A DISEASE PREDICTIVE MODEL FOR CHEMOTHERAPY OF KARNAL BUNT OF WHEAT By Muhammad Abdul Shakoor M.Sc. (Hons) Agri. A thesis submitted in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY IN Plant Pathology FACULTY OF AGRICULTURE, UNIVERSITY OF AGRICULTURE, FAISALABAD 2009 To The Controller of Examinations, University of Agriculture, Faisalabad We, the supervisory committee, certify that the contents and the form of the thesis submitted by Mr. Muhammad Abdul Shakoor Regd. No 85-ag-1234 have been found satisfactory and recommend that it be processed for evaluation by the External Examiner(s) for the award of degree. CHAIRMAN ___________________ Dr. MUHAMMAD ASLAM KHAN MEMBER ___________________ Dr. NAZIR JAVID MEMBER ___________________ Dr. MUHAMMAD JALAL ARIF Contents CHAPTER Title Page 1 INTRODUCTION 1 2 REVIEW OF LITERATURE 5 2.1 History and geographical distribution 5 2.2 Survey and economic losses 8 2.3 Taxonomic status 15 2.4 Symptoms of the disease 15 2.5 Host range 16 2.5.1 Biology of Tilletia indica 16 2.5.2 Dispersal of teliospores 16 2.5.3 Teliospore dormancy 17 2.5.4 Nature of teliospores and their survival in the soil 18 2.5.5 Teliospores germination 20 2.5.6 Dispersal of sporadia from soil level to ear head 21 2.6 Infection process 22 2.6.1 Epidemiology and disease prediction models 24 2.7 The Humid Thermal Index (HTI) model as a tool for 28 determination of potential disease distribution 2.8 Sources of genetics resistance against the disease 35 2.9 Chemical control of the disease 40 3.0 MATERIALS AND METHODS 45 3.1 Survey of Karnal bunt disease of wheat 45 3.1.1 Field survey 45 3.1.2 Survey of grain markets 47 3.1.3 Sampling 47 3.1.4 Survey of agricultural farms 48 3.2 Screening of wheat germplasm for the source 48 of resistance 3.2.1 Isolation and preparation of mass culture of 48 Tilletia indica 3.2.2 Field evaluation of wheat germplasm lines/ varieties 50 for the source of resistance against Karnal bunt disease under artificial inoculation conditions 3.3 Epidemiological studies 55 3.3.1 Cultivation of susceptible germplasm lines/ varieties 55 for epidemiological studies 3.3.2 Development of disease predictive model 56 3.4 Chemotherapy of Karnal bunt disease of wheat 56 3.4.1 In vitro evaluation of fungicides at four dosages 56 rates 3.4.2 In vivo control of Karnal bunt disease of wheat 57 by protective and eradicative spray of fungicides 3.4.3 Protective spray 57 3.4.4 Curative spray 59 4.0 RESULTS 60 4.1 Survey of Karnal bunt disease of wheat 60 4.2 Screening of wheat germplasm for the source of 67 resistance 4.3 Epidemiological studies 67 4.3.1 Correlation of environmental conditions with 67 Karnal bunt disease development 4.3.2 Disease predictive models 73 4.4 Chemotherapy of Karnal bunt disease of wheat 75 4.4.1 In vitro evaluation of fungicides against the mycelial 75 growth of Tilletia indica 4.4.2 In vivo control of Karnal bunt disease of wheat by 77 protective and curative (eradicative) spray of fungicides 5.0 DISCUSSION 80 5.1 Survey of Karnal bunt disease of wheat 80 5.2 Screening of advanced lines / cultivars of wheat 82 against the disease 5.3 Weather conditions and disease predictive models 84 5.4 Evaluation of fungicides against the Karnal bunt 86 disease of wheat 6.0 SUMMARY 89 7.0 CONCLUSIONS 90 8.0 RECOMMENDATIONS 91 9.0 FUTURE STUDIES 92 10.0 REFERENCES 93 LIST OF FIGURES FIG# TITLE PAGE 1 After centrifugation teliospores sedimented into pellet 49 in conical centrifuge test tubes 2 Disinfested teliospores by centrifugation with 5 % 49 chlorex in capped test tubes 3 Germination of teliospores after 10 days on plain agar 51 petri-plates 4 Germination of teliosporse after 20 days in petri-plates 51 containing plain agar 5 Germination status of teliospores after 30 days 6 Germinating teliospores with long slendrical primary 51 sporadia growing from tip of promycelium (basidium) under power microscope 100x 60) 7 Pattern of spore shedding (secondary sporadia) 52 on PDA slants in conical flasks after second day of inoculation 8 (A and B) Pattern of sporadia shedding on PDA 52 slants after third day of inoculation 9 (A & B) production of secondary sporidia on 53 PDA slants after 6 days of inoculation. 10 Screening of wheat germplsm lines/ cultivars’for 54 the source of resistance against Karnal bunt disease under artificial inoculation condition and field evaluation of fungicides. FIG # TITLE PAGE 11 Mean inhibition zones of Tilleta indica by the 76 action of various fungicides at various ppm 76 (A) Control (water) 76 (B) Alert plus at 100 ppm 76 (C) Antracal at 100 ppm 76 (D) Dolomite at 100 pp 76 (E) Shelter at 100 ppm 76 (F) Shelter at 80 ppm 76 LIST OF TABLES TABLE # TITLE PAGE 1 Rating scale used to calculate the severity of Karnal 46 bunt of wheat 2 An example of the calculation of coefficient of 46 infection 3 Rating scale used to determine level of 55 resistance/ susceptibility 4 Fungicides evaluated against in vitro colony growth 58 of Tilletia indica and in vivo control of wheat grains by foliar spray 5 Analysis of variance for the assessment of bunted seeds 60 in grain markets of various districts and locations of the Punjab 6 All pairwise comparisons tests for Karnal bunt disease of 61 wheat for districts ingrain markets of the Punjab 7 Percent wheat sample infection, range of infection 62 and average infection in grain markets and storage godowns of 17 districts of the Punjab by Karnal bunt disease during 2006. 