Plant material and experimental design
Squash seeds (Cucurbita pepo L., cv. Eskandarani) were sown in a clay loam soil and grown under greenhouse conditions at Sakha Agricultural Research Station, Agricultural Research Center, Egypt. The experiment was conducted in a randomly complete block design (RCBD), with three replicated plots for each treatment. Squash seeds were cultivated at spacing of 60 cm between plants in rows and 80 cm between plant rows at the rate of three seeds “per hill.” The seedlings were thinned to one per hill after 15 days after sowing (DAS). Squash plants in all plots received all the recommended agricultural practices.
Tested treatments
The efficacy of the bio-agents (two fungal and five bacterial strains) comparing to the fungicide (Topas-100) for controlling squash powdery mildew disease was tested under greenhouse conditions. Bacillus subtilis, Bacillus pumilus, and Bacillus chitinosporus were previously isolated from the surface of healthy cucumber and squash leaves and identified according to Kamel (2003), while B. megaterium, Bacillus polymexa, and fungal strains (Trichoderma harzianum and Trichoderma viridi) were obtained from the Microbiology Department, Soil, Water and Environment Research Institute, Agriculture Research Center (ARC), Giza, Egypt. Trichoderma harzianum and T. viridi were grown on PDA medium for 10 days, then their spores and mycelial suspensions were separately prepared and adjusted to about 107 spore ml−1 with sterilized water, using a hemocytometer slide, while B. subtilis, B. polymexa, B. pumilus, B. chitinosporus, and B. megaterium were separately grown in nutrient liquid medium in 250-ml flasks and kept on an orbital shaker at 150 rpm for 3–4 days. The pellets of each bacterium were separately suspended in tap water, and number of cells was adjusted to 109 cell ml−1, using a hemocytometer slide. For comparison with the tested bio-agents, plants were sprayed by distilled water as a negative control (C). In addition, widely used fungicide, Topas-100® (10.0% Penconazole “w/v” [(R, S-1-(2-(2, 4-dichlorophenyl) -Q pentyl)-1H-1, 2, 4-triazole]), was sprayed, at the recommended dose of 0.25 ml l−1 as a positive control.
Spore germination of Podosphaera xanthii as affected by tested bio-agents
Conidial spores of squash powdery mildew were obtained from young sporulating lesions. To avoid the old unviable conidia, the lesions were gently shaken by a glass rod, and 24 h later (as recommended by (Godwin et al. 1987), new conidia were deposited 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) before microscopically examined, at × 100 magnification, to determine the spore germination. 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. Three slides were examined for each treatment. Agar-free culture filtrate slides were used as a control treatment.
Pathogenic fungal inoculation
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.
Disease assessment
The disease severity of squash leaves at 45 DAS was assessed as described by Descalzo et al. (1990). Nine random plants were used for each replicate. Severity of powdery mildew was estimated based on the percentage of affected leaf area. The mean of area under disease progress curve (AUDPC) for each replicate was calculated as follows (Pandey et al. 1989).
AUDPC = D [1/2(Y1 + Yk) + (Y2 + Y3 + ……. . + Yk − 1)]
where D = time interval; Y1 = first disease severity; Yk = last disease severity; Y2, Y3, and Yk−1 = intermediate disease severity.
Biochemical assays of antioxidant enzymes
For enzyme assays, 0.5 g of leaf material collected after 15 days from treatment with bio-agents were homogenized at 0–4 °C in 3 ml of 50 mM TRIS buffer (pH 7.8), containing 1 mM EDTA-Na2 and 7.5% polyvinylpyrrolidone. The homogenates were centrifuged (12,000 rpm, 20 min, 4 °C), and the total soluble enzyme activities were measured spectrophotometrically in the supernatant (Hafez et al. 2014a). All measurements were carried out at 25 °C, using the model UV-160A spectrophotometer (Shimadzu, Japan). The enzymes’ assay was tested three times. Activity of catalase (CAT) was determined spectrophotometrically according to Aebi (1984). Polyphenol oxidase (PPO) activity was determined according to the methods described by Malik and Singh (1980). Changes in the absorbance at 495 nm were recorded every 30 s intervals for 3 min. Enzyme activity was expressed as the increase in absorbance min−1 g−1 fresh weight. Activity of peroxidase (POX) was directly determined of the crude enzyme extract, according to a typical procedure proposed by Hammerschmidt et al. (1982). Changes in absorbance at 470 nm were recorded at 30 s intervals for 3 min. Enzyme activity was expressed as increase in absorbance min−1 g−1 fresh weight.
Microscopic examination
The infected leaves, treated by the most effective bio-agents, were selected to investigate the growth status of powdery mildew fungus after 24 h of treatment. Control plants were microscopically observed for comparison and for any differences on the tested pathogen P. xanthii. For scanning electron microscopy (SEM), sections (0.5 cm) were taken from untreated and treated leaves. The samples were prefixed in mixture of 2.5% glutaraldehyde and baraformaldehyde at room temperature for 24 h. The fixative was washed three times by phosphate buffer solution (pH 7.2–7.4). The specimens were postfixed in osmium tetroxide (1% w/v in phosphate buffer 0.07 M, pH 7.2) at room temperature for 1.5 h. After washing with phosphate buffer solution three times, the samples were hydrated in ethanol series (30 and 50% for 30 min, and 70% for 24 h), then dried at critical point of CO2 (Balzers CPD-020) and covered with gold (30 nm) in a sputter coater (Balzers SCD-040). The specimens were examined and photographed by the TESLA BS-300 electron microscope. In all processes, SEM observations and photography were carried out at Electron Microscope Unit, Fac. of Medicine, Tanta Univ., Egypt.
Chlorophyll content and plant growth assessment
Plant growth parameters were determined in the experimental plants at 60 DAS. Total chlorophyll contents, using the SPAD-501 portable leaf chlorophyll meter (Minolta Corp) for greenness measurements in the fifth apical fully expanded leaf (Yadava 1986). Average number and weight of fruits per plant were measured by harvesting fruits at marketable size. The squash fruits from each replicate of each treatment were collected twice a week, from 45 to 90 DAS, and the accumulated yield was expressed as the number and weight of fruits per plant. Leaf area was determined, using the dry weight method. The leaves of a plant were cleaned from dust and then representative 10 disks were taken, using a test tube known for its area (1.76 cm2). The leaf area was calculated, using the following formula:
Leaf area = (weight of leaves × 10 n)/weight of 10 disks
where n = the area of one disk.
Statistical analysis
The data were subjected to statistical analysis by ANOVA, using wasp software (Web Agriculture Stat Package). The values presented are the means of all measurements, and comparisons of means were determined by Duncan’s multiple range tests, at P ≤ 0.05 (Gomez and Gomez 1984).
