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. (M. SHAKOOR)

DEDICATIONS

Dedicated to Holy Prophet MUHAMMAD (Peace be upon him) the greatest social reformers of human beings.

To my mother and father, who always prayed for my success in this life and hereafter.

Chapter 1 INTRODUCTION Bread wheat (Triticum aestivum L.) is the main staple food crop and major source of nutrition for the people of Pakistan. It is grown in winter months of November to April on an area of 27.69 m. ha. (Anonymous, 2007a). Average wheat yield of Pakistan is 2.38 mt/ha, which is extremely low as compared to other wheat producing countries of the world such as Ireland, Denmark, United Kingdom, France, Egypt and Saudi Arabia having 7.86, 7.83, 7.78, 6.23, 6.15 and 4.48 mt/ha, respectively (Anonymous, 2007b). Many factors contribute to low yield in Pakistan, diseases being one of them. Karnal bunt (partial bunt) of wheat caused by Tilletia indica (Mitra) Mundkur causes considerable losses in the yield of wheat. The disease not only reduces the weight of grains, but also deteriorates its quality and makes it unacceptable for human and animal consumption (Gopal and Sekhon, 1988). According to Pamela et al., (2004) it fulfills the following criteria of its inclusion in the list of emerging infectious diseases (EIDs) of plants, (i) it has increased incidences, geographical distribution and host range (ii) it has changed its pathogenesis (iii) it has been newly evolved or newly recognized. The emergence of plant EIDs is similar to those of humans, wildlife and domestic animals (Pamela et al., 2004). Modern changes in the agricultural pattern, implementation of modern farming techniques, ecosystem, intensification and globalization stimulate the attacking mechanisms of the pathogen, which pose threats to agriculture or biodiversity conservation. According to Dobson and Foufopoulos, (2001) understanding of factors driving the plant EIDs require knowledge of host-parasite biology, use of multiple-host system models parameterized for a large number of environmental, ecological and biological factors. Karnal bunt was first detected in 1931 at Karnal in Haryana, India and hence it is called Karnal bunt (Mitra, 1931). It bears many names such as Karnal bunt, new bunt, partial bunt, incomplete bunt, Indian bunt and stinking smut. Singh et al., (1989) reported that Karnal bunt was a disease of wheat, durum, rye and triticale (a hybrid of wheat and rye). Though the disease is native to South Asia but subsequently it has been reported from Iran, Syria, Afghanistan, Iraq, Mexico (Joshi et al., 1983), Nepal (Singh and Dhaliwal, 1989) and United States (Ykema et al., 1996). The disease remained less damaging till late 1970 but subsequently severe epidemics started occurring coinciding with the change over to high yielding, irrigated, semi dwarf and high fertilizer input farming. In early 50s, Karnal bunt disease was considered to be a disease of minor importance and it was confined to hills of Pakistan. In 1971, the disease was reported from Sialkot, Gujranwala and Mardan districts during the crop years of 1966-71 (Hassan, 1971) and the frequency of disease ranged from traces to 2.0 percent. The disease remained endemic for considerable period of time in the Northern area of Pakistan and later it spread to south and was reported as far as Jhang, Khanewal and Muzaffargarh district of the Punjab (Bhatti and Ilyas, 1986). A little later the disease became wide spread throughout the Punjab and was prevalent in 23 districts with a frequency range of 0.32 to 3.50 per cent (Ilyas et al., 1989a). At present almost all commercial varieties of wheat under cultivation are susceptible to Karnal bunt and the disease incidence is aggravating with the passage of time. The disease reduces wheat yields and can cause a fishy, unpalatable odor and taste in wheat flour, reducing flour quality (Bonde et al., 1997; Sekhon et al., 1980; Singh and Bedi, 1985). Since karnal bunt affects the international trade of commercial wheat grain and movement of germplasm, the presence of the disease can cause economic loss to wheat exporting countries (Bonde et al., 1997). The yield and quality losses are generally minor; the most economic loss can be attributed to the quarantine status of the pathogen (Babadoost, 2000; Butler, 1990). The pathogen infects the ovaries in the emerging wheat heads and converts the grains partially or completely into dark colored powdery masses of teliospores. The diseased fields emit a foul smell like that of rotten fish due to production of trimethylamine. Karnal bunt can reduce wheat yields. There is no estimate of losses, due to this disease, occurring in Pakistan; however, survey in India conducted during the years of heavy disease revealed a total loss of 0.5 percent, but in some fields where 89 percent of the kernels were infected, the yield losses ranged from 20-40 percent in highly susceptible varieties (Anonymous, 2004). Brennan et al., (1990) estimated the economic losses from Karnal bunt of wheat in Mexico to be US $ 7.02 million per year. Besides yields losses, Karnal bunt can reduce wheat flour quality due to fishy, unpalatable odour and taste, if a grain lot contains 1-4 percent infected seed (Bonde et al., 1997; Hussain et al., 1988; Mehdi et al., 1973; Sekhon et al., 1980). If in a grain lot 5 percent of the grain is infested, the quality of the flour recovery and chemical changes in composition of flour and gluten contents cause poor dough strength (Sekhon et al., 1980; Gopal & Sekhon, 1988). Karnal bunt is also a disease of quarantine interest and it affects the international trade of commercial wheat grain and movement of wheat germplasm through out the world. Thus presence of diseased grain in wheat lots can cause economic loss to wheat exporting countries (Bonde et al., 1997, Babadoost, 2000; Butler, 1990). Karnal bunt differs from other diseases of wheat in that the pathogen infects plants during anthesis and it sporulates on the same generation of the host which it infects. Neither all spikes of plant nor all grains in spike are affected by the disease and usually a few irregularly distributed kernels are bunted (Mitra, 1935; Bedi et al., 1949; Dhaliwal et al., 1983). Infection of individual kernels varies from small points of infection to completely bunted kernels but completely infected ones are rare (Chona et al., 1961). The embryo of the infected kernels usually remains undamaged except when infection is severe (Cashion and Luttrel, 1988) but the endosperms of the kernels get shrunken to varying degrees. At maturity severely infected kernels are filled with teliospores of the pathogen which serve as primary source of inoculum. The spikes of infected plants are generally reduced in length with less number of spikelets (Mitra, 1937). During harvest, infection sori are broken resulting in contamination of healthy seeds, soils, equipment, machinery and the vehicles with the liberated spores. The spores may be blown by wind for long distances. The teliospores of the Karnal bunt fungus can survive in soil for more than 5 years (Krishna and Singh, 1983b; Babadoost et al., 2004; Bonde et al., 2004). Although many control strategies have been suggested for the management of Karnal bunt disease and the strategies include seed treatment with hot water and solar energy, seed treatment with fungicides and soil drenching with fungicides (Anonymous, 2005), however, the results were not convincing. The cheapest and the most feasible method of Karnal bunt control is the use of host resistance and breeding for varieties resistant to Karnal bunt disease. Control of Karnal bunt has now become a major concern in Pakistan due to scarcity or non-availability of resistance in commercial wheat varieties under cultivation. The gravity of the situation of the disease calls for evaluation of fungicitoxicants against the disease for its management. As the pathogen is soil, seed and air-borne, it can penetrate locally into host plant, so application of spray fungicides is very critical. Epidemiological factors have great influence on the epidemic development of karnal bunt disease. Wheat is vulnerable to Karnal bunt fungus only during a 2-3 week windows of its’ physiological development stages if the environmental conditions happen to be conducive during this short period for successful infection and the weather favourable for the disease development does not exist every year (Workneh et al., 2008). Therefore spraying for the disease every year would be unnecessary waste of time and resources. Characterization of environmental conditions conducive to partial bunt disease is required to decide the timing of fungicide application. Development of a disease predictive model for the chemotherapeutic control of this disease was the main objective of the studies which was accomplished with following line of work: 1. Survey for the incidence of Karnal bunt disease of wheat in major wheat growing areas of the Punjab. 