8 Analysis of variance for the incidence of Karnal bunt disease 63 of wheat under natural condition in various hot spot areas of the Punjab during field survey 2007 9 All pairwise comparisons test for Karnal bunt disease of 64 wheat for districts recorded in hot spot areas (field). 10 Survey of Karnal bunt disease of wheat under natural 65 conditions in different hot spot areas of the Punjab during the crop year 2007. 11 Range of infection and percent wheat grains infection 66 in hot spots of various research stations/ research institutes, model farms and seed corporation farms during wheat crop of 2007 12 Level of resistance/ susceptibility of advanced wheat 68 lines and commercial cultivars against Karnal bunt disease of wheat 13 Analysis of variance of Karnal bunt disease incidences 69 recorded on wheat varieties during 2006 and 2007 14 Correlation of environmental factors with Karnal bunt 69 disease of wheat on different lines/ varieties. 15 Comparison of monthly environmental conditions 72 and karnal bunt incidence recorded on wheat varieties during 2006 and 2007. TABLE # TITLE PAGE 16 Summary of stepwise procedure for independent variables 74 influencing Karnal bunt disease of wheat during growing season 2005-07 17 Summary of stepwise procedure for independent variables 74 influencing Karnal bunt disease during growing season 2005-06. 18 Summary of stepwise procedure for independent variables 75 influencing Karnal bunt disease during growing season 2006-07 19 Mean inhibition zones of colony of Tilletia indica by various 77 fungicides at 4 dosages rates amended on PDA medium 20 Percent decrease in wheat kernels infection by Tilletia indica 78 by protective and eradicative spray of fungicides Abstracts Karnal bunt disease caused by (Tilletia indica) is sporadic in nature. The fungus attacks the wheat crop in Feb. and March which are the susceptible stages (booting stage) of the host. During the survey it was estimated the disease is extending from north to south Punjab that are hot and dry areas of the province. Epidemiological studies revealed that range of minimum air temperature between (11-16 oC), maximum air temperature (27-31 oC) and rainfall more than (1.15 mm) during the vulnerable growth stages produced the critical circumstances for the invasion of the fungus on the wheat crop. Based upon the environmental conditions of two years data combined stepwise regression indicated that all the employed environmental factors explained 84 % of the in disease development, Out of 200 germplasm lines/ varieties only 16 lines were highly resistant and 9 were resistant. Protective spray of fungicides (Dolomite and Shelter) reduced the disease 62 to 63 % respectively. ACKNOWLEDGEMENT All appreciations and acclamations are for Almighty Allah, the Compassionate, the ever Merciful, the only Creator of the universe and bestowed the mankind with knowledge and wisdom. The humblest thanks to the Holy prophet Hazrat Muhammad (peace be upon him), for the directive “Acquisition of knowledge is obligatory upon every Muslim man and women”. The author feels great pleasure and honour to express profound gratitude and appreciation by heart and soul to Dr. M. A. Khan, Professor , Department of Plant Pathology, University of Agriculture, Faisalabad, whose affectionate supervision, scholastic guidance, encouragement and constructive criticism enable the author to plan, execute and complete this task. Heartful thanks are extended to Dr. Nazir Javed, Associate Professor, Department of Plant Pathology, University of Agriculture, and Faisalabad for his valuable suggestions to improve this dissertation. Special thanks are also due to Dr. Muhammad Jalal Arif, Associate professor, Department of Entomology, University of Agriculture, Faisalabad, who spared the time without any hesitation for kind guidance, encourageous suggestions to improve the dissertation. Sincerest thanks to S. T. Sahi, Associate Professor and Principal; Agricultural College Depalpur who contributed a lot of moral support during this study and experiments. Lastly the author is grateful and lacks words to express his heartful thanks to parents who prayed for my success, along with economic supports and inspired from time to time.