In-vitro efficacy of plant extracts and fungicides on Alternaria tenuissima inciting leaf blight of Kodo millet (Paspalum scrobiculatum L.) and their effect on conidial characters

Introduction

Kodo millet (Paspalum scrobiculatum L.) is a very ancient crop which is believed to be originated in Africa and was domesticated in India 3000 years' ago and is distributed widely, grown by farmers of low income group, it is resource poor soil under the traditional agriculture systems [1] . Kodo millet is a principal member of millet, is a self-pollinating, C 4 crop, has chromosome number 2n = 4x = 40 and belongs to family Poaceae. Besides India, the crop is extensively grown in tropical and sub-tropical regions which include countries viz., Indonesia, Philippines, Thailand, Vietnam, Bangladesh, Myanmar and western part of Africa. In India, crop is extensively grown in states of Madhya Pradesh, Chhattisgarh, Tamil Nadu, Telangana, Maharashtra, Gujarat, Karnataka, Uttar Pradesh and Jharkhand [2] . Kodo millet is a small seeded, low water demanding, hardy, climate resilience crop, fertilizer responsive, which can be grown in various cropping systems under different ecological situations. Being C 4 plant it has high efficacy of using CO 2 . Kodo has gained a lot of attention these days due to its nutritional value which is very rich in numerous health benefits and agronomic practices with low maintenance. Presence of good amount of proteins, carbohydrate, minerals and vitamins makes it very famous. Grains of kodo also contain high amount of lecithin, an important factor for enriching the nervous system with very less or negligible gluten content. Kodo millet increases the insulin sensitivity for people who are suffering from diabetes. Low glycaemic index, rich in antioxidants like polyphenols are unique properties of the kodo grains. Consumption of kodo reduces the problems of constipation, stomach cramping, bloating, flatulence and lowers the problems of cardiovascular diseases [3,4,5] . Different types of pathogens have been reported in kodo millet viz., fungi, bacteria, nematodes, viruses, some partial root parasites, etc. which limits the production of kodo [6] . Leaf blight caused by Alternaria spp. and Helminthosporium spp. were reported long time ago in kodo millet as a minor disease but now leaf blight is becoming an emerging problem in kodo millet [7] very few studies were undertaken regarding characterization of the pathogen, identification of the resistant sources and management of the disease, hence needs more studies on different aspects of pathogen as well as disease.

Materials and Methods

An experiment was carried out in the laboratory of the plant pathology department, College of Agriculture, Jawaharlal Nehru Krishi Vishwa Vidyalaya, Jabalpur, Madhya Pradesh, India. In this experiment, a total of eight available plants (Table 1) were collected from the locality and was tested against the fungus using poisoned food technique [8] . One hundred grams of the plant was taken and washed with NaOCl and water to surface sterilize (2 to 3 times) and was allowed to dry at room temperature for some time. Plant parts (100g) were crushed in 100 ml of sterilized water in sterilized pestle and mortar, then the extract was filtered with double layered muslin cloth under aseptic conditions. The stock solutions of 5% and 10% were prepared and mixed with 95 and 90 ml of potato dextrose agar medium (PDA) to get 5% and 10% of the concentrations respectively. PDA supplemented with different types of plant extracts was poured in Petri plates of 90mm at rate of 10ml per plate and the plates were allowed to solidify. Disc of 5mm from the test fungi (10 day old) was taken with the help of cork borer and forceps and placed in the center of the plate. For comparison, a separate plate was maintained without any extract (control). All the treatments were replicated thrice in completely randomized design (CRD). Plates were placed in Biological Oxygen Demand (BOD) incubator (25 ± 2 ºC); the readings were taken after 48 hours (hrs.) of inoculation at the interval of 24 hrs. Inhibition of the tested fungus was measured in (mm) and was calculated by the formula given by (Vincent 1947) [9] ;

Where, I = Per cent inhibition (%). C = Radial growth of the fungus in control (in mm). T = Radial growth of the fungus in treatment (in mm).

