2. IN-VITRO EFFICACY OF DIFFERENT FUNGICIDES, BOTANICAL EXTRACTS AND BIOCONTROL AGENTS AGAINST HELMINTHOSPORIUM ORYZAE, THE CAUSAL AGENT OF BROWN SPOT OF RICE BY GHULAM HUSSAIN JATOI 2K13-PP-324 Supervisor DR. MANZOOR ALI ABRO Assistant Professor Department of Plant Pathology Faculty of Crop Protection Sindh Agriculture University, Tando Jam

3. 1. SUPERVISOR DR. MANZOOR ALI ABRO Assistant Professor Department of Plant Pathology Faculty of Crop Protection Sindh Agriculture University Tando Jam. 2. CO-SUPERVISOR-I DR. REHANA NAZ SYED Assistant Professor Department of Plant Pathology Faculty of Crop Protection Sindh Agriculture University Tando Jam. 3. CO-SUPERVISOR-IIDR. JAVED ASGHAR TARIQ Senior Scientist at Nuclear Institute of Agriculture (NIA) Tando Jam SUPERVISORY COMMITTEE

5.  Importance of Rice:  (Oryza sativa L.) is one of the most important crop in the world and a staple food for more than half of the world’s population.  Production of Rice :  P roduction during economic year 2014 was 741.3 million tonnes (FAO STAT. 2014). Around 90% of total rice is produced by Asia continent alone (Mahy et al., 2008).  Major rice producer countries:  China, India, Indonesia, Bangladesh, Philippines and Pakistan. As a cash crop, it contributes 4.49% to the foreign exchange earnings and accounts for 1.3% GDP (Anonymous, 2014).  Diseases of Rice crop:  Rice crop is attacked by 50 diseases i.e., fungal, bacterial, parasitic (nematodes) and viral diseases.

6.  Major Diseases:  The three most important diseases of rice 1. Brown spot, Helminthosporium oryzae, 2. Rice blast, Pyricularia oryza 3. Bacterial leaf blight, Xanthomonas oryzae Among them, Brown spot of rice caused by Helminthosporium oryzae is the most devastating disease in the world

7.  Brown Spot of Rice:  The first documented case of Helminthosporium oryzae was reported in Bengal, India in 1942-43 where 50-90% of the rice crops were destroyed that causes a major famine in which two million people died of starvation (Padmanabhan, 1973).  Symptoms:  Symptoms are observed on:  Tillering stage  Round foliar spots  Originally dark brown to purple-brown.  Spots are circular to oval with a light brown to gray (Vidyasekaran et al., 1986).

8.  Management:  Different approaches including the use of fungicides, biocontrol, use of Botanicals are being practiced to control the development of this disease.  Chemical control: Application of chemicals for the control of H. oryzae is the most effective management option, but under high disease pressure effective control is not achieved (Lore et al., 2007). Mencozeb Melody duo Thiomil Bavistin  Other management Practices:  Biological control (bacteria, Fungi)  Resistant cultivars  Agronomic practices  Biotechnological methods (Ribot et al., 2008).

9.  Botanical extracts: The presence of plant chemicals in medicinal plants have involved a great contract of notice, concentrate on their role in prevent diseases 1. Neem (Azadirachta indica) 2. Akk (Calotropi sprocera) 1. Garlic (Allium sativum) 1. Datura (Datura stramonium) 2. Ginger (Zingiber officinale) (Harpyaree et al., 2010 ).

10.  Biological control: Mechanisams : Mycoparasitism, Antibiosis, competition for nutrients or by developing plant resistance (Saikia et al., 2006). Agrobacterium spp Pseudomonas Trichoderma

11. OBJECTIVES Sampling from paddy seeds for the isolation and identification of the causal fungus. To evaluate the efficacy of selected fungicides on the linear colony growth of the Helminthosporium oryzae in-vitro. To evaluate the efficacy of different botanical extracts on the linear colony growth of the Helminthosporium oryzae in-vitro To study the isolation, morphological characterization and identification of antagonistic bacteria from rizhospheric soils of rice To screen potential bacterial biocontrol agents against the Helminthosporium oryzae under in-vitro conditions

13. Survey and sampling :  Diseased samples consisting of seeds of Winner Hybrid, Supper Karnal, IR-6, IR-8, DR-59, Dokri- Basmati  Localities : Rice varieties were collected from district Larkana & surrounding areas.  Isolation of seed borne fungi: Pathogens were isolated by Standard blotter Method and PDA media.

14. Purification of Helminthosporium oryzae: Fungus was purified on PDA plates for Identification and studying colony characteristics. Technical Bulletin method is used for studying shape and size of seed born disease and health testing of rice (Agarwal, (1989) and Ellis, 1980). The pure cultures of PDA slants were preserved at 25 0 C for further studies.

15.  Inoculum multiplication:  Inoculum of Helminthosporium oryzae was multiplied on susceptible variety of Basmati 385.  Paddy rice was soaked in water for over-night, autoclaved, inoculated with the pathogen and kept at 28°C for 7 days.  Pathogenicity:  Pathogenicity test of the most frequent fungus i.e. Helminthosporium oryzae was conducted in the green house on apparently 4 healthy varieties and 2 highly susceptible variety basmati 270. and Bangalo.

