Biological control of important fungal diseases of potato and raspberry by two Bacillus velezensis strains

Bacillus strains used for experiments

The B. velezensis BZR 336 g (Assembly: GCF_009683125.1, GenBank: NZ_WKKU00000000.1) and B. velezensis BZR 517 (Assembly: GCF_009683155.1, GenBank: NZ_WKKV00000000.1) strains were originally isolated from the winter wheat rhizosphere in the Krasnodar region (45°1′N, 38°59′E). The strains belonged to the Federal Research Center for Biological Plant Protection’s (St. Petersburg–Pushkin, Russia) “State Collection of Entomoacariphages and Microorganisms” bioresource collection (No. 585858). The material and technical facilities of the unique scientific installation «New generation technological line for developing microbiological plant protection products» of the Federal Research Center for Biological Plant Protection’s (St. Petersburg–Pushkin, Russia) were used in the research (https://ckp-rf.ru/usu/671367/).

The strains were stored at 4 °C in test tubes with potato-glucose agar (50% potato broth, 2% glucose, 2% agar). For each experiment, fresh liquid cultures were prepared on the original optimized nutrient medium in New Brunswick Scientific Excella E25 (Framingham, MA, USA) incubator shakers (180 rpm). The optimized culture medium was created using Czapek’s medium for bacteria. Molasses was used as a carbon source and corn extract was used as a nitrogen source (Asaturova et al., 2015). The B. velezensis BZR 336 g culture was cultivated at 25 °C for 48 h, and the B. velezensis BZR 517 culture was cultivated at 30 °C for 36 h. These parameters were obtained from a previous study (Sidorova et al., 2020).

In vitro antifungal activity of Bacillus strains

We evaluated the antagonistic properties of B. velezensis strains against the phytopathogenic fungus R. solani, isolated from infected potato tubers, and against fungus D. applanata, isolated from infected raspberry canes in Western Siberia. B. velezensis BZR 336 g and B. velezensis BZR 517 strains were used at concentrations of 10⁴–10⁶ CFU/mL. The experiment was conducted using the agar block techniques (Shternshis, Shpatova & Belyaev, 2016).

Strains’ liquid cultures were introduced into potato glucose agar and Czapek medium for R. solani and D. applanata, respectively, at the indicated concentrations and poured into sterile petri dishes.

The solution was allowed to solidify before a 10 mm diameter agar block with a phytopathogenic fungus was placed in the center of the dish. The plates were incubated at 25 °C for 14 days in darkness. Petri dishes with a medium served as the control. Experiments were replicated five times and the entire experiment was conducted twice.

Antagonistic activity was evaluated by measuring the diameter of the fungal colonies after 3, 5, and 7 days. The fungal inhibition (I, %) was determined based on the data obtained:

$I,\mathrm{\%}=(Dc-Dt)/Dc\times 100$

where Dc is the diameter of fungal colony in control and Dt is the diameter of fungal colony in treatment.

Weather conditions in the summer months of 2015 and 2016

The average summer temperature in 2015 was 18.6 and 19.1 °C in 2016. The amount of precipitation for the same period in 2015 was 207 and 135 mm in 2016. The lowest precipitation occurred in June in 2015, and June and August in 2016. The wettest month was July in 2015. Higher temperatures were noted in July 2016 compared to July 2015.

Field trials of B. velezensis strains on potato plants

Field trials were conducted on experimental plots at the Novosibirsk State Agrarian University (55°1′N, 82°55′E). Plant growth conditions included weeding, inter-row cultivation, and hilling. We used Kemira potato complex fertilizer (Fertika, Russia) (50 g per m²) consisting of nitrogen (11%), phosphorus (9%), potassium (17%), magnesium (2.7%), and sulfur (2.7%). We cultivated two potatoes from different maturity groups: early maturity cv. Yuna and mid-early cv. Svitanok kievskiy.