2. Screening of advanced lines/varieties received from Wheat Research Institutes, Faisalabad by artificial inoculation. 3. Determination of correlation of environmental factors with Karnal bunt disease of wheat. 4. Development of a disease predictive model to determine the relationship of environmental factors with disease incidence. 5. In vitro and in vivo evaluation of spray fungicide for the management of Karnal bunt disease of wheat

Chapter 2

REVIEW OF LITERATURE History and geographical distribution Karnal bunt was formally recorded in 1930 at the Botanical Research Station, Indian Agricultural Research Institute (IARI), Karnal, Haryana in northwest India (Mitra, 1931), now in the Punjab region of India. The first report of Karnal Bunt from a non-Asian country was received from Mexico in 1972, but the disease has been confined to localized areas within the state of Sonora (Anon. 1991a). The Karnal bunt pathogen (Tilletia indica) was not identified outside Asia until 1972, when it was reported from the state of Sonora in northern Mexico (Fuentes-Davila, 1998; Sansford, 1998). At that time, the disease was restricted to the Yaqui and Mayo valleys in Sonora and was found only in traces in farmers’ field. However, in the early 1980s, during surveys in these valleys, Karnal bunt disease was found in 64 % of the farms (Charles et al., 2005). According to Boratynski et al., (1985) and Marshal et al., 2003, Karnal bunt entered in the United States and Canada from Mexico, because in October 1983 the pathogen was intercepted, as bunted kernels, at the Calexico, CA, and Port of entry in Mexican boxcars. Subsequently, Mexican boxcars were found frequently contaminated with bunted wheat grains, and viable teliospors of T. indica were found in dust and debris in 9 of 20 randomly selected boxcars. It was concluded that Mexican boxcars destined for United States and Canada were the best potential path way for the artificial movement of Karnal bunt pathogen into the United States. On 8 March 1996, the first report of disease in the United States was documented on durum wheat in Arizona, and a few weeks later it was confirmed on bread wheat also in California (Ykema et al., 1996). Samples of wheat, maintained by the Arizona Department of Agriculture, were tested for the presence of Karnal bunt pathogen, and teliospores of T. indica were found in grains harvested in 1993, indicating that the disease had been in Arizona in 1992. It was soon discovered that bunted wheat grains also had been shipped to, and planted in, New Mexico and Texas (Ykema et al., 1996). The disease has later been established in the New World in northwest Mexico (Warham, 1986) and has been reported in southern California, Arizona and Texas in the US (Rush et al., 2005). Da Luz et al., 1993 reported the presence of T. indica in a seed lot produced in Rio Grande do Sul state, in 1989 but there have been no reports since and establishment has not been proven. T. indica was first noted from Brazil in 1993 in the southern part of the Rio Grande do Sul and some efforts were made practically to get rid of it (Da Luz et al., 1993). However, no further publications of its existing status in Brazil have been made. It entered into the Brazil through imported wheat germplasm for research purposes during accessions between 1990 and 1992 (Mendes and Ferreira, 1994, Oliveira et al., (2002) . Karnal bunt also occurs in an area of Northern Cape Province in South Africa (Crous et al., 2001). However, between 2002 and 2004 it was found to have spread to a number of new areas (Naudé, 2002, Anon., 2004a). All locations where pathogen (Tilletia indica) has been found to lie on or close to latitude 30o north and 30o south and declared as having arid or semi-arid climates with mild/cool winters and hot summers. In the main, they have been recognized as dry environments ranging from desert with little or no rain to ‘arid steppe’ or ‘semi-desert’ with a short rainy season. In the plains of northwest India, the climate is termed sub-tropical summer rain; Karnal bunt also has been recorded in some fringing environments, but incidence is very low and outbreaks can be sporadic. The climates of these areas are termed ‘subtropical rain’ and ‘tropical summer rain’ (Pearce and Smith, 1990; Stefanski et al., 1994). Regional distribution and gradual dispersal of the pathogen within a country indicated that the disease has a close relationship to the environmental condition specific to that area. The disease is not common in the Indian subcontinent out side the North West region. Disease incidence is very low in central and eastern India. Partial bunt or Karnal bunt disease has never been found in the south or northeast of the Indian subcontinent (Anon. 1996; Joshi et al., 1983; Singh, 2005). It has never been reported in the field in ‘oceanic temperate’, ‘temperate continental’ or ‘sub-tropical winter rain’ environments prevailing in Europe. During domestic survey and investigations research workers from UK had collected 25 samples of wheat grain during the harvesting years 2002, and 2003 and 50 samples during 2004, tested by using the EU/EPPO-recommended T. indica Diagnostic Protocol (Inman et al., 2003, Anon., 2004c), all of which also proved negative for T. indica. The situation regarding specific surveys for the presence of T. indica in other European Union countries is unknown. However, Hungary and Lithuania are preceding inspection of wheat grain and triticale originating in countries affected by T. indica (Talevi et al., 2004). The pathogen has not been reported in southern areas of India and Mexico due to different environments to areas in these countries where the disease is established, although there have been many chances for internal spread (Fuentes-Davila, 1996a; Singh, 2005). In the northwest of India, where incidence of Karnal bunt is relatively high, rain-fed wheat is sown in late October and irrigated wheat is sown in November. Harvesting generally starts in mid-May. Four to six irrigations are recommended with one coinciding with flowering (Anon., 2006). In Pakistan, it was known to occur in Sialkot district of the Punjab in 1950 (Saleem and Akhtar, 1988). Mundkur, (1943) and Bedi et al., (1949) reported this disease from Punjab and NWFP. They also reported that the disease was airborne and increased with excessive irrigation. In the year 1986-87, it appeared in epidemic form in the majority of wheat growing areas of the Punjab and upto 30% disease was recorded in wheat grain samples of various commercial cultivars collected from the 17 districts of the Punjab. (Anon., 1986; Ilyas et al., 1989). During the epidemic years, the disease also caused substantial losses to crop (Bhatti and llyas, 1986). Teliospores of Tilletia indica were also found in 