1.1. Agritek Ltd., Gurgaon, India); Fosetyl-Al (Alitte 80

1.1.        
 MATERIALS AND METHODS

1.2.1.  
Antagonistic activity tested
compounds on P.viticola

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1.2.1.1.      Compounds

Commercially
available formulation and technical grade, azoxystrobin (Amistar, active
ingradient (a.i.) 23 SC %, Syngenta India Ltd.); kresoxim methyl (technical
grade 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 (technical
grade, a.i. 98.17 % Dupont, India); Mancozeb (Dhanuka M-45, 75 % WP, Dhunka
Agritek Ltd., Gurgaon, India); Fosetyl-Al (Alitte 80 % WP, Bayer Crop Science
Ltd., 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.   Leaf
disc bioassay

Sensitivity
test was done following the leaf disk assay using the 24-well plate. The 5-7 th
leaf from the apex of healthy growing shoots of Thomson Seedless were
collected, washed under running tap water and 15 mm discs were cut. Deaf disc
treated disc treated with different chemical with different dose and placed
upside down in a well containing 1 ml of solidified 0.5 % water agar. Leaf
disks were inoculated with 10 ?l sporangial suspensions containing 50,000
sporangia ml-1 at the centre of the disc. Plates were incubated at
22°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 was
determined, considering lesion in control as 100% infected area. EC50
value was calculated by plotting the log10 fungicide concentration
against the percent infected leaf area.

Table. List of
compounds 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 for
detection of QoI and CAA fungicide resistant gene Plasmopara viticola

1.2.2.1.   QoI
Resistance detected using Nested PCR-RFLP method: DNA
was extracted from seven P.viticola isolates by using RED Extract-N-Amp
plant PCR kits (Sigma). PCR amplification carried out in a Gene Amp PCR System
(Applied Biosystems) using first primer sense (5′-GCCGGTATCATGTTAGTAGT-3′) and
antisense (5′-GACCTAAAGTATTAGGGTAG-3′) which corresponded to bases 106-125 and
757-738 of   P. viticola cyt
b gene, 20 µl  final volume
First 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 of
each dNTP and 2 µl  of DNA extract
solution, 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 and
626-607 of cyt b gene respectively, with 1 unit of Taq polymerase 1, 50
mM KCl, 10 mM Tris-HCl, 2 µM of each primer, 0.2 mM of each dNTP and 2 µl  of first PCR product, programme : initial
incubation at 95 °C for 3 min, followed by 35 cycle of 95 °C, 60 s, 57 °C, 60
s, 72 °C, 60 s, the final extension  at
72 °C for 7 min. Nested PCR products were digested with
restriction enzyme Fnu4HI (New
England Biolabs, Ipswich, MA) RFLP analysis was performed on 2.0 % agarose gel
by electrophoresis of digested PCR products.

1.2.2.2.   CAA
resistance gene detection using PCR-RFLP method

PCR-restriction fragment length polymorphism
(PCR-RFLP) method developed by Aoki et.al. (2011) used for G1105S mutation in PvCesA3
gene in seven P.viticola isolates.
PCR amplification was carried out using primer set. Sense primer
5′-TTTGGCTTCTTCGTCATGAG- 3′, Anti-sense5′-CTGCACAAACACGACAATGT-3′ which
corresponded to bases 3308-3328 and 3451- 3431 PvCesA3 gene of P.
viticola. 20ul of PCR reaction
mixture containing 50 mM of KCl 10 mM Tris HCl (pH 8.0), 0.2 mM of each dNTP, 2
um of each primer, 0.5 of Taq DNA polymerase and 2 ul of DNA extact, amplification
was carried out in a Gene Amp PCR system. PCR programme, initial incubation at
95° 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 144
bp size of PvCesA3 gene were digested
with restriction enzyme Alu I (Thermo
Fisher Scientific Inc.) and the restriction digested product was analyze on 2.5
% agarose gel by electrophoresis.

1.2.3.        
Field Experiment

1.2.3.1.   Locations

Field
experiments were conducted in farmer fields at Dhondgavan wadi, District Nashik
(20°.2550″ N and 73°.8964″ E). Trials were conducted after fruit
pruning from October 2014 to March 2015.

1.2.3.2.   Variety

Study
was conducted on Vitis vinifera cultivar Thompson Seedless (clonal
selections, Sonaka) which is the most popular table grape cultivars in
Maharashtra. The vines were grown at spacing of 10′ between rows and 6′ between
vines. The vines were trained on extended ‘Y’ trellises system. Methods of
fertilization, irrigation, and other cultural practices were carried out as per
regular commercial practices.

1.2.3.3.                    
Experiment

Table.
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

 

The
vineyard was pruned on 28 October 2014 and ten treatments were evaluated for
control of downy mildew under natural epiphytotic conditions. Trials were
conducted in randomized block design with four replications that composed of
two vines each. The treatment vines were surrounded by guard vines. The weather
data was monitored on an automatic weather station and treatment applications
were initiated as soon as the weather became conducive for disease development.
Subsequent sprays were given whenever the weather was found favorable for
disease development.  Ten application of
each 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 sprays
was calculated based on requirement of 1000 L ha–1.

1.2.3.4.    Assessment of downy mildew on leaves and
bunches in the field.

Disease
incidence/ severity on leaves and bunches were recorded by adopting 0-4 disease
rating 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 by
following formula of McKinney (1923).

                              Sum of numerical ratings
×  100

PDI   =            

                  Number of leaves observed ×
Maximum rating  

 

The
ratings were recorded on ten leaves and a bunch on five randomly selected canes
on each vine at different time intervals and designated as PDI 1, 2, 3, 4 and
5. The harvestable yield was recorded at optimum physiological maturity by
considering bunches which had market value. The PDI data was transformed using
arcsine transformation. AUDPC (area under the disease progress curve) was
calculated according to the equation of Campbell and Madden (1990):

Where
n is the number of evaluations, y is disease severity, and t denoted the days
on which disease observations were recorded.

1.2.3.5.  
Statistical analysis

Statistical
analysis was done using SAS ver. 9.3. Means were compared by Tukey’s
Studentized Range (HSD) Test. The results significant at P 0.05% only
are discussed.