E. D. Mytsa, M. A. Pobedinskaya, L. Yu. Kokaeva, S. N. Elansky
Late blight of potatoes and tomatoes, caused by the mushroom-like organism, the oomycete Phytophthora infestans (Mont) de Bary, is common in almost all areas of the world where these crops are cultivated. Under epiphytotic conditions, the loss of potato yield from late blight can reach 10–30% or more, and of tomato, up to 100% (Ivanyuk et al., 2005).
One of the main sources of primary inoculum Ph. infestans, leading to plant infection, are thick-walled resting reproductive structures - oospores. Hybrid oospores formed as a result of crossing genetically different parental strains contribute to an increase in genotypic diversity in the population, as a result of which the process of adaptation of strains to new varieties and applied fungicides is accelerated. Formation of oospores Ph. infestans in the field was noted in many countries of the world: Russia (Smirnov et al., 2003), Norway (Hermansen et al., 2002), Sweden (Strömberg et al., 2001), the Netherlands (Kessel et al., 2002) and other regions. There is evidence that oospores of the causative agent of late blight can survive for more than 2 years in the soil in a viable state (Hermansen et al., 2002; Bшdker et al., 2006) and cause plant infection after overwintering (Lehtinen et al., 2002; Ulanova et al. al., 2010).
The main method of combating late blight today is chemical protection, which consists in treating plantings with fungicidal preparations. Many chemicals used to protect potatoes and tomatoes from late blight have a strong inhibitory effect on oospore formation (Kessel et al., 2002; Kuznetsov, 2013). However, other drugs are widely used on potatoes that do not directly affect Ph. infestans, and their effect on oospore formation is unknown. Therefore, the purpose of this work was to study the effect on the formation of oospores of some drugs widely used on potatoes, but not registered against late blight.
We used 9 Ph. infestans of different types of mating, isolated by us from infected potato leaves in Moscow, Leningrad, Ryazan regions. To study the effect on the growth of mycelium and the formation of oospores, the following drugs were used: fungicides Maxim (active ingredient fludioxonil from the class of phenylpyrroles) and Scor (difenoconazole, triazoles), Aktara insecticides (thiamethoxam, neonicotinoids) and Tanrek (imidacloprid, neonicotribidinoid) (herbicotribidin , triazines). All pesticides are registered in the "State Catalog of Pesticides and Agrochemicals" for 2014. To study the effect of the pesticide concentration on the growth of oomycete colonies, each strain was inoculated with an agar block in the center of a Petri dish with a dense oatmeal medium. The test pesticide was preliminarily added to the medium at concentrations of 0.1, 1.0, 10.0, and 100.0 mg / L (in terms of the active ingredient - DV). As a control, we used a medium without added pesticide. The inoculations were incubated at 18 ° C for 12-15 days until the colony diameter of the pesticide-free control was 70-80% of the diameter of the Petri dish, after which the diameter of the colonies was measured in the control and experimental variants.
The experiments were carried out in 3 replicates. The study of oospore formation was carried out on an agar oat medium (30 ml in a Petri dish) with the addition of a fungicide at concentrations of 0.1, 1.0, 10.0, and 100.0 mg / L and on a medium without fungicide (control). For this, agar blocks with mating type A1 and A2 isolates were placed in pairs on the surface of the nutrient medium at a distance of 5 cm from each other. The inoculations were incubated at the optimum Ph. Infestans at a temperature of 18 ° C for 20 days. After cultivation, the nutrient medium with spores was resuspended with a mixer in 30 ml of distilled water, and preparations for microscopy were prepared from the resulting suspension. In each variant, 180 fields of view were viewed (3 replicates, 60 fields of view). Then the concentration of oospores was recalculated (pcs / μL of medium).
Effect of pesticides on radial colony growth. Difenoconazole, thiamethoxam and imidacloprid had no statistically significant effect on the radial growth of Ph. infestans (Table 1). The herbicide metribuzin caused a slight growth retardation in the initial period (5-7 days of growth); however, by the 10th day, the diameters of the colonies became similar in size. Fludioxonil statistically significantly inhibited the development of Ph. infestans at a concentration in the medium of more than 10 mg / l.
Table 1
Effect of pesticides on radial growth of colonies Phytophthora infestans
Fungicide-DV (drug) | Colony diameter at different concentrations (mg / L) DW in the medium, mm | ||||
0.0 | 0.1 | 1.0 | 10.0 | 100.0 | |
Thiamethoxam (Aktara drug) | 82 6 ± | 81 7 ± (99%) | 82 6 ± (100%) | 81 6 ± (99%) | — |
Imidacloprid (Tanrek) | 792 6 ± | — | 76 9 ± (96%) | 77 8 ± (97%) | 76 5 ± (96%) |
Fludioxonil (Maxim) | 82 6 ± | — | 74 12 ± (90%) | 56 10 ± (68%) | 46 3 ± (56%) |
Metribuzin (Zenkor) | 88 12 ± | — | 85±12 (97%) | 86 9 ± (98%) | 80 5 ± (91%) |
Difenoconazole (Scor) | 82 7 ± | — | 76 9 ± (93%) | 84 4 ± (102%) | 81 6 ± (99%) |
Note. The “±” sign is followed by the confidence interval for a significance level of 0.05. Values in parentheses express the ratio of the diameter of the colonies in the experimental variant to that in the control without pesticides. A “-” sign means that no research was carried out.
