Collection and maintenance of the infected plants
As the leaf crinkle disease is highly seed transmissible, symptomatic plants in the experimental farms at Tamil Nadu Agricultural University were identified, tagged during summer 2020 and seeds from those infected plants were collected at the time of harvest and were sown in the glasshouse during
kharif 2020. Symptomatic leaves developed were used as inoculum for further use.
Optimization of mechanical inoculation protocol
The present protocol was optimized by modifying the method described by
Biswas et al., (2012) in the inoculation procedure by adding abrasives and additives to obtain high symptomatic transmissibility and to attain the infection on urdbean plants at the earliest. The present study was performed during
rabi 2020-21 using VBN 8 cultivar of urdbean where in, two methods of incubating sprouted seeds in the leaf crinkle infected leaf sap were followed (i) with pin prick of the sprouted seed and (ii) without pin pricking, where in the virus suspension was grinded with 0.05 M PPB (pH 7.0) with different combinations of additives and abrasives. In pin prick method of sap inoculation, 2-3 gentle pricks were made to the emerging meristem by wounding with a fine needle (Fig 1A).
To evaluate the effect of antioxidants, four different combinations of inoculum was prepared
viz., 0.05M PPB (pH 7.0) containing 0.2% Sodium Sulphite (SS), PPB containing 0.01M 2-Mercaptoethanol (2-ME) and PPB with 0.15% SS + 0.01M 2-ME. Similarly, to determine the role of abrasives on percent transmission of virus, the sap was divided into three parts
i.e., only 2% carborundum (320 grit), only 1% celite (545) and both 2% carborundum + 1% celite. Seeds soaked in infected leaf suspension were sown in pots (20 x 20 cm) filled with potting mixture consisting of red soil, farm yard manure, vermiculite and sand in the ratio of 3:2:2:1 (W/W). Total 30 plants for each parameter (6 pots: 5 plants/pot) for three replications along with respective healthy control were maintained under 28±2°C and percent transmission was recorded from 14 to 55 Days post inoculation (DPI).
Validation of the optimized protocol
Following the optimization of the mechanical inoculation to urdbean by sprout seed abrasion method, the experiment was extended for protocol validation. Nine cultivated urdbean genotypes
viz., VBN4, VBN5, VBN6, VBN9, VBN10, VBN11, MDU-1, CO 6 including the susceptible check variety CO5 were obtained from National Pulse Research Centre, Vamban, Tamil Nadu and Department of Pulses, Tamil Nadu Agricultural University (TNAU). All the sprouted seeds of different genotypes were mechanically inoculated with standardised method and buffer control (without any inoculation) was maintained for all the genotypes separately in the glasshouse.
Statistical analysis
Analysis of variance was performed on the data (ANOVA). For analysis, the per cent data was arcsine transformed. A completely randomised design was used to calculate the standard error of mean (SEm), standard error of deviations (SEd) and critical difference (C.D.) values (
Gomez and Gomez, 1984).