The diseased organs (leaves, shoots, buds and fruits) of different varieties of apple (Malus domestica M.) were selected in the orchards and fruit farms of the Chui and Issyk-Kul provinces (Table 1) to isolate a pure culture of scab disease

pathogen (Venturia inaequalis (Cooke) Wint).

A sample consisted of different plant parts selected from one tree. Each sample contained at least five copies of plant parts with disease symptoms (the affected leaves, ovary, young shoots, the slices of damaged tissue branches and trunks), which were placed in a safe package with a label with the name of the culture, varieties, age, place, time of collection and an indication of the type of lesion.

Samples were placed in a 150 ml container with a lid, before a 30 ml phosphate buffer or phosphate-buffered saline was added. The container was placed on a rotary shaker and then incubated at 200 rev/min from 1.0 to 1.5 hours. For samples with symptoms, the appropriate amount of macerate was selected for qualifying analysis. For asymptomatic samples, a suspension concentrated by centrifugation was used. The sample was also frozen at −18˚C. Macerate was carefully poured directly into a 50 ml centrifuge tube, leaving the pulp in a container or pre-filtered through filter paper, then centrifuged for 10 minutes at 8000 g and for 15 minutes at an acceleration of 7000 g, at a temperature below 10˚C. The supernatant was discarded without damaging the sediment. The pellet was resuspended in 1 ml of phosphate buffer and transferred to a sterile microtube.

The following culture media were used for the primary isolation of scab pathogens:

1) Potato agar (g/distilled water): potatoes―200 g; Agar―15 g; water―1 L; pH―6.2;

2) Apple agar: chopped apple fruit weight―200 g; Agar―15.0; water―1 L; pH―6.5;

3) Czapek’s medium; pH―6.2;

4) Wort agar: wort―50 ml; agar―20 g; water―1.0 L; pH―6.5.

A special technique was used to determine the morphological features of Venturia inaequalis proposed by [18] [19] for other fungi. This technique is simple, cheap and does not require much time. It analyses the following morphological features: the size of spores, the diameter of the hyphae and culture function; the growth of the colonies and the colour of the pathogen colonies were studied accurately.

Biocontrol antagonistic microorganisms (Streptomyces and Trichoderma) from our laboratory collection were used in this study.

N.S. Egorov’s [20] perpendicular stroke method was used. The antagonist culture was plated on the diameter of Petri dishes and after four to five days, test cultures (Venturia inaequalis) were plated.

Also, the agar blocks method was used. Biocontrol antagonistic microorganisms (Streptomyces and Trichoderma) were plated onto the surface of the agar medium, formed during the growth of a continuous lawn in a Petri dish. Cultures are incubated at a suitable temperature for four to five days. Then, a sterile cork drill (6 - 8 mm diameter) was cut from the layer agar blocks and transferred to the surface of the agar medium, inoculated only that the test organism (Venturia inaequalis). Agar blocks were placed in increasing (lawn) up at an equal distance from each other and from the edge of the cup, connected tightly to the agar plate.

The potential of candidate antagonists to suppress the conidia production of Venturia inaequalis on infected leaves was tested on the local “Aychurok” variety of apple seedlings, highly susceptible to scab disease. Seedlings were sprayed with conidial suspensions of Venturia inaequalis (1 × 10⁵ ml⁻¹) until runoff and placed in a moist chamber consisting of a plastic tray enclosed by a transparent plastic top. After two days of incubation at 15˚C with diffuse light, the tops were removed from the trays and seedlings were further incubated for five days at 85% RH, 15˚C and with 16 hrs light per day. Thereafter, V. inaequalis-inocu- lated seedlings were sprayed with antagonist suspensions (containing 1 × 10⁶ spores or cells ml⁻¹) or water (containing 0.01% Tween 80) as control. Two seedlings were used for each replicate of each treatment. Touching of leaves of neighbouring plants or the polyethylene was avoided. Seedlings were grown for nine to 12 days at 15˚C, with 16 hrs light per day at 138 µE・s⁻¹・m⁻². From both seedlings of each replicate, the youngest five true leaves were carefully removed and put into Duran bottles (100 ml) containing 35 ml of tap water with 0.01% Tween 80. Bottles were shaken with a flask shaker and the concentration of conidia of V. inaequalis was determined for each suspension with the aid of a haemocytometer [21] .