Table 2
Effect of pesticides on oospore formation Phytophthora infestans in agar environment
Fungicide-DV (drug) | The number of oospores in the medium at different concentrations (mg / l) DV, pcs / μl | ||||
0.0 | 0.1 | 1.0 | 10.0 | 100.0 | |
Thiamethoxam (Aktara drug) | 79.6 3.6 ± | 79.8 3.8 ± (100%) | 79.1 3.9 ± (100%) | 71.4 3.7 ± (90%) | — |
Imidacloprid (Tanrek) | 79.6 3.6 ± | — | 70.0 3.4 ± (88%) | 66.0 3.1 ± (83%) | 35.8 2.8 ± (45%) |
Fludioxonil (Maxim) | 112.7 6.9 ± | — | 98.4 8.6 ± (87%) | 73.6 5.4 ± (65%) | 42.3 3.7 ± (36%) |
Metribuzin (Zenkor) | 135.0 9.5 ± | — | 103.0 9.8 ± (70%) | 118.2 9.3 ± (88%) | 74.8 8.1 ± (55%) |
Difenoconazole (Scor) | 79.6 3.6 ± | 72.5 3.6 ± (91%) | 82.2 3.7 ± (103%) | 54.9 2.8 ± (69%) | 35.8 2.3 ± (45%) |
Study of the effect of pesticides on the formation of oospores Ph. infestans in a nutrient medium. It was found that a statistically significant decrease in the number of oospores at certain concentrations was caused by all investigated drugs (Table 2). At an active substance concentration of 1.0 mg / l, all pesticides, with the exception of Aktara and Skor preparations, led to a noticeable decrease in the amount of formed oospores (by 12–24% compared to the control). A further increase in the concentration of active substances in the medium led to an increase in the inhibitory effect. Preparations based on thiamethoxam and difenoconazole caused a statistically significant decrease in the number of oospores when the concentration of the active substance in the medium was more than 10 mg / l.
Discussion and conclusion. The study of the effect of pesticides not registered against late blight of potato on the radial growth of mycelium showed, as expected, a weak suppression of growth (fludioxonil) or no effect on growth (other studied pesticides).
Table 3. Concentrations of active substances in the working fluid
Preparation (fungicide-DV) | Used in the work of the concentration of DV in the nutrient medium, mg / l | Concentration of DW in the working liquid during potato processing, mg / l |
---|---|---|
Aktara (thiamethoxam) | 0.1, 1, 10 | 37-75 * |
Tanrek (imidacloprid) | 1, 10, 100 | 50-100 |
Maxim (fludioxonil) | 1, 10, 100 | 1000 |
Zenkor (metribuzin) | 1, 10, 100 | 1630-4900 |
Scor (difenoconazole) | 0.1, 1, 10, 100 | 188-625 |
* The values are presented according to the "State catalog of pesticides and agrochemicals" for 2014.
All studied pesticides caused a decrease in the formation of oospores in the nutrient medium. The tested concentrations of pesticides in the medium were lower or approximately corresponded (for imidacloprid) to the permitted concentrations in the working fluid (Table 3). In our experiments, the suppression of the formation of oospores intensified as the dose of the drug increased, which suggests an increase in the effect upon contact with a more concentrated working fluid. Difenoconazole caused a significant decrease in the concentration of oospores not only in experiments on a nutrient medium, but also in tests on cut-off potato leaves placed in a liquid containing a fungicide. Thus, in the Vektar Belorussian variety, 32 oospores per 1 mm2 of leaf area were observed in the control, at a concentration of difenoconazole in water of 10 mg / l - 24, and at 100 mg / l - 12 oospores / mm2. The difference in the concentrations of oospores at 100 mg / l of the fungicide and in the control is statistically significant (Elansky, Mytsa, unpublished).
Pesticides can affect a wide variety of processes in fungal cells. In the literature, we were unable to find information that to some extent explains the possible effect of the studied drugs on the formation of oospores. Let's try to make some assumptions regarding the action of difenoconazole. The mechanism of its fungicidal action is to inhibit the C14-dimethylase enzyme, which plays a key role in sterol biosynthesis. Sterols are synthesized by fungi, plants and other organisms and are part of their cell membranes. Oomycetes of the genus Phytophthora, in the absence of sterols, are only capable of vegetative growth; the formation of oospores is completely suppressed (Elliott et al., 1966).
Oomycetes are unable to synthesize sterols on their own; they incorporate into their membranes the sterols obtained from the host plant, modifying them. In our experiment, we used oat agar medium rich in â-sitosterol and isofucosterol (Knights, 1965), i.e., substances that stimulate the formation of oospores. It is possible that difenoconazole inhibits the work of enzymes involved in the modification or utilization of sterol compounds obtained from plants. This can, in turn, reduce the intensity of oospore formation.
In small concentrations, as shown in our work, difenoconazole had a weak stimulating effect on the growth of mycelium and the formation of oospores.
The suppression of the formation of oospores in the nutrient medium has been previously shown for antiphytophthora fungicidal preparations. Thus, in the work of Kessel et al. (2002) investigated more than 10 antiphitofluoroids commercial drugs. Fluazinam, dimethomorph, and cymoxanil in non-lethal concentrations completely suppressed the formation of oospores in an agar medium; metalaxil, maneb and propamocarb showed moderate efficacy; Mancozeb and Chlorothalonil had practically no effect on oospore formation. In the work of S.A. Kuznetsov (Kuznetsov, 2013), inhibition of the formation of oospores on a nutrient medium by non-lethal concentrations of metalaxyl was shown.
Our experiments showed that pesticide preparations used on potatoes, which did not even have a direct inhibitory effect on the growth of the late blight pathogen, suppressed the formation of oospores. Thus, a properly carried out chemical protection of potatoes using fungicides, insecticides and herbicides reduces the likelihood of oospores formation in plant leaves.
This work was supported by the Russian Science Foundation (project No. 14-50-00029).
The article was published in the journal "Mycology and Phytopathology" (Volume 50, Issue 1, 2016).