23.8% (5 of 21 samples) of germplasm lots from Pakistan in 1984 (Diekmann, 1987). Mirza (2005), during a survey collected 254 Karnal bunt samples from Punjab and 96 from North West Frontier Province (NWFP) during growing season 2003-04. Samples of the varieties MH-97, Inqilab-91 and Wattan which were mostly grown in the wheat growing areas of the Punjab were infected with Karnal bunt disease. Almost 83.33% from Kasur and Okara, 66.66% from Lodhran, 60% from Pakpattan, 50% from Sialkot, Rawalpindi and Khushab, 47.6% from Gujranwala, 44.44% from Faisalabad, 32.14% from Bahawalpur and 15% from Multan were infected with Karnal bunt disease of wheat. With the passage of time, the spread and geographical distribution of Karnal bunt disease of wheat increased. Mustafa, (1965) reported the disease from Iraq. The disease had also been recorded in southern Nepal (Singh, et al., 1989), and reported from southern Iran (Torarbi et al., 1996). Wheat from Afghanistan (Locke & Watson, 1955), Lebanon, Syria and Turkey (Lambat et al., 1983) has been found contaminated with teliospores of T. indica, although the pathogen has never been in the field in these countries (Warham, 1986, 1992). Later wheat germplasm consignments from Lebanon, Syria and Turkey were found free of teliospore contamination (Diekmann, 1987). The status of the disease in these countries was unclear. However, an international survey using a standardized protocol has been suggested as the only way of determining the exact distribution of the Karnal bunt disease (Babadoost, 2000). Survey and economic losses Karnal bunt disease caused Tilletia indica (Mitra) Mundkur) was reported from Karnal, India for the first time; hence it was named as Karnal bunt. In India and Pakistan, variations in losses have been noted during unusual and uneven surveys carried out in different years. However, there is trend of increasing incidences with passage of time. Royer and Rytter, (1985) stated that semi-dwarf cultivars of wheat imported from Mexico were widely-grown in India during 1965. These cultivars were more susceptible than those of used earlier and were the major cause of the increase in disease incidence between 1969–1975. Indian farmers apparently replanted harvested seed irrespective of whether conditions, thus perpetuating T. indica in the soil. Agarwal et al., (1976) reported that Karnal bunt disease was considered endemic in India until the 1969–70 wheat growing season when most of the imported Mexican cultivars grown were affected by the disease with a maximum of 7.5% seed infection in the cultivar S-331 in the foothills of the Himalayas of Uttar Pradesh. Joshi et al., (1983), highlighted the occurrence of Karnal bunt in the ‘plains of undivided India’ as being at ‘trace levels’ in the early years. During 1969–70 it was widespread in Delhi, Punjab, Haryana, Rajasthan and western Uttar Pradesh. By 1974–75 it was reported as severe in many areas of northern India, especially in the foothills of the Himalayas and the Tarai region of Uttar Pradesh, Punjab and Himachal Pradesh; disease severity was described as especially high in Hempur in Uttar Pradesh (15–23%). Percentage of lots of 8 wheat cultivars falling into different ranges of seed infection; the maximum being 8.3% of the lots of cultivar HD-2009 falling into the 20–50% seed infection category. Aujla et al., (1977) described that the ‘infection % of the bunt’ during 1975– 76 on different cultivars ranged between 2.2 and 52.9% in Jammu and Kashmir and 1.4 and 25.1% in the Punjab. They found that the incidence of disease was highest on late flowering cultivars which coincided with temperatures of 18–22°C and >70% relative humidity. A survey conducted in 1981 revealed that the disease was present in areas where it had previously been unreported including new locations in central India (Zhang et al., 1984). Singh, (1994) summarized the distribution, incidence, and severity of Karnal bunt disease in Uttar Pradesh containing 36% of the area of land under wheat production at that time in India. A Karnal bunt distribution map was arranged for surveys. It was noted that Karnal bunt was commonly distributed in various western and eastern districts of Uttar Pradesh with northern hill and southern dry regions being generally free. It was severe at many places in northern India especially in the foothills of the Himalayas, Uttar Pradesh, Punjab and Himachal Pradesh. Disease severity was as high as 15–50% at Hempur and Pantnagar in Uttar Pradesh. During 1975–76 maximum disease incidences in India were found in the Punjab where 39% of grain samples were infected as compared to 9% in Haryana. During 1977–78, 38% of field samples from Haryana were infected as compared to 25% in Himachel Pradesh and Kashmir. The disease increased in 1978–79 when the incidence of field samples infected in Punjab, Himachel Pradesh and Kashmir were observed 78%, 71% and 89% respectively. Karnal bunt was also reported from West Bengal, Bihar, Madhya Pradesh and Gujarat covering eastern, central and western parts of India. Singh et al., (1996) conducted post-harvest surveys from 1986–1994 in north- western India and pointed out a high level of infection in the climatically favourable years of 1986, 1987, 1990 and 1991 (34, 25, 21 and 16% of samples infected). Beniwal et al., (2000) reported that in the state of Haryana (south of the Punjab) Karnal bunt infection ranged from 0.05–9.90% and 0.05–0.30% during the 1995–96 and 1996–97 cropping seasons, respectively. Indu-Sharma et al., (2004) highlighted the position of different grain markets of Punjab in India with regards to status of Karnal bunt disease of wheat. Less affected areas during the last 11 years (1994-2004) from where wheat may be procured for foreign exchange at the grain market level or by contract farming were identified. Every year variations were noted in the disease as an evident from the percent disease free samples recorded in different years. Amongst different districts, the Karnal bunt disease incidence was least in Patiala district where 85% samples were free from Karnal bunt disease followed by samples from Amritsar (81.12%). Average Karnal bunt free samples were as high as 90% and above in seven grain markets of the Punjab (Amritsar, Bagharian, Bhadson, Muktsar, Kotshmeer, Delhon, and Bhasor). Varshney et al., (2004) during monitoring the incidence of the disease rejected the certified seeds due to infection caused by Tilletia indica in 1025 seed samples of 15 wheat cultivars from main wheat producing areas in India and the number of discarded grains varied from seed lot to seed lot of the same cultivar. The rejection for cultivar HD-2009 was 0.25% in 1995-96, but was 100% in cultivar HS-240 in the same year. Singh et al., 2002, collected the wheat grain samples from Aligarh, Faizabad, Varanasi, Gorakhpur, Uttaranchal, Meerut, Dehradun, Udam, Singh Nagar, and Uttar Pradesh, India during 1997-98 and 2000-01 to assess the scope of the disease incidence in wheat caused by Tilletia indica in the two provinces of India. In an associated experiment, seed grains of 15 bread wheat varieties cultivated by farmers as certified seeds were evaluated for the confirmation of disease in Uttaranchal and Uttar Pradesh India, during 1998-2001. Udam Singh Nagar indicated the highest number of diseased seed grains in 1998 (57.4%) and 1999 (53.3%), whereas Dehradun recorded the highest number of infected seed grains in 2000 (35.7%). The Maximum number of wheat seeds plots discarded due to karnal bunt was recorded during 1999; however, wheat seed plots from Gorakhpurh revealed the highest karnal bunt incidence during 1998. Seeds of cultivar Sangam recorded the highest karnal bunt infection (36.4%), whereas HP1633 recorded the lowest (10.5%). Only the seeds of cultivar HP1633 were not rejected due to karnal bunt. In India Gill et al., (1993a), reported that the percentage