Four isolates of the test pathogen were isolated from infected root, leaf, stem tissues of strawberry, bush bean, and pea. The infected plant specimens were washed with tap water and were cut into small pieces (5 mm). The pieces were surface sterilized with 1.0% chlorox for 5 min and then rinsed in sterilized water for three times. The sterilized pieces of the diseased tissue were placed on 1.5% water agar amended with streptomycin sulfate. After 3 days, hyphal tips of isolates were transferred into Potato Dextrose Agar (PDA) plates, which were incubated in the laboratory at room temperature (25 °C). Sclerotia were removed by elutriation and sieving onto screens with 600- and 150-micrometer openings [16]. Isolates from sclerotia were obtained by germinating sclerotia on water agar and transferring hyphal tips to PDA. The isolates were purified following hyphal tip technique [17], identified using standard key [18], and stored in PDA slants in test tubes at 10 °C.

Inocula of the four isolates of R. solani were prepared on autoclaved-moist wheat grain. Wheat grain was soaked in water for 12 h. After soaking, excess water was drained off and water-soaked wheat grains were poured into 500-mL Erlenmeyer flasks. The inoculum of each isolate was prepared in a separate flask. Five-millimeter-diameter mycelial discs were cut from the edge of 3-day-old PDA cultures of the pathogen. Ten to 12 mycelial discs from each isolate of the pathogen were added to the flasks containing autoclaved wheat grains in the flasks and incubated at 25 °C for 21 days. They were shaken by hand at 2–3 days interval for even colonization. The colonized wheat grain was air dried for 1 week and stored at 10 °C before being used as an inoculum of the pathogen.

The isolates of Rhizoctonia spp. were evaluated and compared with regard to their pathogenicity by the soil inoculation technique using strawberry plants in cultures out of pot. Earthen pots were filled with sterilized soil (500 g/pot). The inocula of R. solani isolates were thoroughly mixed with the soil at the rate of 20 g/kg soil. The treatments consisted of four isolates including an uninoculated control where only sterilized soil was used and were replicated three times. Three seedlings of the ‘BARI strawberry-1’ variety were sown in each earthen pot. The plant-containing pots were arranged in a completely random design. Disease development was observed regularly and recorded 15, 30, 45 and 60 days after sowing to estimate the lethal effect of pathogens at the vegetative and reproductive stages. The causal agent of mortality was confirmed after re-isolation of the pathogen from infected roots, leafs, and fruits of strawberry.

A total of 20 isolates of T. harzianum were screened against R. solani to select the most effective biocontrol agent. All the isolates were isolated from the rhizosphere and rhizoplane soils of bean, rice, chilly, tomato, sunflower, strawberry, and wheat. The soil dilution plate technique and root washing methods were used for isolation of the fungus isolates [17]. In the dilution plate technique, 10 g of composite soil collected from the rhizosphere and the rhizoplane of the selected plant species were taken in a 250-mL Erlenmeyer flask. Sterilized water was added to flasks (100 mL/flask). The flasks were agitated on a vortex for 2 min for thorough mixing and 1-mL sub-samples were transferred from each flask to another one containing 9 mL of sterile water. In this way, a 5-fold serial dilution of the soil suspension was prepared. Then, 0.1 mL of each dilution was incorporated into the PDA plate. The isolates of T. harzianum were purified in acidified water agar using hyphal tip culture technique; the cultures were maintained in PDA slants at 10 °C for further use.

An in vitro test was conducted to find out the comparative antagonistic potential of 20 selected Trichoderma isolates against R. solani isolate SR1 on PDA by dual culture technique [19]. Discs of mycelium (5-mm diameter) of each of the selected fungal isolates were cut from the edge of an actively growing fungal colony with a cork borer (5-mm diameter). One mycelial disc of individual isolates of Trichoderma and one disc of a test fungal pathogen were placed simultaneously on the edge of each PDA Petri plate at opposite directions. Three replicate plates were used for each isolate of Trichoderma and a test pathogen. The plates were arranged on the laboratory desks following a completely randomized design. The plates receiving only discs of R. solani served as controls. The plates were incubated in the laboratory at an ambient temperature of 25 ± 3 °C. Thereafter, inhibition percentages of the R. solani were calculated based on the growth of the pathogen on PDA plates following the formula suggested by Sundar et al. [20].

Three fungicides, namely Bavistin 50 WP (Carbendazim), Ridomil (Metalaxyl) and Provax-200 (Carboxin), were tested in vitro to evaluate their effect on colony growth and sclerotia formation of R. solani following the poison food technique on PDA Petri plates [19]. All fungicides were used at 100, 250, and 500 ppm. The details of the fungicides are presented in Table 1.

