Efficacy of different fungicides against the in-vitro growth of Pyricularia oryzae causing Rice blast disease

Rice blast disease caused by Pyricularia oryzae is a major threat to the farmers as the disease severity may lead upto 80% crop loss in severe conditions. A laboratory experiment was conducted at National Plant Pathology Research center of NARC, Khumaltar,Lalitpur for testing the efficacy of different fungicides adopting poisoned food method against the pathogen Pyricularia oryzae causing rice blast disease. The experiment was carried out in a completely randomized design with 10 treatments including control following 3 replications of each. Two concentrations (50 ppm and 100 ppm) each of different fungicides viz. Dithane M-45 (Mancozeb 75%W.P), Bavistin (Carbendazim 50 % W.P), Antracol (Propineb 70%W.P) , Diamethmorph 50%WP, Sectin ( Fenamidone 10%+ Mancozeb 50%W.G),Protector ZN ( Chlorothalonil 75% W.P), Safaya (Carbendazim 12%+ mancozeb63% W.P) , Nativo 75% W.G (Tebuconazole 50%+ Trifloxystrobin25%W.G) and Topcare ( Azoxystrobin 50% W.D.G) were evaluated against the growth of the pathogen. Results showed that lower concentration (50ppm) of Bavistin and Safaya fully inhibited the growth of the pathogen while Nativo at higher concentration (100 ppm) was shown to exhibit 100% inhibition of the pathogen. The fungicides significantly inhibited the mycelia growth with the increase in concentration of fungicide. However, the fungicides found effective for mycelia inhibition of P.oryzae should be further tested in field conditions to verify their efficacy as well the optimum dozes of fungicide should be re-evaluated. Keywords— Efficacy, Fungicide, Inhibition, in-vitro, Poisoned food technique.


INTRODUCTION
Rice (Oryza sativa L.) is the main cereal crop of the world and is a staple food for more than a half population of the world (Ahmad et al., 2020, Singh et al., 2019.More than 2.7 billion people of the world have rice as a major source for their food (Kulmitra et al., 2017). Rice is also the major source of food for the people of Nepal. Rice, maize and wheat are the three major food crops grown in different parts of Nepal. However, rice has the highest productivity in Nepal. Rice is grown in all three ecological belts of Nepal ranging from the plain terai to the high Himalayan region (Joshi et al., 2011).The terai region of Nepal has high productivity of rice contributing to 70% total domestic production of the country.
The productivity of rice in the country is hindered by many factors among which disease is the prime one. The crop loss due to diseases and pest is recorded 37% annually in rice (Moktan et al., 2021). Blast is the major fungal disease attacking the rice plant and is caused by the fungus Pyricularia oryzae. This disease is prevalent in humid areas in all the rice growing region of the world as the conidia are not developed below 88% R.H (Neelakanth et al., 2017

II. MATERIALS AND METHOD
A laboratory experiment was conducted at NARC, Khumaltar with 10 treatments (50 ppm and 100 ppm) including control and experiment was arranged in completely randomized Design (CRD) to test the efficacy of different fungicides against the in-vitro growth of pathogen P. oryzae causing rice blast disease.The experiment was carried out fromJuly 2020 to October 2020.
Culture of P.oryzae, previously isolated from rice, Mulpani was obtained from the National Plant Pathology Research Centre NARC, Khumaltar and pure culture was maintained on potato dextrose agar (PDA) under incubated condition at 25 o C for 7 days.
Different fungicides (Table. 1) were evaluated in-vitro for testing their efficacy to inhibit the mycelium growth of fungal pathogen P.oryzae. Stock solution was prepared for each fungicide by diluting 1gm in 100 ml of distilled water and required amount was incorporated into the conical flask containing Oat meal agar to get 50ppm and 100ppm concentration and mixed thoroughly before autoclaving.
After autoclaving, 20 ml of the poisoned media was poured aseptically into the sterilized petri plates each of 9 cm diameter under laminar air flow chamber and allowed to solidify. As for control, only OMA media was used without addition of any fungicide. 5mm radial disc of the pathogen from the 7 days old culture plate was placed on the center of the petri plate containing OMA media and incubated at 24 0 C.
The diameter of the pathogen was taken from two directions after 2 day after inoculation (DAI) and recorded for 10 days after. Percent inhibition of fungal growth was calculated using the formula (Vincent, 1947).