of bunted wheat kernels increased with passage of time. Infected kernels that were noted 25 % in 1997-78, increased up to 86 % in 1982-83 cropping season. The percentage of wheat seed samples with bunted seeds were 9, 34, and 17 in Haryana during the 1874-75, 1977-78, and 1978- 79 cropping seasons, respectively, and 60% of samples had infected kernels in 1982 (Singh et al., 1986). In 1989-90, 1990-91 and 1991-92, 62, 63, 94 and 62 % of wheat samples respectively (Gill et al., 1993). However, data recorded by survey teams in northwest India revealed that even during the worst epidemic years, the loss was only 0.2 to 0.5 of total yields (Joshi et al., 1980). According to Singh, (1994), the state of Utter Pradesh in northeast India exhibited less than 1% losses even in the worst years. A detailed analysis of wheat samples collected from 15 districts of eastern Uttar Pardesh in 1987, a year of particularly high epidemics in India, revealed that an average of 3.79 % of seeds per samples were infected (Rai and Singh, 1989; Rai et al., 1988). In 1978-79 one of the wheat fields in Madhya Pradesh had 23.5 % of the seeds infected. (Singh and Prasad, 1980). Singh et al., 2001, conducted a survey to collect the samples from different wheat grain markets in Punjab, India during the period of bumper crop yield with maximum wheat production, i.e. from April to mid-May in 1999 to Karnal bunt disease. Analysis of the samples revealed that the Karnal bunt disease was well-spread in Punjab. From a total of 552 samples collected, 42.7% samples were infected by T. indica. The maximum bunted samples were found in Faridkot district where the prevalence of the disease was 92.8%, whereas the minimum prevalence of bunt was 3.4% in Amritsar district. The overall disease incidence in Punjab was 0.24%. The disease incidence ranged from 0.002% in Kapurthala district to 1.34% in Ropar district. The samples from Ropar (2.53% incidence) and Sangrur (2.13% incidence) revealed the endemic occurrence of the disease in certain areas of Punjab. Wheat cv. HD 2329 showed higher susceptibility to Karnal bunt compared to cv. PBW 343, which occupies the maximum area in Punjab. The prevalence and incidence of the disease were 53.6 and 0.36%, respectively, on HD 2329, and 35.8 and 0.20%, respectively, on PBW 343. The survey also indicated the existence of low bunt areas in Punjab, which need to be more specified by continuous extensive surveys in these areas. Singh et al., 1999, reported that during 1989-90 to 1996-97 wheat grain samples were collected from markets in different districts of the Indian Punjab. No completely Karnal bunt free area was found in the state but some areas of low disease severity were observed in Bathinda. In Pakistan, Ilyas et al., (1989b) conducted a survey of 29 districts of Punjab during the crop season of 1987-88 to determine the Karnal bunt infection. Grains samples were collected randomly from grain markets and farmer’s fields. A composite sample of 25g was drawn from each main sample to work out the percentage incidence and range of infection of karnal bunt in different districts of the Punjab. The disease was found to be widespread in 23 districts of Punjab. The disease was; however, completely absent in six districts namely Multan, Muzaffargarh, Layyah, R.Y. Khan, D.G. Khan and Rajanpur. Ehsan et al., (2002), conducted a survey in 78 wheat growing localities of Pakistan to assess the prevalence of Karnal bunt disease. The highest disease incidence was observed on cv. WL-711 (46.0%) grown in Hathion. High disease incidence was also recorded for Pak-81 (0.47-6.32%) and Pirsabak-85 (0.41-5.57%) and for the districts of Mangora (2.53-23.42%), Mardan (0.41-46.00%), Malakand (6.32%), and Swabi (0.54- 5.85%). Wheat-growing areas in Manshera (0.42-1.04%), Peshawar (0.64-2.36%), Abbottabad (0.00-1.37%), and Attock (0.00-1.25%) had low to medium disease incidence. Bhutta et al., 1999, conducted a survey in Punjab. A total of 730 wheat seed samples were tested to assess the incidence of karnal bunt using the dry inspection method from 1993/94 to 1996/97. High infection percentage (3%) of karnal bunt in various seed lots was noted. Seventy three % and twenty five % of the samples from Central Punjab and northwest areas of Pakistan respectively were found contaminated with bunted grains. Southern parts of the country were found free from 1994/95 to 1996/97. Mirza, (2005) conducted a comprehensive survey and on farm disease assessment in Punjab and North West Frontier Province (NWFP). During his survey he collected three hundred and fifty Karnal bunt samples from both the provinces during 2003-04, of which 254 were from the Punjab and 96 from NWFP. Samples from the varieties MH-97, Inqilab-91 and Wattan which were mostly grown in wheat growing areas of Punjab were found infected with Karnal bunt disease. Almost 83.33 % samples from Kasur and Okara 66.66 % from Lodhran, 60.00 % from Pakpattan, 50.00 % from Sialkot, Rawalpindi and Khushab, 47.6 % from Gujranwala, 44.44 % from Faisalabad, 34.48 % from Sahiwal, 33.33 % from Lahore and 32.14 % from Bahawalpur were affected. Singh, (1998) assessed the yield losses upto 0.16 % in an epidemic year in the foothills of the Himalayas. Brennan et al., (1992) divided the losses from Karnal bunt into two types: direct losses and indirect losses. Direct economic costs caused by disease affecting quality included :- (a) value of the yield loss; (b) value of quality loss (c) the cost of handling and marketing infected and infested product and (d) economic cost of the loss of markets through quarantine or marketing restrictions imposed following the presence of the disease (loss of seed and grain exports). Indirect loss or control costs aimed at preventing the spread of disease or reducing its’ severity includes: (a) cost of in- crop control measures. (b) cost of quarantine or regulatory restrictions imposed on the production and marketing of crops (c) regulatory cost associated with monitoring the disease and (d) costs associated with extra processing or fumigation of the output from infested area. Karnal bunt normally has nominal impact on wheat yield. However, comparatively small numbers of infected grain have a major effect on germinability and quality of wheat products (Warham, 1986). The pathogen was not identified outside of Asia until 1972, when it was reported from the state of Sonora in northern Mexico (Fuentes-Davila 1998; Fuentes-Davila and Duran, 1986). The yield losses reported in the literature vary from <1 % to 20 % and average losses due to Karnal bunt disease are less than 1 % in effected areas of India, Pakistan and Mexico (Gordan and Brennan, 1998). Brennan and Warham (1990) estimated the yield loss in north-western Mexico to be 0.12 % per year and quality loss at 0.69 in the value of crop in infested area. Fuentes-Dávila, (1996a) during a survey reported that levels of Karnal bunt were ‘noticeable’ in 1981–82 in the Yaqui and Mayo valleys in the state of Sonora, Mexico.The disease remained limited to north-western Mexico reaching around 400 km north, south and west with incidence varying annually depending upon meteriological conditions. Disease dominated in years when high relative humidity and rainfall coincided with anthesis of the crop. Low disease incidence developed in the Yaqui valley in 1982, 1984, 1987, 1988, 1990, 1994 and 1996 with surveys showing 85.0 to 99.7% of samples tested to be disease-free. The highest levels of infection established in 1983 and 1985 with 7.5 and 11.2% of samples with >1% infected grain. Due to the favorable environmental conditions at the vulnerable growth stages of wheat Karnal bunt disease has a considerable potential to establish in Australian wheat belt. Sixty seven suitable sites were found at risk from Karnal bunt disease during survey and modeling. They also expected a loss of $A 491 million per year