowest efficiency was shown by the Infinito SC, Consento SC, Ordan SP, Thiovit Jet WG agents. Their use did not have any effect on the mycelium development, sporulation was observed abundant, oospores and antheridia were formed, the appearance of zoospores with a high infection rate was noted. It is obvious that the active substances of these drugs do not have fungicidal effectiveness against Albugo candida.
       
Since the preparations are not registered for use on horseradish, we set up a field experiment to study the phytotoxicity and fungicidal activity of pesticides in the field. The main results of the experiment are presented in Table 2. No phytotoxic effect was found in any of the agents. The maximum effectiveness against Albugo candida  was noted for the Ridomil Gold MZ WG (70-91%), Zummer (65.5-74.2%), Proton (53.3-66.8%), Acrobat (50.1-66.8%), Topaz (41.1-46.5%), Rajok (40.1-45.5%), Bravo (40.2-54.2) fungicides. Infinito, Consento and Thiovit Jet agents had biological effectiveness, which is consistent with the results obtained on the Petri dish.
 

Thus, it was found that the use of Ridomil Gold MZ WG, Zummer SC, Proton EW, Acrobat WG, Topaz EW, Rajok EW and Bravo SC in the horseradish protection system against white rust in the monsoon climate of the South of the Russian Far East significantly reduces the harmfulness of Albugo candida in all agent consumption rates. The observed effect is a consequence of the fungicidal activity mancozeb, mefenoxam, oxadixyl, dimethomorph and other fungicides active ingredients. It was noted that the use of Ridomil Gold MZ WG (had the maximum effectiveness against the white rust causative agent. Both in Petri dishes and in the vegetative experiment, Ridomil Gold MZ WG suppressed the development of mycelium, did not allow sporulation to develop and inhibited symptoms. The proposed treatment schemes are one of the possible options for Albugo candida. management.

The authors gratefully acknowledge Dr. of biological sciences Slabko Y.I. (FSBSI ”Far East Federal Research Center of agrobiotechnology n.a. A.K. Chaika”) for methodological assistance with the experiments and critical comments during this article preparation. The authors also thank Romanciuc Riceard (https://orcid.org/0000-0002-0654-7854) for his help in this article translation.