International Research Journal of Biological Sciences ___________________________________ ISSN 2278-3202Vol. 4(10), 48-51, October (2015) Int. Res. J. Biological Sci. International Science Congress Association 48 In vitro study of Fungicides in controlling Helminthosporium oryzae causal organism of Leaf brown Spot of Rice Pandey Sandeep School of Environmental Biology, A.P.S University, Rewa, M.P. INDIA Available online at: www.isca.in, www.isca.me Received 12th September 2015, revised 25th September 2015, accepted 6th October 2015 Abstract Leaf brown spot is a serious disease of rice causing considerable yield losses. Among various control measures the uses of chemical fungicides have been found highly effective. In vitro studies were carried out to evaluate the efficacy of fungicides in inhibiting the mycelial growth of H. oryzae pathogen causal organism of leaf brown spot of rice. All the fungicidal formulations at various concentrations were found significant in reducing the radial growth of the fungus. Among the tested fungicide Bavistin @ 1500 ppm was found significantly effective in inhibiting the radial growth of mycelium and second best treatment was Hinosan that was found at par with each other at the same concentration after 144 hrs of incubation. Keywords: Leaf brown spot, rice, fungicide, in vitro. Introduction Brown spot disease of rice caused by Helminthosporium oryzae (Cochliobolus miyabeanus) also known as Dreschslera oryzaecauses severe yield loss that reaches up to 90% in certain areas. Brown spot pathogen can infect both seedlings and mature plants and thus a serious menace to the rice crop. Brown leaf spot disease considered seed borne, and air borne, leaf spot vary in size and circular to oval in shape depending upon the environmental conditions. The smaller spots are dark brown to reddish brown and larger spots have dark brown margin and reddish brown to grey centers. Damage by brown spot is particularly noticeable when crop nutritionally deficient in potassium. The various methods prevail for rice disease control but the most important control tactics used worldwide includes use of resistant varieties and chemical control. Rice disease management strategies mainly aim at prevention of outbreak or epidemics through the use of host plant resistance and chemical pesticides. Fungicide treatment is one of the low cost control measures available for brown spot management but host plant resistance is most economical. Synthesizing and characterizing a new molecule to be used as fungicide involves several steps. Initially the new lead molecule is tested in-vitro for its efficacy against the target pathogen and then it is characterized under field condition to ascertain its efficacy against the target disease and to finalize the most effective dose/rate that can be used for the control of the target disease. Therefore the study has been carried out to evaluate the efficacy of fungicide in least possible dose concentration to inhibit the mycelial growth of H. oryzaeunder laboratory conditions. Material and Methods Single spore isolation from leaf surface: The leaves of highly susceptible traditional variety Dehula were collected showing the typical lesion of brown spot from Agriculture field for single spore isolation. The spores were picked up from the spots of the leaf surface with the help of sterilized needle and a suspension of the spores was prepared in sterilized water. About 15 ml of the suspension of brown spot pathogen was poured into petri dishes containing potato dextrose medium at 24. During this period, the germinated spores were marked by glass marker pen on petri dishes by examining under low power microscope. Small blocks of the medium containing single spores were cut by means of an inoculating needle and transfer into petri dishes containing medium and pure fungal culture was maintained for in vitro study. In vitro experiments: In vitro studies of five fungicides viz. Bavistin, Hinosan, Aureofungin sol, Danzole plus and Propineb 70 WP (table-1) were conducted over untreated check for evaluating the efficacy against H.oryzae. Poisoned food techniques were employed for evaluating the sensitivity of test fungus. The fungicides were taken in concentrations of 1000 and 1500 ppm and were incorporated aseptically in potato dextrose agar medium. The amended medium was then poured in sterilized Petri dishes. 