due to the Karnal bunt disease of wheat. These sites were through the southern wheat belt from Western Australia to central to New South Wales. (Gordon and Brennan, 1998) Murray et al., 1996, estimated the losses due to karnal bunt in Australia including direct and indirect as 54.46 $A/ ton of grain. A national Karnal bunt survey programme was arranged in 1996 for the confirmation of Karnal bunt disease only in localized areas and not widespread in the U. S. wheat crops. The fundamental motto of the survey was to provide U. S. certifying official the data to issue phytosanitary certificates demanded by the countries which import U.S. wheat. Approximately 650 counties in 39 states participate in the national survey each year, with samples ending by the middle of September. Bread wheat, durum wheat and triticale were incorporated in the survey. Samples were collected from countries where the vulnerable cultivars were grown (Charles et al., 2005). When national survey was initiated in 1996 a considerable numbers of samples were compulsory to confirm the limited distribution of Karnal bunt disease (caused by Tilletia indica) in the United States. During the first year of the national survey, a field had been quarantined at the discovery of a single teliospore of T. indica, but in 1997, United State Department of Agriculture (USDA) and Animal and Plant Health Inspection Services (APHIS) adopted the bunted kernel standard. Primarily the monitoring of wheat grain samples for bunted kernels was done by visual observation either on a plastic tray or in a vibrating grain inspection machine (Anon. 2002). Since the samples typically consist of several thousand kernels, analysis was tedious. In the beginning of 2002, a high-speed optical sorter was used that selectively removed, based on seed discoloration, suspected bunted kernels from the samples (Dowell et al., 2002). This technology to a large extent reduced the amount of material to be examined manually and is now used for national Karnal bunt survey samples. In May 2001 USDA confirmed that wheat in an elevator in Young County, TX. tested positive for Karnal bunt samples.. Consequently, during survey collection of grain samples by APHIS, in and around the Young County, confirmed the presence of T. indica in the adjoining Texas counties of Archer, Throckmorton and Baylor. In 2002, because of convenience of a Kb positive field to the county line, the regulated area was further extended into neighboring Knox County, but no Kb-positive field had been identified in that county. Vocke et al., (2002) reported that the Karnal bunt regulatory programme allows the US Department of Agriculture to issue phytosanitary export certificates stating that wheat in a given shipment is from an area where Karnal bunt is not known to occur. Charle et al., (2005) during a comprehensive survey found that over 85 % of the 399 fields that have tested positive for Karnal bunt disease. The maximum number of bunted kernels collected from individual field has 478 in Arizona in 2004, 209 in California, in 2004 and 224 in Texas in 2001 with 1.0, 0.46, and 0.5 % respectively.

Taxonomic status Tilletia indica (Mitra) Mundkur (synonym Neovossia indica Mundkur) belongs to class Ustilaginomycetes, phylum Basidiomycota, order Ustilaginales, family Tilletiaceae and genus Tilletia specie indica. Black, dusty-appearing teliospors of the fungus gave it the name “smut” (Boned et al., 1997). The class name is derived from “ustulatus”, meaning burned, in suggestion to the blackened appearance of the infected plants (Carris, et al., 2006). Cereal-infecting species of Tilletia that produce teliospores within the ovaries of their hosts plants are generally called bunt fungi, also considered to be derivation of word burned (Duran and Fischer, 1961). Mundkur (1938, 1940) stated that T. indica “probably belongs to the genus Neovossia” based on the large number of non-conjugating basidiospores produced by the fungus. Based on a detailed taxonomic study Krishna and Singh, (1982) justified its placement in Neovossia indica. However, western scientific literature prefers to designate the name of causal agent of Karnal bunt as Tilletia indica (Duran and Fischer, 1961; Duran, 1972). Symptoms of the disease The Partial bunt is too much difficult to detect under field conditions, and generally careful examination revealed evidence of disease (Morris et al., 1997, and Bonde et al., 1997). Only a few kernels of some wheat heads become partially infected, except, in severe cases in which whole of the kernels in an ear are replaced with fungal sorus. There may be a slight swelling or darkening of infected florets. Therefore, Karnal bunt is easiest to detect by observing the harvested grains carefully. The pathogen converts the infected ovary into a sorus where a mass of dark brown coloured teliospores are produced. Small sori are generally formed in longitudinal furrow, leaving the rest of seed unaffected. However, in severe cases the major part of the endosperm along the longitudinal furrow may get spoiled. Partially infected grains occur in clusters of spikelets and many such aggregate clusters may be present in a spike (Nagarajan et al., 1997). In a standing wheat crop, infected spike can be detected by the shiny silvery black spikelets with glumes spread apart and swollen ovaries. The infected grains emit a fishy odour due to trimethylamine and wheat products from severely infected grains are unpalatable. Cashion and Luttrell, (1988) reported that the pathogen (Tilletia indica) did not invade the embryo and the mycelium growth was limited to the pericarp. Transmission electron microscopes (TEM) study revealed that the mycelium proliferated in the pericarp by disintegrating the middle layers of parenchymatous cells and prevents fusion of the outer and inner layers of pericarps, with the seed coat. The mycelial mat formes a compact hymenium-like structure which gives rise to short, septate stalks with single teliospores (Roberson and Luttrell, 1987). Host range Tilletia indica infects bread wheat (Triticum aestivum L.), durum wheat (Triticum turgidum Desf.), and Triticale ( x Trititicosecale Wittm.) under natural conditions (Fuentes-Davila et al., 1996). The wild wheat species Aegilops geniculata Roch (as T. ovatum), Aegilops sbaronensis Eig., A. pereggrina (Hack.) Maire and Weiller var. peregrine (as T. variabilis), and “Triticum scerrit” are reported as hosts for Tilletia indica, (Aujla et al., 1985). With artificial inoculation, T. indica infects triticale, 3 species of Triticum L., 11 species of Aegilops L., 2 species of Bromus L., 3 species of Lolium L., and Oryzopsis miliacea (L.) (Royer and Rytter, 1988). Host range lists for Tilletia species contained naturally occurring hosts and artificially inoculated hosts (Warham et al., 1986) and hosts resulting from synonymy of similar species (Duran, 1987; Duran and Fischer, 1961). Biology of the Tilletia indica Dispersal of teliospores: Dispersal of Karnal bunt pathogen is documented by means of its’ teliospores. The long distance dissemination of teliospores contaminating wheat seeds or grains is believed to be in a manner similar to other bunt disease of wheat. (Wilcoxson & Saari, 1996). These bunted seeds are known as reservoir of teliospores. Besides being seed- borne the spores can be carried to new areas through machineries and hand tools. Teliospores cling to plant parts, clothes, farm equipments, vehicles, threshers, and combine harvesters. They may also be dispersed by rain water and animals, including insects and birds, both as surface contaminants and through faeces. Teliospores may be transferred to new areas in the form of wind-blown inoculums (Bonde et al., 1987). Teliospores also germinate successfully after ingestion by livestock and insects like grasshoppers, providing another mean of dissemination (Smilanick et al., 1985b).