After 264 hrs. of incubation, the conidia per microscopic field (100 X) were counted and length, width of the conidia was measured. Number of longitudinal and transverse septa in 25 conidia was recorded. Micrometry of the pathogen was done to ensure proper details regarding length and width of the pathogen in the treatments which showed spore count with microscope (Leica company) and software (Leica Application Suite) at 100X. In order to check the efficacy of the fungicides the experiment was carried out in the same laboratory mentioned above. Seven available fungicides namely, Tebuconazole 5.36%, Metalaxyl 35% WS, Carbendazim 50% WP, Carboxin 37.5% + Thiram 37.5% DS, Carbendazim 12% + Mancozeb 63% WP, Metalaxyl 8% + Mancozeb 64% WP and Difenoconazole 25% EC along with control were used. These fungicides were tested against the test fungus using poisoned food technique [8] at 100, 250 and 500 ppm. Different types of fungicides are listed in Table 2. Different concentrations which were required were taken in sterilized flask containing sterilized water to make stock solution then required amount of fungicide was poured into the PDA flask. Then flask having PDA was agitated lightly to mix solution properly and for the uniform distribution of fungicides into the medium. About 10ml of PDA was poured into 90mm Petri plates and was allowed to solidify. Test fungus from seven days old was taken from cork borer and forceps. Then was placed at the center of plate under aseptic conditions. All the treatments were replicated thrice in completely randomized design (CRD). Separate control was maintained without any fungicide for comparison. Inoculated plates were kept in BOD (25 ± 2 ºC). Readings were taken after 48 hrs. of incubation at 24 hrs. of interval. The inhibition percent and micrometry was done as in above experiment of plant extracts.

Scale of sporulation

Statistical Analysis

Data obtained was analyzed using OPSTAT online software at 5% level of significance and is presented as the mean of the triplicates.

Results

Observations on the radial growth and conidial characteristics as influenced by different aqueous extracts (Table 3 At 10% concentration (table 3; figure 2; plate 1), best inhibition was observed in aqueous extract of clove of garlic (41.5mm with inhibition of 51.1%) followed by neem (59.7mm with inhibition of 29.7%) while minimum was seen in marigold (80.0mm with inhibition of 5.8%) after 216 hours of incubation. No sporulation was seen in garlic, ginger, turmeric, neem and Parthenium. Good sporulation was seen in marigold while fair in Aloe vera. On other hand, it was excellent in control. In Aloe vera, mean length and width of conidia was 11.66 µm and 4.34 µm, 35.17 µm and 12.81 µm in Calotropis and 28.29 µm and 8.60 µm in control respectively. Range of longitudinal septa found were 2-4 in control and Calotropis while 3-5 in Aloe vera while range of transverse septa was 0-2 in control and Aloe vera respectively (table 5; plate 3). To evaluate the different fungicides at (table 2) 100, 250 and 500 ppm. Data depicted in table 6 presents the growth and inhibition of the fungus at all the three different concentrations. Difenoconazole 25% was most effective in suppressing mycelial growth and showing inhibition with mean of 25.6mm with inhibition of 69.5%, 23.8mm with inhibition of 71.9% and 13.3mm with inhibition of 84.7% when compared to control (84.1mm, 84.9mm and 85.0mm) at 100, 250 and 500 ppm respectively after 192 hours of incubation. This was followed by Tebuconazole 5.36% (45.8 with inhibition of 45.5%, 27.3mm with inhibition of 67.8% and 26.0mm with inhibition of 69.4%) when compared to control (84.1mm, 84.9mm and 85.0mm) at 100, 250 and 500 ppm respectively after 192 hours of incubation. On other hand, least inhibition and maximum growth of pathogen was observed in Metalaxyl 35% and Carbendazim 50% (77.3mm with inhibition of 8.08%) at 100 ppm when compared to control (84.1mm) after 192 hours of incubation (table 6; figure 3; plate 4). At 250 ppm, Carbendazim 12% + Mancozeb 63% showed least inhibition (71.1mm with 16.2% inhibition) when compared to control (84.9mm), whereas Carbendazim 50% (68.6mm with inhibition of 19.2%) closely followed by Carbendazim 12% + Mancozeb 63% (67.3mm with inhibition of 20.7%) at 250 ppm when compared to control after 192 hoyrs of incubation respectively. Treatments of Carbendazim 12% + Mancozeb 63% and Difenoconazole 25% did not showed any sporulation. Poor sporulation was observed in Metalaxyl 35%, Carbendazim 50% and Metalaxyl 8% + Mancozeb 64%. Fair sporulation was in Tebuconazole 5.36%, whereas good sporulation in Carboxin 37.5% + Thiram 37.5% and excellent sporulation was observed in control at 100 ppm.

