1.1. MATERIALS AND METHODS1.
2.1. Antagonistic activity testedcompounds on P.viticola 1.
2.1.1. CompoundsCommerciallyavailable formulation and technical grade, azoxystrobin (Amistar, activeingradient (a.
i.) 23 SC %, Syngenta India Ltd.); kresoxim methyl (technicalgrade a.i. 94 % and Ergon a.i.
44.3 % SC, TATA Rallis India); dimethomorph(a.i. technical grade 97.50 % and acrobat 50%WP, BASF India Ltd Mumbai India);mandipropamid (Revus a.i 23.4 % SC, Syngenta India); Metalaxyl-M(techinical grade a.i.
97.0 %, Syngenta India Ltd.); Cymoxanil (technicalgrade, a.i. 98.17 % Dupont, India); Mancozeb (Dhanuka M-45, 75 % WP, DhunkaAgritek Ltd., Gurgaon, India); Fosetyl-Al (Alitte 80 % WP, Bayer Crop ScienceLtd., Mumbai, India); Activated potassium salt of long chain phosphorus 96%(APSP 96%, Privi Nutrifight, Privi Life Sciences Pvt.
Ltd., Mumbai, India); Chitosan 70 % Degree of deacetylation (DD) ; Amisulbrom ( Kirari, a.i. 23 % SC Dhanuka , India). 1.2.1.
2. Leafdisc bioassaySensitivitytest was done following the leaf disk assay using the 24-well plate. The 5-7 thleaf from the apex of healthy growing shoots of Thomson Seedless werecollected, washed under running tap water and 15 mm discs were cut. Deaf disctreated disc treated with different chemical with different dose and placedupside down in a well containing 1 ml of solidified 0.
5 % water agar. Leafdisks were inoculated with 10 ?l sporangial suspensions containing 50,000sporangia ml-1 at the centre of the disc. Plates were incubated at22°C with alternating periods of 12 h light and dark. After six to eight days,the lesion diameter was measured and the percent infected leaf area wasdetermined, considering lesion in control as 100% infected area. EC50value was calculated by plotting the log10 fungicide concentrationagainst the percent infected leaf area.Table. List ofcompounds used for sensitivity assay and concentration Sr.No Compound Concentration ( µg/mL) 1.
Azoxystribin 0, 0.1, 1, 10, 100, 1000 2. Kresoxim methyl 0, 0.1, 1, 10, 100, 1000 3.
Dimethomorph 0, 0.1, 1, 10, 50, 100 4. Mandipropamid 0, 0.1, 1, 10, 50, 100 5. Amisulbrom 0, 0.1, 1, 10, 50, 100 6. Cymoxanil 0, 0.
1, 1, 10, 100, 1000 7. Metalaxyl 0, 0.1, 1, 10, 100, 1000 8. Mancozeb 0, 0.
1, 1, 10, 100, 1000 9. Fosetyl-Al 0, 100, 250, 500,750,1000 10. Chitosan 0, 1, 10, 100, 1000, 10000 11. APSP 0, 500, 1000, 2000, 4000, 10000 1.2.2. Molecular method fordetection of QoI and CAA fungicide resistant gene Plasmopara viticola1.2.
2.1. QoIResistance detected using Nested PCR-RFLP method: DNAwas extracted from seven P.viticola isolates by using RED Extract-N-Ampplant PCR kits (Sigma). PCR amplification carried out in a Gene Amp PCR System(Applied Biosystems) using first primer sense (5′-GCCGGTATCATGTTAGTAGT-3′) andantisense (5′-GACCTAAAGTATTAGGGTAG-3′) which corresponded to bases 106-125 and757-738 of P. viticola cytb gene, 20 µl final volumeFirst PCR reaction mixture containing 1 unit of Taq polymerase(Thermo Scientific), KCl 50 mM, Tris-HCl 10 mM, 2 µM of each primer, 0.
2 mM ofeach dNTP and 2 µl of DNA extractsolution, the programme : initial incubation at 94 °C for 5 min, followed by 95°C, 30 s, 55 °C, 45 s, 72 °C, 45 s, the final extension at 72 °C for 7 min. Nested PCR reaction 20 µl mixture containing, second primer sense(5′-GGGGTTTGTATTACGGATCT-3′) and antisense (5′-GGATTATTTGAACCTACCTC-3′) which corresponded to bases 295-314 and626-607 of cyt b gene respectively, with 1 unit of Taq polymerase 1, 50mM KCl, 10 mM Tris-HCl, 2 µM of each primer, 0.2 mM of each dNTP and 2 µl of first PCR product, programme : initialincubation at 95 °C for 3 min, followed by 35 cycle of 95 °C, 60 s, 57 °C, 60s, 72 °C, 60 s, the final extension at72 °C for 7 min. Nested PCR products were digested withrestriction enzyme Fnu4HI (NewEngland Biolabs, Ipswich, MA) RFLP analysis was performed on 2.0 % agarose gelby electrophoresis of digested PCR products. 1.2.