Teliospore dormancy: Most freshly collected teliospores are dormant and unable to germinate (Mitra, 1931; Banasal et al., 1983; Smilanick et al., 1985a). The highest rate of germination occurres with one year old teliospores (Mathur and Ram, 1963; Kiryukhina and Shcherbakova, 1976). Three types of teliospore dormancy have been noted in T. indica. Teliospores taken from freshly harvested grains germinate poorly compared with several months to a year or more old bunted grains. (Bansal et al., 1983; Mitra, 1935; and Rattan and Aujla, 1990). This type of dormancy is known as postharvest dormancy that occurs in T. horrida also (Chahal, et al., 1993). The second type of dormancy is long- term dormancy, in which germination rarely exceeded 50% under optimal laboratory conditions and it remained between 15%–30% in most reports, although teliospores were stored for more than one year. This type of dormancy, contributes to teliospore survival under field conditions. The third type of dormancy can be induced by cold temperature. Dry teliospores of T. indica kept at −18◦C progressively lost the capability to germinate over a period of 12 weeks of treatment (Zhang et al., 1984). The teliospores in these experiments were plated immediately after treatment and non-germinability was thought to indicate lethality. However, the inhibition of germination was minimized if teliospores were plated after a 20-day “thawing” period at 22◦C rather than immediately after the cold treatment (Chahal and Mathur, 1992). Cold-induced dormancy had also been observed in experiments performed over 4 to 8 days of freezing at 0◦C (Sidhartha et al., 1995). Bedi et al., 1990 noted that with the passage of time germination of teliospores is affected, 4-14 month old teliospores had maximum germination which declined with further aging. Similar dormancy also occurs in Tilletia horrida (Chahal et al., 1993). Cold-induced dormancy of the dwarf bunt pathogen T. controversa Kuhn also occurs annually in natural field environments of temperate climates (Hoffmann, 1982; Hoffmann and Goates 1981).

Nature of teliospores and their survival in the soil. Due to the hardy nature of teliospores of T. indica it remains viable in the soil for a long period of time. Teliospores of T. indica, T. horrida, and other bunt species that have been examined have relatively thick cell walls with four distinct layers, an endosporium, a thin middle layer, an exosporium from which the ornamentation develops, and an outer sheath (Hess and Gardner, 1983; Nawaz and Hess, 1987; Roberson and Luttrell, 1987). The teliospore wall resists toxic gases and liquids including methyl bromide and chloropicrin (Smilanick et al., 1988), hydrogen peroxide (Smilanick et al., 1994), propionic acid, ozone (Rush et al., 2004), and degradation in the digestive tract of animals (Smilanick et al., 1986). This enables teliospores of T. indica to survive for several years in hot desert to cold temperate field environments (Babadoost et al 2004; Bonde et al., 2004; and Chib et al., 1990). Under typical laboratory conditions, T. indica teliospores can survive for at least 16 years (Bonde et al., 1997).. Teliospores are globose to subglobose in shape, 22-49µm in size, reticulate with spines and are surrounded by a thick cover sheath of 2-4 microns (Nagarajan et al., 1997). The peridium or sheath is fragile, fractured structure and is easily identifiable under the electron microscope. The second sheath episporium is reticulate and has numerous curved spines while the third layer is endosporium. Mature teliospores are dark brown and immature spores are light brown Bonde et al., 1996.) Some hyphal fragments on mature teliospores may be present. Pale orange to mainly dark, reddish brown to opaque-black and densely ornamented with sharply pointed to truncate spines, occasionally with curved tips make the distinguishable morphological characteristics of T. indica compared with other two similar species T. horrida and T. walkri (Anon. 2004). The teliospors of Tilletia indica are very resistant to adverse environmental conditions and have been reported to survive in the contaminated soil for 2 to 5 years as reported by various research workers (Chib et al., 1990; Krishna and Singh, 1983; Singh et al., 1990). Teliospore, being the only source of survival in the soil provides the basis of pathogen spread and play a vital role in the perpetuation of disease. Bonde et al., 2004, conducted an experiment to study the survival of teliospores of Tilletia india in different types of soils. A teliospore longevity study was initiated in Kansas, Maryland, Georgia, and Arizona. Soil from each location was with T. indica teliospores and placed in polyester mesh bags. The bags were placed within soil from same location with in polyvinyl chloride pipes. Pipes were buried in the respective plots such that the bags were at 5, 10 and 25-cm depths. Each pipe was open at the both ends to allow interaction with the outside environment; however, it was fitted with screens preventing possibility of teliospore escape. In the Karnal bunt–quarantine area of Arizona, bags of infested soil also were placed outside the pipes. Teliospore-infested soil from each location was well furnished with dry conditions in a laboratory. During the first 2 years, viability of the teliospores of Karnal bunt declined more rapidly in pipes than outside pipes, and more rapidly in fields in Kansas and Maryland than in Georgia or Arizona. After 2 years, viability declined equally. In the laboratory over 3 years, viability decreased significantly more rapidly in dry soil from Kansas or Maryland than in dry soil from Georgia or Arizona, while pure teliospores remained unchanged. Consequentley Bonde et al., (2004) concluded that soils, irrespective of weather, affect teliospore longevity. Teliospores of T. indica are introduced into the soil at harvest and may persist there for 45 months (Krishna and Singh 1983; Bonde et al., 1997; Singh, 1994 and Warham, 1986). Teliospores on the soil surface germinate and produce primary and secondary sporidia which, under favourable environmental conditions, infect plants at flowering (Bonde et al., 1997; Singh 1994 and Warham 1986). Chib et al., and 1990, and Gill et al., 1993a, reoorted that in India, T. indica teliospores survived for 3 years on soil surface in the field, but spores buried 20 cm deep decreased to low viability after 2 years. In another experiment Krishna and Singh 1983, investigated that in India, survival of teliospores on the soil surface and at depths of 7.5 and 15 cm was 45, 39, and 27 months, respectively. It was reported that viability of the teliospores of T. indica decreased with increasing burial depth from 5 to 20 cm (Rattan and Aujla, 1990; Sidharta et., al 1995). Teliospores survived better in loamy sand soil than in clay and sandy loam soil (Rattan and Aujla, 1990). Singh, 1994, and Smilanick et al., 1989, demonstrated that the survival of pathogen is longer in dry soil than in wet soil in the form of teliospores. In laboratory conditions storage teliospores retained their viability for 5 to 7 years (Kiryukhina and Scherbakova, 1976; Mathur and Ram, 1963 and Zhang et