Each plot measured 30 m² and included 120 plants. Potato seed tubers were dipped for 30 min in a BZR 336 g or BZR 517 (10⁶ CFU mL⁻¹) suspension prior to planting. The growing season was 3 months, during which the potato plant height, the number of above-ground stems, the biomass of daughter tubers, and potato yield were measured at 4, 6, and 10 weeks after planting. The experiment was arranged using a completely randomized block design. Four plot areas (10 m², 40 plants per plot) were subjected to each treatment. Tubers treated with water were used as control. Two independent experiments were performed. We did not observe any diseases except R. solani.

We assessed potato stems infected by R. solani and the plants’ morphometric characteristics (height and number of stems per plant) 4, 6, and 10 weeks after planting. The disease incidence (DI%) was calculated by the formula: DI (%) = (T × 100)/N, where T is the number of infected plants and N is the total number of plants. Disease severity (DS, %) was estimated using the Townsend–Heuberger formula according to the following scale: 0 = no disease symptoms; 1 = stem canker up to 25 mm; 2 = stem canker up to 50 mm; 3 = stem canker more than 50 mm; 4 = canker surround the whole stem; five = stem completely nipped (Frank, Leach & Webb, 1976).

For daughter tubers the disease incidence (DI, %) was calculated using the formula:

$DI(\mathrm{\%})=(T\times 100)/N$

where T was the number of infected daughter tubers and N was the total number of daughter tubers.

We determined tubers’ biomass distribution by their weight. The daughter tubers were categorized as: small (less than 35 g), medium (36–180 g), or large (more than 180 g). Yields were determined by weighing all daughter tubers from each plot.

Field trials of Bacillus strains on raspberry plants

Raspberry plants were also represented by two cultivars: cv. Kirzhach is non-resistant to Didymella applanata and cv. Kolokolchik is resistant to this fungal pathogen. The raspberry cultivar Kolokolchik was selected at the Siberian Research Institute of Horticulture (Barnaul, Russia), and the raspberry cultivar Kirzhach was selected from the All-Russian Institute of Horticulture and Nursery (Moscow, Russia). Raspberry plots were located at the Siberian Research Institute of Crop Production and Breeding’s experimental station. Plant growth conditions included weeding and inter-row cultivation until the raspberry flowering stage. In these experiments, plots were not fertilized. During our study, we did not observe raspberry diseases other than D. applanata.

Plants were located in rows 40 cm wide; rows were spaced 2.5 m apart. The area of each plot was 10 m² and included 50 raspberry canes in four replicates. The size of the buffer zone between plots was 2 m. B. velezensis BZR 336 g and B. velezensis 517 strains were suspended at a concentration of 10⁶ CFU mL⁻¹ and used to manage raspberry disease. B. velezensis suspensions were applied at a volume application rate of 0.1 L/m² using a hand-held Orion-6 sprayer (Quazar Corp. Warsaw, Poland). The control plots were treated with water. Two independent experiments were performed. Bacterial strain treatment timing was determined at the appearance of the first disease symptoms. Observations were carried out throughout the growing season for 2 years.

Disease severity (DS, %) was estimated using the Townsend–Heuberger formula according to the following scale: 0 = healthy cane; 1 = patches with a smooth surface and a diameter less than 0.5 of the cane girth, appearance of splits was possible; 2 = patches with a smooth surface and the diameter more than 0.5 of the cane girth, appearance of splits was possible; 3 = patches with a deformed surface and the diameter more than 0.5 of the cane girth, splits or tissue deformation that come up to the vascular cylinder; 4 = patches with a deformed surface that completely cover the whole cane girth, splits or tissue deformation that come up to vascular cylinder (Shternshis et al., 2002). The disease incidence (DI, %) was calculated using the same formula used for potato plants.

A randomized complete block design was used to assign treatments to four replicates for both potato and raspberry cultures. We manually harvested the crops and the yields were determined by weighing all potato daughter tubers or all harvested raspberry fruits from each plot.

