Collection of disease samples :
Asalio is cultivated medicinal crop. Fristly, I took permission through head of plant pathology department of Rajasthan College of Agriculture, Udaipur from Asalio’s farmers to enter selected their farm around the Udaipur district in Rabi season of 2017. Asalio Alternaria leaf spot disease samples were collected from severely Alternaria leaf spot infected farmer’s field during survey of department during disease inspection. These farmers were growing local land races of Asalio on their fields as a cultivated crop. The fungal culture of pathogen recovered from these samples was used in present study.
Isolation, purification and pathogenicity tests of the pathogen.
Fungal pathogen was isolated from collected diseased samples of Asalio using standard methodology on potato dextrose agar (PDA) medium. Small bits of infected portions were surface sterilized for 1 minute in mercuric chloride solution (0.1%) and washed thrice in sterilized distilled water under totally aseptic conditions in a laminar air flow. These were then dried by keeping in two folds of sterilized filter papers then aseptically transferred to PDA in Petri plates. The plates were incubated at 27±1ºC for 7-8 days and identified by ITCC (New Delhi) as a Alternaria alternata culture which caused Alternaria leaf spot disease in Asalio and the culture morphological characters were same as previously reported by Utikar and Padule (1980)10. He reported that conidiophores of A. alternata were simple, light brown, variable in length ranging from 17.10 to 61.56 μm and mostly 2-3 septate rarely 4-5 septate. Conidia were found light to dark brown in colour, uniform with 0-2 longitudinal septa and 1-6 transverse septa, and variable in shape and size, mostly oval shape with rudimentary beak and in size measureing about 10.26-77.52 x 4.56-14.82 μm. Simmons and Roberts (1993)11 observed three-dimensional sporulation patterns of A. alternata in electron microscope at 50 magnification.
For proved pathogenicity and fulfilling the Koch’s postulates an inoculum with load of 1×103 conidia ml-1 concentration of the spores was inoculted in pot grown Asalio. The typical concentric ring symptoms appeared within 7-10 days after inoculation and the symptoms are same as previously reported by Melkania (1980)12 that Alternaria alternata caused Alternaria leaf spot on leaves of cress at Almora (H.P.) in India.
The fungal culture of pathogen re covered from these samples was used in present study. The pot experiments were carried out at cage house of Plant Pathology, Rajasthan College of Agriculture, Maharana Pratap University of Agriculture and Technology, Udaipur, the study on “Management of Alternaria leaf spot of Asalio Caused by Alternaria alternata.” was undertaken during Rabi 2018-2019. Present studies on the aspects viz., symptomatology, isolation, pathogenicity test, effect of age of the host and effect of inoculum density on disease development on pot grown Asalio plants
Effect of plant age on disease development :
A pot experiment was laid out with five replications of each treatment following completely randomized design (CRD). A Soil mixture containing soil from fields of RCA, Udaipur and FYM (3:1) was used to fill 30 cm earthen pots. Seeds of susceptible local landrace of Asalio were obtained from Agronomy farm of RCA, Udaipur. These seeds were sown in these pots. Asalio plants of different ages were achieved by staggered sowing at 10 days interval so as to obtain 10, 20, 30, and, 40 days old plants for simultaneous inoculation at one time. Five pots for each plant age group (having 10 plants each) were maintained in cage house of Department of Plant Pathology, RCA, Udaipur. The conidial suspension (1×103conidia ml-1) of pathogen was used for inoculation of different age groups of plants. Inoculation was made by spray inoculation technique using a hand held atomizer. The inoculated plants were kept in humid chamber for 24 hrs and also these were periodically sprayed with distilled water and covered by polythene bags having 2-3 pores and then transferred to cage house and high humidity was maintained throughout the disease development period by frequent irrigations. Observations for disease severity of Alternaria leaf spot were recorded when plants reach the physiological maturity using 0 to 5 scales given by Gawande and Patil (2003).( (Table 1)
Table No. 1: Standard disease rating scale (0-5 scale) Gawande and Patil (2003) for accessing PDI of Alternaria leaf spot of Asalio
Scale
|
Description of the symptom
|
0.
1.
2.
3.
4.
5.
|
No infection.
0.1-10.0 % Leaf area infection.
10.1-25 % Leaf area infection.
25.1-50 % Leaf area infection.
50.1-75 % Leaf area infection.
75.1-100 % Leaf area infection.
|
The per cent disease index (PDI) was calculated using the formula of the McKinney (1923)
Sum of all individual disease rating
Per cent disease index (PDI) = ____________________________________________________ × 100
Total No. of plants assessed × maximum rating
Effect of inoculum concentration on disease severity :
A pot experiment was laid out with five replications of each treatment following completely randomized design (CRD). A Soil mixture containing soil from fields of RCA, Udaipur and FYM (3:1) was used to fill 30 cm earthen pots. Seeds of susceptible local landrace of Asalio were obtained from Agronomy farm of RCA, Udaipur. These seeds were sown in these pots. Five pots for each plant age group (having 10 plants each) were maintained in cage house of Department of Plant Pathology, RCA, Udaipur. The serial dilution technique was uses with simultaneously adjusting four different inoculum densities viz., 1×101, 1×102, 1×103, 1×104 conidia ml-1 using a haemocytometer. The spray inoculation technique was used for inoculations on pot grown plants different concentrations of conidial suspension. The inoculated plants were kept in humid chamber for 24 hrs and also these were periodically sprayed with distilled water and covered by polythene bags having 2-3 pores and then transferred to cage house and high humidity was maintained throughout the disease development period by frequent irrigations. The PDI was calculated by using the formula given below :
Sum of all individual disease rating
Per cent disease index (PDI) = _____________________________________________________ × 100
Total No. of plants assessed × maximum rating
Genomic DNA extraction and PCR amplification:-
The genomic DNA was extracted from representative isolate of A. alternata with standard protocol of PrepMan®Ultra sample preparation reagent 13. The reagents containing DNA fragments of isolate (100 ML) was shifted to new sterile 1.5 mL Eppendorf tubes for purification. The fungal colonies (3 mm diameter) was picked from the pure culture and suspended into reagents. The mixture was incubated for 10 minutes at 100 °C and centrifuged for 2 minutes at 13000 rpm. A 50 μL supernatant was shifted to new tubes and DNA was stored at -20 °C 14. The ITS from morphologically confirmed A. alternata isolate was amplified through PCR with the help of forward and reverse primers 15. The PCR reaction was carried out according to standard protocol 16. The genomic DNA was replaced with sterile distilled water in the negative control.
All methods were carried out according to Indian Council of Agricultural Research (ICAR) guidelines and legislation.