2.2. CAAresistance gene detection using PCR-RFLP methodPCR-restriction fragment length polymorphism(PCR-RFLP) method developed by Aoki et.al. (2011) used for G1105S mutation in PvCesA3gene in seven P.viticola isolates.PCR amplification was carried out using primer set. Sense primer5′-TTTGGCTTCTTCGTCATGAG- 3′, Anti-sense5′-CTGCACAAACACGACAATGT-3′ whichcorresponded to bases 3308-3328 and 3451- 3431 PvCesA3 gene of P.
viticola. 20ul of PCR reactionmixture containing 50 mM of KCl 10 mM Tris HCl (pH 8.0), 0.2 mM of each dNTP, 2um of each primer, 0.5 of Taq DNA polymerase and 2 ul of DNA extact, amplificationwas carried out in a Gene Amp PCR system. PCR programme, initial incubation at95° C for 5 min, 35 cycles using the following programme 95° C, 20s; 54°C, 20s;72 °C, 30s, the final extension was at 72 °C for 7 min. The PCR products of 144bp size of PvCesA3 gene were digestedwith restriction enzyme Alu I (ThermoFisher Scientific Inc.
) and the restriction digested product was analyze on 2.5% agarose gel by electrophoresis.1.
2.3. Field Experiment184.108.40.206. LocationsFieldexperiments were conducted in farmer fields at Dhondgavan wadi, District Nashik(20°.
2550″ N and 73°.8964″ E). Trials were conducted after fruitpruning from October 2014 to March 2015.1.
2.3.2. VarietyStudywas conducted on Vitis vinifera cultivar Thompson Seedless (clonalselections, Sonaka) which is the most popular table grape cultivars inMaharashtra. The vines were grown at spacing of 10′ between rows and 6′ betweenvines. The vines were trained on extended ‘Y’ trellises system. Methods offertilization, irrigation, and other cultural practices were carried out as perregular commercial practices.1.
2.3.3. ExperimentTable.Treatment List Treatment Fungicide Dose (g or ml / L) T1 Activate potassium salt of phosphorous (APSP) 96 % 2 T2 Mancozeb 75 % WP 2 T3 Fosetyl Al 80% WP 2 T4 Chitosan 70 % DD 2 T5 Amisulbrom 20% SC 0.
3 T6 Dimethomorph 50% WP 0.5 T7 Famoxadone 16.6% + Cymoxanil 22.1% 2.25 T8 Azoxystrobin 23 % SC 0.
5 T9 Kresoxim methyl 44.3 % SC 0.8 T10 Untreated Control Thevineyard was pruned on 28 October 2014 and ten treatments were evaluated forcontrol of downy mildew under natural epiphytotic conditions. Trials wereconducted in randomized block design with four replications that composed oftwo vines each. The treatment vines were surrounded by guard vines. The weatherdata was monitored on an automatic weather station and treatment applicationswere initiated as soon as the weather became conducive for disease development.
Subsequent sprays were given whenever the weather was found favorable fordisease development. Ten application ofeach treatment were applied with a knapsack sprayer i.e. 12, 16, 20, 22, 28,33, 36, 40, 45and 50 days after fruit pruning and water volume used for sprayswas calculated based on requirement of 1000 L ha–1. 1.
2.3.4. Assessment of downy mildew on leaves andbunches in the field.Diseaseincidence/ severity on leaves and bunches were recorded by adopting 0-4 diseaserating scale, where 0 = nil, 1 = trace to 25, 2 = 26 to 50, 3 = 51 to 75, and 4= more than 75 per cent leaf area infected (Horsfall and Heuberger, 1942;Horsfall and Barratt, 1986). Percent Disease Index (PDI) was calculated byfollowing formula of McKinney (1923).
Sum of numerical ratings× 100PDI = Number of leaves observed ×Maximum rating Theratings were recorded on ten leaves and a bunch on five randomly selected caneson each vine at different time intervals and designated as PDI 1, 2, 3, 4 and5. The harvestable yield was recorded at optimum physiological maturity byconsidering bunches which had market value. The PDI data was transformed usingarcsine transformation. AUDPC (area under the disease progress curve) wascalculated according to the equation of Campbell and Madden (1990):Wheren is the number of evaluations, y is disease severity, and t denoted the dayson which disease observations were recorded.1.2.3.
5. Statistical analysisStatisticalanalysis was done using SAS ver. 9.3. Means were compared by Tukey’sStudentized Range (HSD) Test.
The results significant at P 0.05% onlyare discussed.