Statistical analysis

We processed our data using Excel 2010 (Microsoft Corporation, Redmond, WA, USA). All results are expressed as the mean. We used STATISTICA 13.2 EN (trial version; Tibco, Palo Alto, CA, USA) to statistically analyze the data. Multiple comparisons of the means were performed using Duncan’s tests with a significance level of P = 0.05.

Screening B. velezensis against R. solani and D. applanata in vitro

The studied strains inhibited the growth of R. solani and D.applanata under laboratory conditions (Fig. 1). The inhibition ratio depended on the concentration of bacteria in the nutrient medium (Tables 1 and 2). The progressive reduction of fungal growth was noted with increasing suspension concentration of B. velezensis strains. After 7 days, the diameter of the R. solani colonies under the action of two strains of B. velezensis significantly decreased at least five times (P < 0.05), and the percentage of the bacterial strains’ inhibition activity at concentrations of 10⁵ and 10⁶ CFU/mL was rather high (Table 1). The same tendency was observed for D. applanata (P > 0.05) although the inhibitory effect was not as evident (Fig. 1; Table 2). In both cases, the inhibitory effect increased with time (Tables 1 and 2).

Potato field trials

The disease incidence on potato stems in the control permanently increased during the growing seasons of in 2015 and 2016. The disease incidence in both years was no less than 80% on two potato cultivars in the control group 10 weeks after planting (Table 3). Using B. velezensis strains for 2 years reduced the disease incidence and severity on potato stems, the degree of which depended on the strain, potato cultivar, and weather conditions that year. In 2015, a statistically significant decrease of the disease incidence (P < 0.05) was observed in both potato cultivars in all cases 6 weeks after planting (Table 3). The disease severity on the stems decreased by 79.8% under BZR 336 g (P < 0.05), and by 23.1% under BZR 517 (P > 0.05) on cv. Svitanok kievskiy, and cv. Yuna by 69.5% and 50%, respectively (P < 0.05) (Table 3). During the same period in 2016, a statistically significant decrease in disease severity of 74.1% was observed on cv. Yuna, under the action of the BZR 336 g strain and of 50% by BZR 517 (P < 0.05) (Table 3).

The greatest decrease in the disease’ incidence and severity on the stems after application was achieved 10 weeks after treatment by bacilli in both 2015 and 2016. In 2015, 10 weeks after treating Svitanok kievskiy tubers with the B. velezensis BZR 336 g strain, the disease incidence decreased by 71.5%, and on cv. Yuna by 61.1% (P < 0.05). In the same year, the strains’ influenced on disease severity was even stronger (P < 0.05) (Table 3), and a smaller effect was observed on cv. Yuna. In 2016, with less air humidity, bacilli’s effect on the disease incidence and severity was statistically smaller (P < 0.05) than in the previous year (Table 3).

The stem damage during the growing season in the control group resulted in a large number of daughter tubers infected by R. solani sclerotia. Damage was especially prominent in 2016, when the disease incidence in the control daughter tubers was 86.7% (Table 3). However, treating tubers with B. velezensis strains significantly reduced the disease incidence in daughter tubers. This value decreased by 71.5% in 2015 and by 93.5% in 2016 in cv. Svitanok kievskiy when using the BZR 336 g strain (Table 3). The BZR 517 strain decreased the disease incidence by 49.6% and 89.6%, respectively (P < 0.05). In cv. Yuna daughter tubers, the data for both strains differed from the control significantly in both years. In 2015, the strain BZR 517 was 3.3 times more effective compared to BZR 336 g. In 2016, BZR 517 was significantly less effective than BZR 336 g (P < 0.05) (Table 3).

The bacterial strains also contributed to the potato plant growth stimulation during the growing seasons. In 2015, the seed treatment led to significant increase in plant height (P < 0.05) in the most cases (Table 4). Under the influence of both strains, in cv. Svitanok kievskiy the number of stems per plant significantly increased in 4, 6, and 10 weeks after planting. A similar result was observed in cv. Yuna (P < 0.05) (Table 4). The effect of these strains on plant height in 2016 was less for cv. Svitanok kievskiy (Table 4). Ten weeks after planting, treating the two potato cultivars with both strains over 2 years led to a significant increase in the morphometric characteristics of the plants (Table 4).

