Introduction

The Colorado potato beetle (Leptinotarsa decemlineata Say) and phytopathogenic fungus (Rhizoctonia solani Kuhn.) are the main pests that damage potato (Solanum tuberosum L.) in Western Siberia. The Colorado potato beetle is the main potato phytophage worldwide¹. In Western Siberia, it appeared much later, from the end of the XX – beginning of the XXI century and quickly became the most dangerous phytophage over the short time period. It is known that the chemical insecticides were the first most effective means to control the Colorado potato beetle². However, studies carried out in various countries revealed gradually acquired resistance of this insect towards chemical insecticides^3,4. Besides, the adverse effects of chemicals on the environment and human health are well known^5,6. This has led to the search for alternative environmentally safe methods to control the population of the Colorado potato beetle^7-9. The use of natural biological agents of bacterial and fungal origin seems to be the most attractive means. Thus, as early as in the middle of the XX century it was proposed to suppress the population of the beetle larvae by spraying potato plants with fungi Beauveria bassiana Bals. Vuil10^,11 and later with Metarhizium spp.^12,13. Compared to fungus-based formulations, the Russian Bitoxibacillin preparation, based on exotoxin-containing strain Bacillus thuringiensis subsp. thuringiensis, turned out to be more effective¹⁴. In addition, in 1983, a new subspecies of Bacillus thuringiensis subsp. tenebrionis¹⁵ were isolated and identified, which was specific against the Colorado potato beetle and served as the basis for the development of biological preparations.

Table 1. Biological efficacy of BtH₁₀ against the Colorado potato beetle larvae in laboratory experiment

Along with the Colorado potato beetle, a serious problem in West Siberia is Rhizoctonia disease of potato. To control disease caused by phytopathogenic fungus Rhizoctonia solani Kuhn., the main method of potato protection in West Siberia, like in other geographical regions of the world,  is the application of chemical fungicides. However, a growing body of research is currently focused on the development of microbiological methods for protection against Rhizoctonia disease^16-18.

At present time, the biological preparations not always are competitive with chemical pesticides, and one of the reasons is the high selectivity of the biological control agent, forcing the use of two or more biological preparations instead of one chemical pesticide. To improve competitiveness it is desirable to use biological preparations of polyfunctional action, possessing, for example, both insecticidal and fungicidal properties.

Table 2. Influence of BtH₁₀ on disease severity and incidence of potato stem canker and stolon canker, %

The biological agents of such effect include bacteria of the genus Bacillus: B. subtilis^{19, 20} or B.thuringiensis^21-24.  Previously, it was shown that strain B. thuringiensis ssp. darmstadiensis (BtH₁₀), isolated from the Colorado potato beetle, exhibited insecticidal activity against the Colorado potato beetle of northwest and south populations²⁵. In addition, this strain has shown antifungal activity against Rhizoctonia disease of potato at a single treatment of potato tubers when planting “Lubava” and “Lugovskoy” potato cultivars²⁶. However, it is important to reveal the dual effect of this bacterium, supplementing the treatment of tubers before planting by spraying with a bacterial suspension during the growing season. Due to the fact that early potato “Yuna” is a promising cultivar for Western Siberia region, it was challenging to use it for carrying out the study on the polyfunctional properties of the entomopathogenic bacterium.

Table 3. Growth-promoting action of strain BtH₁₀ at potato protection

The objective of the present work consisted in evaluation of the insecticidal, antifungal and growth-promoting action of B. thuringiensis subsp. darmstadiensis at treatment of the potato tubers before planting followed by subsequent spraying during the growing season under the natural conditions of Western Siberia.

Materials And Methods

The research objects were the plants of “Yuna” potato cultivar, the Colorado potato beetle, Rhizoctonia disease of potato, Bacillus thuringiensis ssp. darmstadiensis (BtH₁₀), the strain RCAM 01490 taken from the collection of the All-Russian Institute for Agricultural Microbiology.

The efficacy of the bacterial strain against the Colorado potato beetle in a laboratory experiment was tested on larvae of all instars, taken from natural populations. Larvae were planted on the leaves of “Yuna” potato cultivar in Petri dishes and sprayed with a bacterial suspension (10⁶ CFU/ml). The leaves were substituted while drying. Larval mortality was estimated on the 3^rd, 5^th, 7^th and 10^th day. Biological efficacy was calculated by the Abbot formula.

Field experiments were conducted on potato experimental plots, located in the Novosibirsk District of the Novosibirsk Region (55⁰1´N, 82⁰55´E). The experiments were performed in three replicates; the area of each plot was 30 m² and included 360 plants (70х35 cm planting system). The vegetation period in 2014 was characterized as arid, while in 2015 – as humid.

Field experiments included 2 variants in three replications: 1) control (without treatment); 2) treatment of tubers with BtH₁₀, followed by spraying during the growing season with bacterial suspension (10⁶ CFU/ml). The mortality of the Colorado potato beetle larvae was checked 7 and 10 days after spraying the plants.

Table 4. Influence of BtH₁₀ on fractional composition of daughter tubers

The potato stem canker and stolon canker were evaluated 6 and 10 weeks after planting. Disease severity was determined on a Frank scale²⁷, calculating the disease incidence (P) and disease severity (R) by the formulas:  P = R×100/N ,where R – is the number of diseased plants, N – is the total number of record plants;  R= ∑(a×b)×100/N×K ,  where ∑(a×b)– is the sum of the products of the number of diseased plants (a) over the score of the lesion (b), where N – is the total number of record plants, K – is the highest score of the accounting scale. Six weeks after potato planting, samples of potato leaves were collected from the control and experimental sites to determine the peroxidase activity ²⁸.