16. Eff ect of different fungicides on the linear colony growth of Helminthosporium oryzae  Four different fungicides were tested in in-vitro conditions  Name of fungicides 1. Mencozeb 2. Melody duo 3. Thiomil 4. Bavistin  4 treatments and 3 replications  50 ppm  100 ppm  150 ppm  200 ppm  Techniques  Food poisoning Technique used in this experiment  Desig : The experiment was conducted in complete randomized design (CRD)

17. Effect of different botanical extracts on the linear colony growth of Helminthosporium oryzae  Five different botanical extracts were tested in in-vitro conditions  Name of botanical extracts 1. Neem (Azadirachta indica) 2. Akk (Calotropi sprocera) 3. Garlic (Allium sativum), 4. Datura (Datura stramonium) 5. Ginger (Zingiber officinale).  3 Treatments and 3 Replications  5 ml  10 ml  15 ml  Techniques  Food poisoning Technique used in this experiment  Design: The experiment was conducted in complete randomized design (CRD)

18. Isolation and purification of Rhizobacteria isolated from different rice soils  Isolation of Rhizobacteria: was also done from the same soil according to the studies of Kasimpur et al., (2004). Soil was taken from rhizosphere of rice plants.  Gram staining: Gram’s stain is method which is used to separate bacteria into two groups, such as Gram-negative and Gram+positive group.  In this method some bacteria will retain violet dark to red appearance Gram+ve and change blue to litmus to red in color Gram-ve.

19. Antagonistic activity of bacteria against Helminthosporium oryzae in-vitro  Bacterial Isolates:  A total of 25 bacterial isolates were recovered from the cuticular surface and tested for antagonistic effect against H. oryzae.  Screening:  At first, screening was conducted to evaluate the effect of potential antagonists on the fungal growth was carried out by a central disk test assay. (Reynaldi et al., 2004). Scale : (+++ very, good inhibition; + + good inhibition; + poor inhibition; - no inhibition )

21. Graph.1. Response of different rice varieties against Helminthosporium oryzae Diff. Rice cultivars Infection Frequency

22. Table: 1. Frequency of fungi isolated from rice seeds taken from different rice cultivars S.noVariety nameIsolated seed born pathogen Frequency (%) 01Winner, Hyb Helminthosporium oryzae,50 Curvularia30 Alternaria10 02Supper karnal Helminthosporium oryzae.54 Curvularia30 Fusarium16 03IR-06 Helminthosporium oryzae45 Curvularia33 Fusarium10 Penicillium12 04IR-08 Helminthosporium oryzae,58 Curvularia15 Alternaria10 Tillatia ssp17 05DR-59 Helminthosporium oryzae67 Curvularia,33 Helminthosporium oryzae 58 06Dokri-Basmati Alternaria 10 Tillatia ssp32

23. Fungicides Colony diameter (mm) Graph. 2. Effect of selected fungicides on the linear colony growth of H. oryzae

24. Botanicals tested Graph. 3. Effect of selected botanical extracts on the linear colony growth of H. Oryzae

25. Table: 2. Morphological characteristics of bacterial strain isolated from Bakhodero soils

26. Table: 3. Characterization of bacterial strains isolated from Dokri soils

27. Table 4. Morphological characteristics of bacterial strain isolated from Bakrani soils

28. Table 5. Morphological characteristics of bacterial strain isolated from Rashed Wagan soils

29. Table 6. Screening of potential biocontrol agents against Helminthosporium oryzae

30.  Six fungi viz, Helminthosporium oryzae,Curvularia, Alternaria, Fusarium, Penisillium, Tillatia sp were associated with seeds of different 06 rice cultivars  Helminthosporium oryzae was appeared as the. predominant fungus.  Pathogenicity tests indicated that Basmati-270 and Bangalo were found highly susceptible against Helminthosporium oryzae  Mancozeb and Thiomil were found more effective in reducing the linear colony growth of the fungus Helminthosporium oryzae.  Ginger and Garlic and Dhatura were found more effective in suppressing the linear colony growth of the fungus as compared to other botanicals tested  Among the 22 isolates of Rhizobacteria, only 04 were found more effective to antagonize H.oryzae in in-vitro conditions CONCLUSION

31.  Mancozeb and Thiomil could be used against this disease as they were found highly effective against H. oryzae  Keeping in view the results of present research work, it is suggested that hybird varities such as Gard-53 and Danglo hybird should be cultivated in Rice tract areas of Sindh.  Ginger, Garlic and Dhatura could also be used as alternative against this disease as they were highly effective against H. oryzae.  It is suggested that field efficacy of both fungicide and botanicals must be tested at large scale. For very safe and healthy environment, it is suggested that biocontrol agents like Rhizobacteria (Pseudomonas sp and Agrobacterium sp). SUGGESTIONS

32. Acknowledgments  Dr. Javed Asghar Tarique (Senior Scientist at NIA)  Mr. Muhaamed Ismail Buzdar (Senior Scientist at NIA)  Mr. Muhammed Issa Kumbhar (Lab: Supervisor)  Mr. Bhagwandas (Dept. clerk)  Mr. Mazhar Ali Rajper (Lab Assistant )  Muhammed Aslam Bhutto(Faculty clerk)  Muhub Mirjat(Naib Qasid )  Kaim Ali(Naib Qasid )  Rassol Bux Nizamani(Naib Qasid )

33. THANK YOU