The pre-planting treatment of potato tubers with the bacterial strains had a positive effect on the quality of the daughter tubers. Thus, the effect of the strain BZR 336 g appeared to increase with the increasing biomass of the tubers. In 2015, the effect was increased by 37.2% and by 45.0% in cv. Svitanok kievskiy and cv. Yuna, respectively (Table 5). In 2016, the biomass was increased by 31.3–38.0% in both cultivars (Table 5). BZR 517 caused an increase in the medium fraction of tubers (Table 5). As a result, the use of the BZR 336 g strain provided a significant increase of 41.4% in daughter tubers’ biomass in the mid-early cv. Svitanok kievskiy in 2015. In 2016, biomass increased by 62.9%. The biomass was increased in early cv. Yuna by 22.4% and by 27.4% (P < 0.05), respectively (Table 5). Tubers pre-treated with the BZR 517 strain contributed less to the yield formation. In 2015, the daughter tubers’s biomass increased by 10% (cv. Svitanok kievskiy) and by 17.1% (cv. Yuna), respectively (Table 5). In 2016, the tuber biomass increased by 42.2% (cv. Svitanok kievskiy) and by 26.5% (cv. Yuna) compared with the control (P < 0.05) (Table 5). The total potato yield was significantly increased in both cultivars compared to the control (P < 0.05) (Table 5).

Red raspberry field trials

Raspberry cane disease incidence increased in the control differently during the 2 year study period. This increase was more serious during the 2015 season than in 2016 (Table 6) due to increased precipitation in 2015 (207 mm) compared to 2016 (135 mm).

B. velezensis strains were able to reduce the disease incidence. In 2015, we observed a 69.3% (P < 0.05) reduction in disease incidence 4 weeks after BZR 517 treatment on cv. Kirzhach; in cv. Kolokolchik, the reduction was 50.0% (P > 0.05). In 12 weeks, B. velezensis significantly (P < 0.05) reduced this value by 46.4% in cv. Kirzhach, and by 59.7% in cv. Kolokolchik (Table 6). In 2016, 4 and 12 weeks after the treatment both strains significantly reduced disease incidence (P < 0.05) to the same degree in cv. Kirzhach (Table 6). The disease incidence was reduced by more than 50% at 4 and 12 weeks in cv. Kolokolchik under the two treatment strains. This value was only significant at 12 weeks (P < 0.05).

In 2015, 4 weeks after treating the raspberry plants, we did not find a statistically significant difference (P > 0.05) in disease severity after both treatments on cv. Kirzhach (Table 6). The tendency in this reduction was observed only in the resistant cv. Kolokolchik (P > 0.05). A significant reduction in disease severity was observed using the B. velezensis 517 strain (69.1% on cv. Kirzhach and 47.8% on the resistant cv. Kolokolchik (P < 0.05)) (Table 6). Spur blight severity decreased by 54.1% in cv. Kirzhach, and 60% in cv. Kolokolchik (P < 0.05) compared to the control.

In 2016, the disease severity on the raspberry plant cv. Kirzhach significantly decreased by 70.8% due to both strains 4 weeks after treatment and by 52.1% 12 weeks after treatment. The disease severity significantly decreased by almost 50% in 12 weeks using the more resistant cv. Kolokolchik (P < 0.05) (Table 6). Fruit yield increased significantly in the cv. Kirzhach under the influence of both strains in 2015 and 2016 (P < 0.05) (Table 6), but there was no increase in this value in the cv. Kolokolchik in these years. This variation could be due to factors such as cvs. Kirzhach and Kolokolchik’s susceptibility to the pathogen or to different weather conditions in 2015 and 2016.