Effect of different fungicides in inhibiting the growth of P.oryzae at 50ppm and 100ppm
Different fungicides at 50 ppm and 100 ppm were tested against the pathogen P.oryzae following the poisoned food technique and their efficacy ispresented in Table 2. The table shows that the fungal mycelia gradually increased in both treated and control plate with the increase in incubation period of the pathogen. All the 9 fungicides of different concentrations (50 and 100 ppm) visibly inhibited mycelial growth over control during the study ( Table 2).
Bavistin and Safaya were found most effective in inhibiting the mycelia growth among the tested 9 fungicides. Bavistin, Safaya and Nativo were individually effective against the pathogen as they showed maximum inhibition of the mycelia growth. At lower concentration (50ppm), Bavistin and Safaya showed complete inhibition of the mycelia growth whereas at higher concentration(100ppm), Bavistin, Safaya along with Nativo showed complete inhibition of the mycelia. They were significantly superior over other fungicides and on par with each other. These were followed by Azoxystrobin and Sectin (100ppm)with more than 70% mycelia inhibition.
Dithane M-45, Dimethomorph and Chlorothalonil showed low inhibition of the fungal mycelia as compared to above fungicides. The fungicide Protector ZN was ineffective against the pathogen with least inhibition percentage. It was noted that the mycelial growth was decreased with the increase in concentration of fungicides.

Comparison of growth of P.oryzae at 50 ppm and 100ppm
Pathogen at different concentration 50 and 100ppm showed different growth from 1 st to 9 th day after inoculation ( Fig.1 and 2). At 50 ppm, Bavistin and Safaya exhibited no growth of the fungal pathogen till the 9 th day after inoculation.
Slow growth of the pathogen was observed in the treated plate of Nativo during the early days and the growth of pathogen was not observed from 5 th day of inoculation. Similar growth of mycelia was observed in case of Sectin and Antracol treated plates. Dithane M-45 and Dimethomorph also exhibited the similar growth pattern of the pathogen .The growth of pathogen in Protector ZN suddenly increased on the 9 th day whereas the growth was similar in other days. Increase in growth of the pathogen was seen every day in Control plate until the plate was fully covered.  The study shows that increasing the concentration of Azoxystrobin can increase the inhibition percentage of the pathogen. In our experiment the inhibition% of Azoxytrobin was found to be 70% at 50ppm which increased to 78% at 100ppm.This is in accordance with Neelakanth et al., 2017 who recorded that the inhibition percentage of pathogen was increased with increase in concentration of Azoxystrobin.
DithaneM-45 has been reported effective against P.oryzae for seed treatment Konda et al., 2016 and Hajano et al., 2012. However, in our in vitro experiment, it was not found effective. Dithane could be effective as a protectant rather than for control of the disease once the pathogen has invaded.

IV. CONCLUSION
The use of different chemical fungicides have been found effective to control various plant diseases. The treatments used in our experiment are easily available in the market. Farmers apply same fungicide for various type of fungal diseases and our findings show that there is difference among the fungicides in controlling the fungal growth.Same concentration of these fungicides is not equally effective against the pathogen.Results of this study show that rice blast can be effectively controlled by application of different fungicides. From the experiment, it is concluded that Bavistin (Carbendazim 50% W.P), Safaya(Carbendazim12%+Mancozeb63%W.P) are the most effective ones among the tested fungicides with cent percent inhibition of mycelia growth. Nativo (Tebuconazole 50%+ Trifloxystrobin25%W.G) is also effective against the pathogen P. oryzae at higher concentration. The percent of inhibition of pathogen is increased with increasing concentration of fungicides. Further, green house trials and application of these treatments in infected host plants should be carried out to find their effectiveness under field condition.