5 mm disc of the test fungus were cutted from the margins of old test culture was then placed centrally in each of the petri dishes. The inoculated plates were incubated at room temperature and the colony diameter was recorded after incubation at an interval of 24 hr. Research Journal of Biological Sciences ___________________________________________________________ ISSN 2278-3202 Vol. 4(10), 48-51, October (2015) Int. Res. J. Biological Sci. International Science Congress Association 49 Table-1 Chemical fungicides tested for mycelial inhibition of H. oryzaeTechnical Name Trade Name a.i (g) Formulation Dosage/ha Dilution in water (l) Carbendazim 5%GR Bavistin 0.62 kg 12.5 kg 00 Ediphenphos 50% EC Hinosan 250-300 (ml) 500-600 (MI) 750-1000 l Aureofungin 46.15 W.V. SP Aureofungin sol 00 0.005% 500 Hexaconazole 5% EC Danzole plus 50g 1000g 500 Propineb 70% WP Propineb 70 WP 1050-1400 1500-2000 500 lvol/ha Statistical analysisAll Data were first subjected to analysis of variance (ANOVA). Comparisons among means were made using Duncan’s multiple range test ( = 0.05). Results and Discussion The bioefficacy of fungicide on radial growth of H. oryzaeindifferent periodic interval (96, 120 and 144 hrs) were carried in vitro. All the tested fungicides and their different concentrations viz 1000 ppm and 1500 ppm, significantly inhibited themycelial growth of H. oryzae in vitro. It was observed that the rate of mycelial inhibition shows significant reduction with the passage of time. The data presented in table-2 and figure-1 reveals that fungicide Bavistin @ 1500 ppm was found highly effective in inhibiting the radial growth of mycelium in 96, 120 and 144 hrs. of incubation 2.6, 3.5 and 4.5mm, respectively. The Hinosan was the second best treatment that inhibited the radial growth in same concentration and periodic interval 3, 5 and 5mm over untreated check. The fungicide Propineb 70 WP and Aureofungin sol were found at par in checking the mycelial growth and Danzole plus shows least influence in inhibiting the mycelial growth of the fungus. Table-2 Effect of fungicides on mycelial growth of H. oryzaeTreatment Conc. (ppm) Period of incubation (growth in mm) 96 Hr 120 Hr. 144 Hr. Propineb 70WP 1000 9 12 13 1500 5 8 10 Hinosan 1000 5 7 7 1500 3 5 5 Danzole plus 1000 15 16 18 1500 11 14.5 16.5 Aureofungin sol 1000 11 12 15 1500 8 11 13 Bavistin 1000 6 5 5 1500 2.6 3.5 4.5 Control 20 27 38.5 SEm 5.31 6.69 9.68 CD (5%) 17.67 22.25 32.18 Research Journal of Biological Sciences _ _____________________ Vol. 4(10), 48-51, October (2015) International Science Congress Association (a) 96 hrs (b) 120 hrs Inhibition of mycelial growth of H.oryzae at various conc. of fungicides Discussion: All tested fungicides were effective against H.oryzae at higher conce ntration. The standard systemic fungicides were more pronounced compare to other products. The results of this research are in agreement with Ahmad et al who evaluated four fungicides viz. Bavistin, Hinosan, Tilt 250 EC and Dithane M-45 against Bipolarisoryzaeand Dithane M- 45 was the best with 100% reduction of the prevalence of the pathogen at 500 ppm as inhibition followed by Tilt 250 EC, Hinosan and Bavistin. Similarly Arshad et al reported that Ridomil (Metalaxyl+Mancozeb) and Dithane M- 45 (Mancozeb), at 50 ppm concentrations were more effective to control the mycelial growth of fungus in vitro. The result also goes in tune with Hunjan et al who reported that new fun trifloxystrobin + tebuconazole, tebuconazole, and propiconazole showed higher level of efficacy against D. oryzae under laboratory conditions, however, thifluzamide (Spencer 24SC) was least effective against the diseases under the condition. The findings are in accordance with Rivero et al vitro effect of different concentrations (300, 500, 700 and 1000 mg/L) of K1 (INCA) and SIGMA chitosans on fungal mycelial growth and observed that both products had an inhi