The degree of black scurf of the daughter tubers was estimated by the ratio of the mass fraction of healthy and diseased tubers²⁹. Fractional composition of tubers was determined by weighing. Depending on mass, tubers were grouped into 3 fractions: small fraction – up to 35 grams, medium fraction – from 35 to 80 grams, and large fraction – over 80 grams. Growth rate and development of plants were monitored during the growing season. The effect of BtH₁₀ on plant height, number of stems and stolons, as well as tubers mass was evaluated during the experiments.

Statistical data processing was performed by standard methods using MS Excel and ANOVA program for Windows. Data were compared by calculating LSD₀₅ (Least Significant Difference).

Results And Discussion

The mortality of 1^st  and 2^nd– instar  larvae under the action of BtH₁₀ in laboratory occurred on the 1^st day, while on the 5^th day reached 100%, significantly differing from larval mortality in the control (p<0.05). Since older larvae are more resistant to bacteria, their mortality, checked on the 10^th  day, was by 2.6 and 1.6 times less as compared to younger instar larvae (p<0.05) (Table 1). Such dependence is typical for populations of the Colorado potato beetle in different geographic regions under the action of different B. thuringiensis subspecies.

During two years of research in the field environment, the mortality of the Colorado potato beetle larvae was not less than 60% seven days after spraying bacterial suspension, and at least 70% ten days after spraying (Fig. 1).

Figure 1: Biocontrol efficacy (%) of BtH10 towards the Colorado beetle larvae in the field. Click here to View figure

At the same time bacterial strain BtH₁₀ at the initial treatment of planting material and subsequent spraying of plants during the growing season caused a significant reduction in the infestation of potato stems and stolons by pathogen Rhizoctonia (Table 2). Thus, in 2014 the average score of infestation by stem canker statistically significantly (p<0.05) decreased by 5-15 times in 6-10 weeks, while in 2015 – by 8.7-2.7 times in comparison to the control. Disease severity on the stems also decreased on average during two years by 9.4-16.6 times over concerned periods. Comparing the data obtained during treatment the tubers of early potato cultivar in 2014 with just the same bacterial suspension without spraying²⁶, it can be stated that the impact of additional spraying during the growing season on Rhizoctonia disease severity is much higher.

Induction of resistance in potato plants under the influence of entomopathogenic bacterium was confirmed by measurement of the peroxidase activity in potato leaves sampled from control and treated plants (Fig. 2).

Figure 2: Peroxidase activity in potato leaves under the influence of BtH10 six weeks after planting. Click here to View figure

Application of BtH₁₀ by described two techniques provided also a reduction in the disease incidence on daughter tubers by 2.9 times in 2014 and has led to almost complete release from the disease in 2015 (Fig. 3).

Figure3: Disease incidence (%) on the daughter tubers under the influence of BtH10 treatment. Click here to View figure

In addition to insecticidal and antifungal activity of bacteria, we revealed also its growth-promoting action on potato. In a field experiment, potato plants have formed bushes faster under the influence of BtH₁₀. In 2015, statistically significant increase (p<0.05) of potato plant height in comparison to control was observed 6 and 10 weeks after planting, while in 2014 a significant increase in the height  was observed 10 weeks after planting. In both studied years, statistically significant increase (p<0.05) in the number of stems in the bush exceeded that of control by 1.2-1.5 times (Table 3). The number of stolons produced was also increased under the influence of BtH₁₀, especially in the year of 2015, which was more favorable in terms of weather conditions (twofold increase).

The proportion of large fraction tubers of the new crop increased under the influence of bacterial strain (p<0.05) (Table 4). The increase in tuber mass was due to formation of larger size fractions (exceeding the control by 1.4-2.0 times) as well as reduction in amount of small tubers (by 2-5 times). The mass of tubers per one bush after treating with BtH₁₀ was 1.4-1.6 times higher than in the control (Fig. 4).

Figure 4: The effect of BtH10  on  biomass of the daughter tubers per one bush, g. Click here to View figure

Our data are consistent with the results on the effect of different B. thuringiensis strains on phytopathogenic fungus R. solani, damaging other plants^30,31. Antifungal action of entomopathogenic bacteria the authors explained by the production of different secondary metabolites, including chitinases³², as well as by inducing systemic resistance of plants³³. In addition, an antagonistic effect may be due to the production of lipopeptide biosurfactants by the entomopathogenic bacterium^34,35. The biosurfactants were found also in strains of antagonistic bacteria of the genus Bacillus, selected for biological control of plant diseases, including Rhizoctonia disease^36-38.

Conclusion

The pathogenic effect of Bacillus thuringiensis subsp. darmstadiensis (BtH₁₀), strain RCAM 01490, on local population of the Colorado potato beetle larvae was shown based on preliminary laboratory experiments. The spraying of planted potato with BtH₁₀ suspension significantly reduced the number of beetle larvae. At the same time, the initial treatment of potato tubers with a bacterial suspension, followed by spraying of plants, resulted in reduced stolon canker, stem canker, and black scurf of the daughter tubers. In addition, induction of systemic plant resistance was confirmed by measuring the peroxidase activity in potato leaves. The growth-promoting action of the entomopathogenic bacterium resulted in increased plant height, number of stems and stolons. Thus, in case of damage to potato plants by the Colorado potato beetle and Rhizoctonia disease under the natural conditions of Western Siberia, the polyfunctional properties including antifungal, insecticidal and growth-promoting action of B. thuringiensis subsp. darmstadiensis, strain RCAM 01490, were revealed by treatment of potato tubers before planting followed by spraying with bacterial suspension during the growing season.

Acknowledgments

The research was supported by the grant of the Russian Science Foundation (Project No. 14-16-00101) for Novosibirsk State Agrarian University.

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