Antagonistic microorganisms
Different biocontrol agents, i.e., Trichoderma harzianum, T. hamatum, and T. viride were kindly obtained from the Central Lab. of Organic Agriculture, CLOA, ARC, Giza, Egypt.
Commercial biofungicide (blight stop)
The recommended biofungicide which consists of (Trichoderma spp. 30 × 106), obtained from CLOA, was used for comparison.
Micronic sulfur
The recommended fungicide micronic sulfur was used also as a comparison with the tested treatments. Manufacture by Kz, Egypt, consistence of Micronized Soreil/ Samark 70% wp sulfur.
Preparations of the biocontrol agents
Trichoderma harzianum, T. hamatum, and T. viride were prepared as follows: each of the antagonistic fungi was grown for 10 days at 25 ± 2 °C on a liquid Gliotoxin fermentation (GF) medium under complete darkness conditions (Ahmed 2013). All cultures were individually blended in an electrical blender for 2 min, used as suspension at concentration of (30 × 106spores/ml) with a dilution 1:100, and then mixed with 5% Arabic gum and 5% potassium soap and wetting the leaves, using a sprayer to increase adhesive capacity and improve distribution of bioagent on the surface of treated plants for all preparations and blight stop the commercial biocide. Mixtures of the three Trichoderma spp. were mixed at the rate of 1:1.
Field applications
The study was conducted for two successive seasons (2016 and 2017) in a private vineyard, called Electricity Station Farm, which is registered at ECOA control body, as a full organic farm, under the code number A812, Wadi El Faregh, Wadi El-Natroun, El Beheira Governorate, Egypt to evaluate using organic products to reduce powdery mildew of Thompson seedless grapevines and their reflects on vine growth, fruits yield, quantity and quality. The chosen vines were 12 years old, grown in a sandy loam soil, spaced at (1.5 × 3.0 m) apart and irrigated, using drip irrigation system, trained to bilateral cordon with spur pruning and trellised by the “Y” shape system. The vines were pruned at the end of January in both seasons according to Fawzi et al. (2010).
All vines received the same agricultural practices applied in the vineyard. Every five vines acted as a replicate and each three replicates were treated by T. harzianum, T. hamatum, T. viride, their combinations and blight stop were used as a suspension at the concentration of (30 × 106spore/ml) with a dilution of (1:100), while micronic sulfur was used at the rate of (250 g/100 L). Arabic gum and potassium soap were added at (5%) for each spray. Only distilled water was applied at the same period as control. Foliar sprays of the treatments were applied at a regular interval and repeated two times every other week. All treatments received the same normal agricultural practice till harvest in May.
Plant parameters adopted to evaluate the treatments
Disease assessment
For powdery mildew assessment, the evaluation on leaves was carried out 15 days post the last application.
% Disease incidence (DI) was determined according to the formula:
$$ \mathrm{DI}\%\kern0.5em =\kern0.5em \frac{\mathrm{Number}\kern0.5em \mathrm{of}\kern0.5em \mathrm{infected}\kern0.5em \mathrm{leaves}}{\mathrm{Total}\ \mathrm{nostudied}\ \mathrm{leaves}}\kern0.5em \times \kern0.5em 100 $$
The disease severity was recorded immediately pre each spray application and 7 days post the last spray. It was rated as a 0–5 scale, where 0 = no infection, 1 = 1–10% leaf area infected with powdery mildew, 2 = 11–25%, 3 = 26–50%, 4 = 51–75, and 5 = 76–100% (Horsfall and Heuberger 1942). The ratings were converted to percent disease index (PDI), using the formula given by Wheeler (1969):
\( \mathrm{D}.\mathrm{S}.\mathrm{I}\%\kern0.5em =\kern0.5em \frac{\sum \left(\mathrm{n}\ \mathrm{xv}\right)}{\mathrm{ZN}}\kern0.5em \times \kern0.5em 100 \), where D.S.I = disease severity index, n = number of leaves in each category, v = numerical value of each category, z = numerical value of highest category and N = total number of leaves in the sample.
In addition to plant height, no. of leaves/plant, fresh weight/plant, and onion bulb yield as kg/plot was weighted at the end of each season.
Yield (kg/vine)
At harvesting, when TSS% of berries reached about (16–17%) in control, 6 clusters/vine were weighted and an average cluster weight was multiplied by the number of clusters/vine to calculation average of yield/vine.
Biochemical characteristics (properties)
Representative random samples of six bunches/vine were harvested at maturity, when total soluble solids (TSS) reached about (16–17%) according to Tourky et al. (1995).
All bio-chemical analysis were conducted at the Central Lab. of Organic Agriculture; CLOA; Agricultural Research Center, ARC; Giza Governorate, Egypt.
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1.
Total soluble solids content (TSS %): fresh preparation of the berry juice samples was used for determination of the total soluble solids (TSS), using a hand refractometer (Model BX-1 and Brix 0–32%).
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2.
Total acidity percentage was determined according to A.O.A.C (2003).
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3.
TSS/acid ratio was calculated by dividing the percentage of TSS on the value of total acidity.
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4.
Total sugars (%) were determined according to Sadasivam and Manickam (1996)
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5.
Total anthocyanin: total anthocyanin of berries skin (% in mg/100 g fresh weight) was determined by using the spectrophotometer at wave length of 535 mm, according to the method of Husia et al. (1965).
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6.
Total phenols: total phenols (mg/g berries as gallic acid equivalent/g FW) was based on Folin-Ciocalteau reagent (Zieslin and Ben-Zaken 1993). Changes in sugar and phenol contents in infected table grapes, 35 days post the storage time were considered. Determinations of phenolic compounds were calorimetrically determined by a spectrophotometer at 760 nm as described by Snell and Snell (1953).
Statistical analysis
The complete randomized block design was adopted for the experiment. Data were subjected to statistical analysis and compared according to the least significant difference (LSD) as mentioned by Snedecor and Cochran (1989). Averages were compared using the new LSD values at 5% level.