on brown spot fungal growth at all evaluated concentrations and SIGMA chitosan showed a total inhibition at 1000 mg/L, besides its fungicide effect. The results also in close conformity with Ibiam et al10 who investigated the use of seed fungicides (Bavistin, Benlate, Fernasan- D, Apron Plus 50 DS and Dithane– M45), and soaking and slurry methods at various concentrations, for the control of seed- borne fungi of rice variety in vitro and observed that all the fungicides significantly inhibited the seed- borne fungi associated with the seeds of the variety at concentrations of 40g/ml, and 50mg/ml (P0.05), in the soaking method, and at all the concentrations in the slurry method (P0.05) along with percentage seed germination not below 71%, at all the concentrations in the two methods used. Shabana et al11 tested the antifungal activity of three phenolic antioxidants (salicylic acid, benzoic acid and hydroquinone) 102030 Propineb … Hinosan Danzole … Aureofun … Bavistin 1500ppm 1000ppm _____________________ _________________________ ____________ International Science Congress Association 96 hrs (b) 120 hrs (c)144hrs Figure-1 Inhibition of mycelial growth of H.oryzae at various conc. of fungicides All tested fungicides were effective against ntration. The standard systemic fungicides were more pronounced compare to other products. The results of this research are in agreement with Ahmad et al 7 who evaluated four fungicides viz. Bavistin, Hinosan, Tilt 250 Bipolarisoryzaeand observed that 45 was the best with 100% reduction of the prevalence of the pathogen at 500 ppm as mycelia growth followed by Tilt 250 EC, Hinosan and Bavistin. reported that Ridomil 45 (Mancozeb), at 50 ppm concentrations were more effective to control the mycelial growth of fungus in vitro. The result also goes in tune with who reported that new fun gicides viz. trifloxystrobin + tebuconazole, tebuconazole, and propiconazole D. oryzae pathogens of under laboratory conditions, however, thifluzamide (Spencer 24SC) was least effective against the diseases under the same The findings are in accordance with Rivero et al who studied in effect of different concentrations (300, 500, 700 and 1000 mg/L) of K1 (INCA) and SIGMA chitosans on fungal mycelial growth and observed that both products had an inhi bitory effect on brown spot fungal growth at all evaluated concentrations and SIGMA chitosan showed a total inhibition at 1000 mg/L, besides its fungicide effect. The results also in close conformity who investigated the use of seed -dressing D, Apron Plus 50 DS M45), and soaking and slurry methods at various borne fungi of rice and observed that all the fungicides significantly borne fungi associated with the seeds of the variety at concentrations of 40g/ml, and 50mg/ml (P0.05), in the soaking method, and at all the concentrations in the slurry method (P0.05) along with percentage seed germination not at all the concentrations in the two methods used. tested the antifungal activity of three phenolic antioxidants (salicylic acid, benzoic acid and hydroquinone) against B. oryzae at different concentrations observed that benzoic acid or salicylic acid at 9 mM completely inhibited the growth of the pathogen. Conclusion Therefore it can be concluded that all the fungicides involved for controlling brown spot pathogen of rice in vitro were found significan t; however the least possible doses of these chemicals are advised for growers for a better results along with a sustainable agriculture. References 1. Sarkar D., Mandal R., Roy P., Taradar J. and Dasgupta B., Management of Brown Spot disease of rice by usin safer fungicides and some bioagents, 437-441 (2014)2. Iqbal M.F., Hussain M. and Waqar M.Q., Evaluation of best fungicide for controlling brown leaf spot in transplanted rice, Int. J. Adv. Res. Biol. Sci. (2015)3.Kumar P.M.K., Sidde Gowda D.K., Rishikant M., Kiran Kumar N., Pandurange Gowda K.T. and Vishwanath K., Impact of fungicides on rice production in India, Fungicides: showcases of integrated plant disease management from around the world (open access chapter), 77-98 (2013) 4. 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