al., 1984.). Study was conducted by Babadoost et al., (2004) for the assessment of survival of T. indica teliospores in a location in the northern United States. Soils differing in texture and other characteristics were collected from four locations, equilibrated to – 0.3 mega pascal (MPa), and infested with teliospores of T. indica to give a density of 103 teliospores per gram of dry soil. Samples (22 g) of the infested soil were placed in 20-μm mesh polyester bags, which were sealed and placed at 2, 10, and 25cm depths in polyvinyl chloride tubes containing the same field soil as the infested bags. Tubes were buried vertically in the ground at Bozeman, in October, 1997. Soil samples were assayed for recovery and germination of T. indica teliospores 1 day and 8, 20, and 32 months after merging of teliospores into soil. Teliospores recovered from soil samples were 90.2, 18.7, 16.1, and 13.3% after 1 day and 8, 20, and 32 months after assimilation of teliospores into soil, respectively, and was significantly (P < 0.01) affected by soil source. The percentage of teliospore recovery from soil was the greatest in loam soil and lowest from a silt loam soil. The rate of teliospores recovered from soil was not significantly affected by depth of burial and the soil source–depth interaction during the 32-month period. The percentage of germination of teliospores was significantly (P < 0.01) affected by soil source and depth of burial over the 32-month period. The mean percentage of teliospore germination at 1 day, and 8, 20, and 32 months after incorporation into soils was 51.3, 15.1, 16.4, and 16.5%, respectively. In another experiment, samples of silty clay loam soil with 5 × 103 teliospores of T. indica per gram of soil were stored at different temperatures in the laboratory. After 37 months of incubation at 22, 4, –5, and –18°C, the rates of teliospore recovered from soil were 1.6, 2.0, 5.7, and 11.3%, respectively. The percentage of spore germination from soil samples was highest at –5°C. Microscopy studies revealed that disintegration of teliospores began after breakdown of the sheath-covering teliospore. The results of this study showed that teliospores of T. indica could survive in Montana for more than 32 months and remained viable. Teliospore germination Most locally infecting bunt fungi have echinulate, verrucose, or tuberculate teliospores that germinate at approximately 200C–250C (Castlebury et al., 2005). Though each teliospore generally produces one promycelium (Mitra 1931), several promycelia may arise from a single teliospore (Krishna and Singh, 1981; Mitra, 1931; Warham 1988a; Rivera-Sanchez and Fuentes-Davila, 1997). Promycelia vary in length 500 µm and bear at the apex a whorl of 32 to 128 or more primary sporidia (Mitra 1931). Variation in the length of promycelial tips may occur (Peterson et al., 1984). The effects of physical factors that influence teliospore germination in T. indica and T. horrida have been studied extensively (Chahal et al., 1993; Dupler et al., 1987; Krishna and Singh, 1983). Dupler et al., 1987, and Smilanick et al., (1985a) reported that germinating teliospores were remarkably durable, strong and resilient during the process even with wide swings in pH, temperature, and soil moisture including freezing and desiccation (Dupler et al., 1987, Biswas, 2003). The basidium (also called a “promycelium”) emerges through the ruptured wall of the teliospore and either produces basidiospores instantly, or elongates to over 500 μm in length, depending on the species and prevailing environmental conditions. Elongating basidia form retraction septa, confining the cytoplasm to the apical region. A terminal whorl of 10 to 150 primary basidiospores, also called primary sporidia, is formed. Castelebury et al., (2005) reported that the number of basidiospores formed per basidium varied considerably among different taxa and not all locally infecting species produce a large number of basidiospores, and the number of basidiospores also varied considerably within a species (Castlebury and Carris, 1999; Mitra, 1931; and Teng, 1931). Basidiospores may elongate slightly after detachment, and become curved or undulate before germinating to form either hyphae or a sterigma-like structure on which a haploid, uninucleate, allantoids sporidium also called “ballistospore” ( Ingold, 1996) was asymmetrically formed ( Singh and Pavgi, 1972). The allantoids sporidia formed by T. indica, T. horrida, and other locally infecting Tilletia species studied had the same morphology and forcible discharge (Ingold, 1996, 1997). Allantoids sporidia may germinate directly via germ tubes or repetitively to produce additional sporidia. In culture, passively dispersed, filiform sporidia were formed from short, lateral sporogenous cells on the hyphae. Allantoids sporidia were the primary infective agents of T. indica but had not been studied in other locally infecting bunts. The role of the passively dispersed, filiform sporidia in the infection process is unclear. Dispersal of sporidia from soil level to ear head. Prescott (1986) reported that the microconidia or crescent shaped allantoids spores are the product of macroconidia produced by the chlamydospors present in the soil. The microconidia get deposited on the lowermost leaves by current of air and splash. The maximum trapping of the microconidia occurres in air samples during early morning hours when 100 % relatively humidity (RH) prevails. Nagarajan, (1991) explained that microconidia in the presence of leaf wetness produce a secondary crop of spores and as the leaf surface dries, these spores get dispersed to higher/upper leaves. Having climbed up the leaves through monkey jumps, they reach the flag leaf, some also get wind deposited and if at the boot emergence stage coincides with a mild drizzle or rain, and the microconidia get washed down into the sheath. Bedi et al., (1949) revealed that with presence of free water, once again crops of the microsporidia are produced. Before complete ear emergence, if more number of rains and favourable weather occurs causing run down, then more severe Karnal bunt develops. Occasionally, microconidia get lodged on the floret parts at the time of anthesis and if conditions are favourable they infect to produce Karnal bunt sorus on individual grains. According to Bains and Dhaliwal, (1988) spikelets of whear crop inoculated with sporidial suspension of Karnal bunt pathogen (N. indica) at the booting stage, produced secondary sporidia after incubation (intact/detached) under favourable moist conditions in the laboratory. Sporidia were also released from inoculated spikes in the field where sporidial release exhibited diurnal periodicity. With the help of electron microscope it was confirmed that viable and infective sporidia trapped in different experiments were invariabily of the allantoid type. Maximum sporidia developed on the outer glumes of florets. More sporidia were captured between 5 to 6 o'clock than later parts of the day but no sporidia were trapped between 14 to 18 o'clock. However, they could be trapped at any time of the day from the detached spikes incubated in the laboratory under favourable