Source of bio-agents
The efficacy of 2 bacterial bio-agents and 4 fungal strains comparing with the fungicide Topas-100 (10.0% penconazole) for controlling squash powdery mildew disease under greenhouse conditions was estimated. Squash seeds ‘cv. Eskandarani’ were grown one seed per 15 cm pot. Bacillus subtilis and Paenibacillus polymyxa were isolated from the surface of healthy cucumber and squash leaves and identified according to Kamel (2003). The bacterial isolates were grown on nutrient broth (NB) for 48 h while fungal strains (Trichoderma harzianum, T. album, T. hamatum, and T. viridi) were obtained kindly from the Plant Pathology Research Institute., Agricultural Research Center (ARC), Giza, Egypt, and cultured on potato dextrose agar (PDA). Fungal strains (Trichoderma spp.) were grown for 10 days on PDA medium separately, then prepared spores and mycelial suspensions and adjusted to about 107 spore ml/l with sterilized water. while, B. subtilis and P. polymyxa were separately grown in (NB) medium in 250-ml flasks and kept on shaker for 3 days at 150 rpm. The pellets of used bacterial bio-agent were suspended in tap water and modified the number of cells to 109 cell ml/1 by using a hemocytometer slide (Hafez et al. 2018). For assessment with 6 bio-agents, plants were sprayed by distilled water as a negative control and sprayed with Topas-100 at the recommended dose of 0.25 ml/l for comparison.
Disease management
In vitro experiment
The experiments were carried out in the laboratory of the Department of Plant Pathology, Faculty Agriculture, Benha University, Egypt. Conidial spores of P. xanthii were obtained from young sporulating lesions, the lesions were softly shaken by glass bar, and new conidia were dropped on glass slides according to Nair et al. (1962): “Slides were previously cleaned by ethyl alcohol and air dried before covering with thin smears of 2% water agar, amended with filter-sterilized culture filtrate of the tested antagonist. Slides were placed on V-shaped glass rods in sterilized Petri-dishes, containing several layers of water-moistened filter papers”. Slides with conidia were incubated at 25 °C for 24 h under continuous light (Reifschneider et al. 1985). Conidia were considered to have germinated, if a germ tube, at least as long as the width, was produced (Menzies et al. 1991). Percentages of germination were calculated for 100 conidia on a slide at × 100 magnification. Three slides were examined for each treatment. Agar-free culture filtrate slides were used as a control treatment.
Greenhouse experiments
Squash seeds (Cucurbita pepo L., cv. Eskandarani) were sown in a clay soil at the rate of 3 seeds per hill under the experimental greenhouse conditions (28 °C, 85% RH, and 12 h photoperiod). The experiment was conducted in a randomly complete block design, with 3 replicated plots for each treatment. Squash plants in all plots received all the recommended agricultural practices. Natural infection with P. xanthii conidia, the causal agent of squash powdery mildew, was conducted under greenhouse conditions. Infected plants used as inoculum source (susceptible host Eskandarani) were uniformly inoculated by freshly collected conidia by placing heavy infected plants of squash which are sensitive to P. xanthii inoculation according to the method described by (Hafez et al. 2018). In addition, plants were sprayed with 2 bacterial bioagents, 4 fungal strains and the fungicide Topas-100 at recommended dose as mentioned before. Plants sprayed with sterilized tap water were used as control treatment.
Disease assessment
Squash plants after 45 days were examined periodically and the disease measures were determined using the devised scale (0–5) according to El-Ghanam et al. (2018) where 0 = no symptoms appear, 1 = 0.1 to 3% of leaf area covered by the infection, 2 = more than 3 to 10% of leaf area covered by the infection, 3 = more than 10 to 25% of leaf area covered by the infection, 4 = more than 25 to 50% of leaf area covered by the infection, 5 = more than 75% of the plant growth covered by the infection and the plants turned to be stunted.
The disease incidence and the severity of the disease were recorded using the following formula:
$$ \mathrm{Disease}\ \mathrm{severity}\ \left(\%\right)=\Sigma\ \left(\mathrm{nxv}\right)/5\mathrm{N}\times 100 $$
where n = number of infected leaves in each category, v = numerical values of each category, and N = total number of the infected leaves.
Disease incidence (%) = no. of infected plants/total no. of the plants assessed × 100
Biochemical assay
Estimation of total phenolic compounds
One gram of squash leaves sample was extracted by 10 ml of 80% methanol at 70 °C for 15 min. Then phenolic compounds were determined using the colorimetric method of analysis by Folin-Ciocalteu reagent described by Zieslin and Ben-Zaken (1993).
Determination of peroxidase
Peroxidase activity was determined according to the method described by Allam and Hollis (1972). Peroxidase activity was expressed as the increase in absorbance at 430 nm/gram fresh weigh/15 min.
Determination of polyphenol oxidase
Polyphenoloxidase activity was determined according to a modification of Ishaaya (1971). The phenol oxidase activity was determined as O.D. units ×103 at an absorbency of 405 nm.
Effect of use bio-agents on total yield/plant of squash plants
Average numbers and weights of fruits plant were recorded after harvesting fruits at marketable size. The squash fruits from each replicate of each treatment were collected twice a week, after 45 to 90 days from sowing, and the accumulated yield was expressed as the number and weight of fruits per plant.
Statistical analysis
Data were statistically analyzed using the (F) test and the value of LSD (at 5%) according to Gomez and Gomez (1984).