Treatments of Tebuconazole 5.36%, Metalaxyl 35%, Carbendazim 50%, and Metalaxyl 8% + Mancozeb 64% resulted in poor sporulation. Whereas good sporulation was shown by Carboxin 37.5% + Thiram 37.5% and excellent sporulation in control. No sporulation was recorded in the treatments of Carbendazim 12% + Mancozeb 63% and Difenoconazole 25% at 250 ppm (table 6; figure 4; plate 4). At 500 ppm, sporulation was completely inhibited in all the fungicidal treatments at 500 ppm and excellent sporulation was recorded in control (table 6; figure 5; plate 4). Regarding conidial characterization at 100 ppm revealed that there is plenty of variations in size of conidia and the septations. Average length of conidia was 21.76 µm, 12.81, 27.51, 22.64, 20.13 and 20.42 µm in Tebuconazole 5.36%, Metalaxyl 35%, Carbendazim 50%, Carboxin 37.5% + Thiram 37.5%, Metalaxyl 8% + Mancozeb 64% and control, respectively. Whereas, average conidial width was 8.89, 7.34, 10.08, 9.78, 8.47 and 9.03 in the same treatments. Slight variation in longitudinal septa varied from 1 to 3, 1 to 2, 2 to 3, 1 to 3, 1 to 3 and 2 to 3 was noted in the treatments of Tebuconazole 5.36%, Metalaxyl 35%, Carbendazim 50%, Carboxin 37.5% + Thiram 37.5%, Metalaxyl 8% + Mancozeb 64% and control, respectively. Number of transverse septa varied from 0 to 2 was recorded in the different treatments (table 7; plate 5). At 250 ppm, average conidial length was recorded in Tebuconazole 5.36% (16.27 µm), Metalaxyl 35% (22.26 µm), Carbendazim 50% (23.77 µm), Carboxin 37.5% + Thiram 37.5% (24.17 µm), Metalaxyl 8% + Mancozeb 64% (24.90 µm) and 26.54 µm in control. Average conidial width was maximum in Carbendazim 50% (11.19 µm) followed by Tebuconazole 5.36% (10.18 µm), Metalaxyl 35% (9.67 µm), Metalaxyl 8% + Mancozeb 64% (8.80 µm) and in control width was 8.25 µm. Whereas minimum conidial width was in Carboxin 37.5% + Thiram 37.5% (7.70 µm). Number of longitudinal septa were 1 to 2 in Tebuconazole 5.36%, 1 to 3 in control and 2 to 3 in Metalaxyl 35%, Carbendazim 50%, Carboxin 37.5% + Thiram 37.5% and Metalaxyl 8% + Mancozeb 64%. Transverse septa ranging from 0 to 1 were recorded only in the treatments of Metalaxyl 35%, Carbendazim 50%, Carboxin 37.5% + Thiram 37.5% and Metalaxyl 8% + Mancozeb 64% (table 8 and plate 7). At 500 ppm, mean length and width of conidia were 23.34 µm and 9.48 µm respectively with 2-3 longitudinal septa and 0-2 transverse septa (table 9). fungicide against leaf blight causing pathogen, different fungicides were tested viz., Tebuconazole 5.36%, Metalaxyl 35% WS, Carbendazim 50% WP, Carboxin 37.5% + Thiram 37.5% DS, Carbendazim 12% + Mancozeb 63% WP, Metalaxyl 8% + Mancozeb 64% WP and Difenoconazole 25% EC. These were tested at 100, 250 and 500 ppm, among them Difenoconazole 25% EC gave the best results 69.5%, 71.9%, and 84.7% at 100, 250 and 500 ppm respectively. Same findings were given by some researchers [14, 15, 16] .

Acknowledgement

AcknowledgementsThe authors thank College of Agriculture, Jawaharlal Nehru Krishi Vishwa Vidyalaya, Jabalpur -482004, India for supporting the research.

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