Two rows of three-year-old apple trees of the Aychurok variety were used to test the antagonists in the Plant Research field at the Agriculture Faculty, KTU Manas, and Bishkek. For each experiment, two to four trees in two replicates were used. Within each replicate, two treatments were carried out.

Data were analysed following the GLIM program of the Royal Society of London [22] . Significant differences between the two mean values, due to different treatments or varieties and their interaction at a crop growth stage were computed by comparing their significant levels at P < 0.05.

To study the phenotypic characteristics of Venturia inaequalis isolates disaccharides as carbon sources, glucose, cellulose, mannitol and sorbitol were used. Natural isolates of Venturia inaequalis were able to utilize the glucose and cellulose, changing the colour of the liquid medium (with indicator) to red and the mannitol and sorbitol to yellow.

For the development of environmentally friendly local control measures against scab pathogen of fruit trees, the biotests were conducted using soil actinomycetes and Trichoderma virida against Venturia inaequalis. The following actinomycetes strains were used as antagonists: Streptomyces sр. С1-4; Streptomyces sр. Pr-3; Streptomyces sр. KK-4; Streptomyces sр. K-4; Streptomyces sр. K-7; Streptomyces sр. SK-3.3, Streptomyces sр. PАТ-3. Antagonist activity is measured by size of the lysis zones of the test object in mm (Figure 2).

As the results have shown, the antagonistic activity of Streptomyces strains against Venturia inaequalis was different. The C1-4 strain showed a strong antifungal effect against the phytopathogens; a lysis zone was 10.0 mm after 96 hours.

The Streptomyces strains, such as PAT-3 and Pr-3, inhibited the growth of the pathogen culture, using its colonies as a source of nutrients. Other strains, such as Streptomyces KK-4, K-4 and SK-3.3 were not active against Venturia inaequalis (Table 2).

Two strains (Streptomyces sp. C1-4 and Streptomyces sp. PAT-3) with strong antagonistic and hyper-parasitic effects were selected for the experiment in a liquid medium. A liquid culture of these antagonists was cultivated with Venturia inaequalis for 48 hours.

The microscopy study of a liquid culture showed a clear picture of the destruction of plant pathogenic conidia and conidiophores, as well as, attached to them, the branched mycelium and spores of antagonists-Streptomyces. The hyper-parasitism mechanism is accurately tracked through these micro-pictures (Figure 3).

Five days after artificial infection of highly susceptible apple seedlings of the local “Aychurok” variety, the first symptoms of the disease emerged on the youngest leaves: oil spots on the leaves’ surfaces. After seven days, necrotic or chlorotic lesions were noted on the edges of the leaves (Figure 4).

Five leaves from each tree were stripped and placed in 100 ml of sterile water. After shaking for one hour, 1 ml of suspension was planted on the nutrient media. Direct microscopy (1000×) of the diseased leaves and the colonies growing on the medium has found mycelium, conidia and conidiophores of the pathogen (Figure 5).

After the appearance of visible signs of the disease, seven days after infection, the seedlings’ leaves were abundantly treated with antagonist cultures. Ten days after the first treatment with antagonist cultures, five leaves of the apple seedling were removed for microscopy and planting on the relevant mediums. Pathogen colonies on a single leaf were counted and the average number of colonies was calculated (Figure 6).

As Figure 6 shows, conidia production of V. inaequalis on apple leaves has been reduced by up to 50% five days after treatment with Streptomyces sp. C1-4. After 10 days, it had been reduced by up to 60% in comparison with the control trees. After 15 days, a second treatment was carried out. On the 25th and 35th days after the second treatment, the number of conidia of the pathogen had been reduced by up to 35%.

On the leaves of seedlings which had been sprayed with Trichoderma virida suspension (1 × 10⁶ conidia ml⁻¹), conidia production of the pathogen decreased sharply, up to 30% after five days, reducing by up to 80% after 10 days in comparison with control seedlings. After 15 days. a second treatment was carried out. 25 and 35 days after the second treatment the number of pathogen conidia had reduced by up to 90% and 95% respectively (Figure 7).