The effect of fungicides on radial growth and sclerotia formation of R. solani isolate SR1 was determined on a PDA medium. PDA was prepared by mixing infusion of 200 g of peeled potato, 20 g dextrose and 17 g of agar in 1000 mL of distilled water. The medium was cooked properly and poured into conical flasks at 100 mL per flask. Before solidification, the requisite quantity of individual fungicide was added to the medium to have concentrations of 100, 250, and 500 ppm. After thorough mixing with the fungicide, the medium was autoclaved at 121 °C under a pressure of 1 kg/cm for 20 min. Approximately 20 mL of melted PDA mixed with fungicides was poured into each Petri dish. After solidification, the plates were inoculated by 5-mm-diameter discs of 3-day-old PDA cultures of R. solani. Three replicated plates were used for each dose of every fungicide. Three replicated PDA plates received no fungicide and were also inoculated as controls. The inoculated plates were incubated at 28 ± 1 °C till the fungus covered the PDA in control plates, and sclerotia formation was recorded after 7 days of inoculation. The inhibition of radial growth was computed based on colony diameter on control plate using the formula shown below [20]. To determine the sclerotia formation at different doses of each fungicide, pathogens were evaluated through visual rating as no sclerotial crust, minimum sclerotial crust, moderate sclerotial crust, and maximum number of sclerotial crust by taking a 1-cm² area of the plate. The evaluation was replicated thrice.

To determine the effect of the fungicides on mycelial dry weight of R. solani, potato dextrose (PD) broth was used. Requisite quantity of each fungicide was added to the broth to have concentrations of 100, 250 and 500 ppm. Three replicated flasks were used for each dose of the three fungicides. The flasks were inoculated with mycelial discs of 5-day-old R. solani cultured on PDA. These mycelial disc were cut and put into the flask. Additional three flasks containing the PD broth receiving no fungicides were used as controls. The inoculated flasks were incubated at room temperature (25–28 °C) for 14 days. At the end of the incubation, the cultures in all flasks were filtered separately through pre-weighted filter paper. The dry weight of mycelium was determined after drying the mycelium on filter paper in an oven at 70 °C for 12 h. The dry weight of mycelium was obtained by subtracting the weight of the filter paper from the combined weight of the filter paper and of mycelium. Inhibition of mycelia dry weight was measured comparing with the control flasks.

Another in vitro test was conducted to determine the effect of organic amendment on the growth of R. solani following the poison food technique [19]. Hundred grams of each organic amendments viz. mustard oilcake (Brassica napus), coconut oilcake (Cocos nucifera), til oilcake (Sesame indicum), and soybean oilcake (Glycine max) were added in 1000 mL of water and preserved in earthen pots for 2 weeks. A requisite quantity of individual oilcake extracts was added to 100-mL conical flasks with PDA medium to have concentrations of 1, 2 and 3% (v/v). After thorough mixing of oilcake extracts, the medium was autoclaved and approximately 15 mL of melted PDA with extracts were poured into each Petri dish. After solidification, the plates were inoculated by placing 5-mm discs of 3-day-old PDA cultures of R. solani. Inhibition of radial growth was computed on colony diameter on control plate using the same formula [20].

The field experiment was conducted in Randomized Block Design with nine treatment combinations replicated three times. The size of the individual plots was 1 m × 1.5 m and the plot–plot distance was 0.5 m. Recommended doses of fertilizers (30:50:20 for N-P-K) were applied. Muriate of potash was sprayed @ 3% solution at the flower bud initiation stage between 30 and 35 days after sowing with a hand sprayer. Field application of T. harzianum isolate STA7 was done by broadcasting on the surface of plots on the day before planting @ 2.0% (WAV) by growing on autoclaved wheat grain. The procedure for the preparation of inocula of Trichoderma was the same as that described earlier for the preparation of inocula of R. solani. Seedlings of ‘BARI-1’ strawberry variety were collected from the Bangladesh Agricultural Research Institute and three seedlings were transplanted in each plot. Three replications were used for each treatment. Provax-200 50WP was applied as foliar spray at 10 days after transplanting using a hand sprayer. The Trichoderma isolate STA7, mustard oil cake and Vitavax-200 was applied in different combinations in nine different treatments viz. T₁ = pathogen + Trichoderma isolate STA7; T₂ = pathogen + Provax-200; T₃ = pathogen + mustered oil cake; T₄ = pathogen + Trichoderma isolate STA7 + Provax-200; T₅ = pathogen + Trichoderma isolate STA7 + mustered oil cake; T₆ = pathogen + mustered oil cake + Provax-200; T₇ = pathogen + Trichoderma isolate STA7 + mustered oil cake + Provax-200; T₈ = healthy seedling in a sterilized soil (healthy control); T₉ = healthy seedlings in a pathogen-inoculated soil (diseased control). Data on the mortality of seedlings were recorded at the vegetative and reproductive stages. Diseased seedlings were counted every alternate day and continued for 28 days after planting. To determine the cause of death of the seedlings, the dead seedlings were uprooted gently. The causal pathogens associated with the dead seeds and seedlings were isolated and identified for the confirmation of the causal agents. The data were analyzed for ANOVA using MSTAT-C program, and mean separations were performed by DMRT.