There has been a pattern of preventing the progression of the disease caused by V. inaequalis. Before the use of antagonists, the diseased seedlings had an average of three to four infected leaves, with either a single necrotic lesion or several lesions scattered over the leaf’s surface. After the first and second treatments with Trichoderma virida, the number of infected leaves had not increased and progression of the disease had completely stopped.

When Streptomyces sp. C1-4 was used in some leaves, there was still some

disease progression. So, while tested Streptomyces sр. C1-4 strains have statistically significantly reduced conidia of V. inaequalis, their efficiency was lower than that of Trichoderma virida. Streptomyces sр. С1 strain has caused a significant reduction of V. inaequalis on leaves, which could be repeated in subsequent independent experiments.

To determine the shelf life of antagonist spores on the leaf surfaces of seedlings after treatment, the treated leaves of seedlings were removed and relocated to the surface of culture media with outer and inner surfaces. The leaves were treated with Streptomyces sp. C1-4 and planted on an ammonium starch medium (for actinomycetes), while the leaves treated with Trichoderma virida were planted on a Czapek’s medium.

The intensity and density of antagonist colonies growing around the leaves in the mediums showed their persistence on leaf surfaces after treatment. The longer they remained on the surface of diseased leaves, the greater their probability of being able to suppress the development of the pathogen.

Results have showed that the conidia of Trichoderma virida were obtained between 10 and 50 days after the second treatment, encouraging intensive growth around the leaves planted on the Czapek’s medium (Figure 8). In some dishes, the growth of one to two colonies of the Venturia inaequalis pathogen within the colonies of Trichoderma virida was observed; after a short time, the pathogen colonies were fully covered by colonies of the antagonist (Figure 9). This suggests that the Trichoderma virida has a double effect: as an antagonist and as a hyper-parasite. It could therefore suppress the development of pathogen mycelium on diseased leaves and use them as a nutrient source, producing chitinase and other enzymes that degrade the cell wall of the pathogen mycelium [23] .

Due to this effect, two applications of Trichoderma virida within 35 days completely stopped the disease in seedling leaves. After 40 days, the seedlings had recovered, with new leaves and some damaged leaves which remained on the crown continuing to function normally (Figure 10(a)). The Streptomyces sp. antagonists were shown to be inferior in effectiveness compared to Trichoderma

virida; nevertheless, it prevented the progression of the disease on the leaves. Seedlings continued to grow new shoots and leaves 50 days after the second treatment (Figure 10(b)).

In the case of applying a biological product based on Streptomyces sp. C1-4; after the second treatment, Streptomyces sp. C1-4 spores remained on the leaf surfaces of seedlings for between 10 and 25 days. During this period, the spores of the antagonist agent grew slowly, but this encouraged dense actinomycetes growth around the leaves planted on the medium (Figure 11). Streptomyces sp. also produces the enzyme chitinase and a number of antibiotic substances, destroying the structure of the phytopathogens mycelium [24] [25] [26] [27] .

Two rows of three-year-old Aychurok apple trees were used to test the antago-

nists in the field. For each experiment, three trees were used (replicates). In the experimental and control plots, the trees were sprayed with a conidia suspension of the pathogen V. inaequalis (1 × 10⁷ conidia / ml) after the complete opening of the leaves and before flowering. After seven days, disease symptoms started to appear on the leaves of the apple trees (Figure 12).

Immediately after the appearance of these symptoms, the diseased leaves of trees in experimental plots were sprayed with a conidia suspension of Trichoderma virida (1 × 10⁷ conidia/ml) and a spore suspension of Streptomyces sp. C1-4 (1 × 10⁸ spor/ml) until runoff at the bottom and upper side of the leaves. Treatment was carried out on a bright day after 17 hours; the air temperature was 18˚C - 20˚C. The disease development was monitored until 40 days after artificial infection was conducted. The damaged leaves and appearance of new leaves with disease symptoms were monitored. The development of the disease on leaves stopped 35 - 37 days after first treatment with the Trichoderma virida antagonist suspension, so that the damaged sections on the leaves did not change size further and the number of new leaves with diseased symptoms did not increase (Figure 13).