Four isolates of R. solani were isolated from infected root, leaf, and stem tissues of strawberry. The isolated R. solani were mostly brown in color, with dense mycelial growth with zonation, but the number of sclerotia formation varied. Based on the number of sclerotia, R. solani isolates were rated as +, ++, +++ and ++++ sclerotial groups, and four R. solani isolates, namely SR1, SR16, SR7 and SR13 representing the lower number of sclerotia to the maximum number of sclerotia, respectively, were randomly selected for the pathogenicity test against strawberry plants in cultures out of pot.

The pathogenecity test of R. solani isolates was done against strawberry plants in cultures out of pot (Table 2). All the isolates developed black root rot disease, causing more than 70% of mortality in strawberry plants. The isolates SR1 showed the highest root rot disease development, causing 95.47% plant mortality followed by SR13, SR7 and SR16, showing 86.63, 83.74 and 73.15% plant mortality, respectively. Based on the present findings, isolates SR1 was selected for further study.

To observe the antagonistic effect of T. harzianum against R. solani, both were tested on PDA using the dual culture technique (Table 3). All 20 isolates of T. harzianum showed more than 50% of inhibition of the radial growth of the test pathogen compared to control. Among the isolates, STA7 showed significantly highest inhibition (71.97%) of the radial growth, followed by STA9 (68.91%) and STA5 (66.85%). The lowest 51.24% radial growth inhibition was observed at STA17. The isolate STA7 was selected for further study.

All the selected concentrations of Provax-200 inhibited above 85% of radial growth and mycelia dry weight of R. solani, even at the lowest concentration (Table 4). The number of sclerotia was also negligible at the lowest concentration of Provax-200. Fungicide Ridomil appeared to be highly ineffective in inhibiting radial growth, mycelia dry weight, and sclerotia formation. Bavistin appeared as moderately effective against R. solani. Complete inhibition of the radial growth of R. solani was observed at the higher concentrations, i.e. 250 and 500 ppm, of Provax-200. The highest concentration of Bavistin was found to inhibit about 90.37% of the radial growth and 95.00% of the mycelia dry weight of the test pathogen, with a minimum number of sclerotia.

The effects of organic amendments on the inhibition of the hyphal growth of R. solani are presented in Table 5. The study revealed that the maximum inhibition (59.66%) of the hyphal growth of R. solani was obtained in mustard oil cake at the highest concentration (3%), which is significantly superior to all other amendments. At the highest 3% concentration of till oil cake and 2% concentration of mustard oil cake, inhibition rates of the radial growth of R. solani of 56.85% and 54.55%, respectively, were obtained.

An integrated control approach has been made to control strawberry fruit rot. A total of three pathogens, Colletotrichum gloeosporioides, Fusarium oxysporum, and R. solani, were isolated from the infected leaves, out of which C. gloeosporioides were the most predominant in T₉, T₃ and T₈. The second highest 26.33% C. gloeosporioides was isolated from infected leaves in T₃, where only healthy seedlings were sown without any control measure. A significant number of the least infected leaves and of the healthiest leaves were observed at T₇. Mustard oil cake appeared to be less effective in controlling the leaf diseases and significantly inferior to other treatments. The integration of different component in the treatment T₇ also appeared significantly superior in controlling leaf diseases of strawberry in comparison to any individual or mixed component of other treatments. Integrated management options (T7) produced healthy leaves and fruits (Fig. 1), while in the disease control treatment, dead plants (Fig. 2) were observed.

The effect of different combinations of Trichoderma, fungicide and mustard oil cake on strawberry leaf and fruit health in field conditions are given in Tables 6 and 7. Among the three pathogens, associated with fruit rot disease of strawberry, C. gloeosporioides was found as the most predominant in each case. The highest 87.00% fruit rot was observed in T₉ followed by T₃ (75.03%). In T₈, only healthy seedlings were grown, significant anthracnose symptoms developed on the fruit. A significantly highest percentage of healthy fruits (76.33%, Fig. 1) was obtained in T₇ followed by T₄. In fact, mustard oil cake alone seems to be less effective in controlling fruit rot disease.