and moist conditions. Sporidia developed at 15 and 20°C but not at 30°C. These findings indicated that repeated cycles of sporidial production in spikes provided more inoculum than expected from soil-borne teliospores of N. indica. Infection process The Karnal bunt disease cycle is initiated with the germination of the teliospores at or near the soil surface, producing basidiospores that form hyphae, allantoids sporidia, and successive generations of sporidia. According to Prescott, (1986) and Sidhartha et al., (1995) the diurnal release of Tilletia indica sporidia occurs in the presence of high relative humidity mainly between 1800 and 0800 h but the most favorable at approximately 0200 to 0300 h . Some sort of “defense mechanism” has been suggested that insures the existence of sufficient basidiospores at the time of heading, such as a factor that triggers teliospores to germinate when the plant is heading (Warham 1988; Whitney and Rrederiksen, 1975). Otherwise, teliospores would undergo a hypothesized “suicidal germination”, if they germinate when the host is not in a susceptible condition (Rush et al., 2005, and Stein et al., 2005). Preliminary infection by T. indica occurs when sporidia accumulate on spikes and germinate to produce hyphae that enter stomata (Goates, 1988). Hyphae then penetrate intercellularly to the base of the floret and enter into the periderm of nascent kernels passing through the funiculus. Scanning electron microscopy studies revealed that the stomata of the rachis could also be essential path way for primary infection (Dhaliwal, 1989). However, solid information collected during the movement of hyphae within spikes indicates the rachis was not a regular site for initial infection (Rattan and Aujla, 1991). Nagarajan et al., (1997) stated that basidiospores or hyphae of T. indica frequently fuse to form a dikaryotic mycelium prior to attack on the host plants, and basidiospore fusion during culture has been reported (Krishna and Singh, 1983). During the study of infection process of T. indica visible hyphal anastomosis was only vary infrequently observed prior to penetration into the wheat plant, was not considered a normal means of dikaryon formation (Goates, 1988). Most probably, haploid hyphae are capable of infecting the host plant but the dikaryotic state must be attained for the development of teliosporogenesis (Duran and Cromarty, 1977). The stage at which the fusion of two nucleuses started in the T. indica was not identified. The method of penetration and timing of dikaryon formation in T. horrida are unknown, although Singh & Pavgi, (1973) suspected that sporidia lodge on the feathery stigma and enter into the style to reach at the chalazal end of ovary. Frequently the embryo is not colonized except in sever infection when the embryo is killed, and infected seed grains are often able to germinate (Singh & Pavgi 1973; Fuentes-Davila, et al., 1996). Teliospores of T. indica arise from sporogenous cells that generate a thin hymenial stratum on the surfaces of the cavities formed by the partition of the inner and outer layers of the pericarp and by the separation of the inner pericarp from the seed coat (Roberson and Luttrell, 1987). Teliospores develop at the terminal portion of sporogenous hyphae, where the dikaryotic cytoplasm becomes surrounded by a septum and karyogamy occurs during enlargement of teliospore of T. indica primary cells (Fuentes-Davila and Duran 1986). This was basically the same procedure observed in Tilletia species infecting systemically (Trione et al., 1989). The process of teliosporogenesis in Karnal bunt pathogen was influenced by temperature. Even after flourishing introduction of infection, utmost diurnal temperatures of 35o–40oC after the grain-filling stage decrease teliosporogenesis significantly. Goates and Hoffmann, (1987) reported that during initial stages of teliospore germination, the diploid nucleus undergoes meiosis followed by several rounds of mitosis, producing abundant haploid nuclei prior to the development of the basidium. In T. indica, about 64 to 128 nuclei were observed migrating into the basidium. Nuclei migrate into developing basidiospores, split synchronously, and one daughter nucleus returns to the basidium. These nuclei in the basidium then either entered empty basidiospores or degenerated. The sorting of 64 or more nuclei that were tightly grouped at the tip of the basidium followed by orderly migration of 1 nucleus into each of approximately 100 basidiospores is a remarkable feat of cellular mechanics. Each haploid cell of the basidiospore can produce hyphae, or form allantoid or filiform sporidia. According to Fuentes-Davila, 1984; Duran and Cromarty, 1977, T. indica is heterothallic and bipolar with four alleles controlling mating and pathogenicity. Variable numbers of chromosomes can be found among monospore isolates from single teliospores of T.

indica, indicating that chromosomal alteration or differential segregation occurs during

meiosis. Epidemiology and disease prediction models Karnal bunt disease spreads from season to season and various stages of disease development begin when teliospores of the disease become dislodged from infected kernels during harvest, become airborne, and settle in the field or spread to adjacent and remote areas with wind currents or through other resources. The classic work of Mundkur, (1943), and Bedi et al., (1949), revealed that natural infection by T. indica occurs through airborne sporadic (inoculum) during heading, but there is considerable disagreement on plant stages that are susceptible, and on the most susceptible stage. Results from a lot of studies provide the justification of susceptibility only during specific periods within boot swelling to anthesis stage (Aujla et al., 1986; Bains, 1994; Nagarajan 2001; Rush et al., 2004). However, Goates, (1988), reported that under natural conditions, the spikes were within the boot and airborne sporidia could not reach the glumes, where hyphal penetration was initiated. According to the recent studies of Goates, (2006) with Karnal bunt disease of wheat revealed that infection from airborne inoculum can occur when florets begin to emerge from the boot up to the soft dough stage of wheat kernel, and infection peak up after spikes had completely emerged, but before the on set of anthesis. These results are contradictory to studies claiming susceptibility prior to spike emergence (Kumar and Nagarajan, 1998; Nagarajan, 2001; Sharma et al., 1998 and Bains 1994) and it was the first report of susceptibility much beyond anthesis. According to Indu-Sharma and Nanda, (2003) teliospores of Karnal bunt when suspended over water, started germinating from 15 October onwards to March under field conditions. Environmental conditions were condusive frequently for the disease at vulnerable stage (ear emergence). Prolonged tenure of mist, fog or rain for 20 days in December postponed the teliospore germination and the sporidia lost the potential to cause the disease after a lapse of 45-50 days. Nutritional component of the medium in which sporidia were cultured, affected the disease inducing potential. Under specific climatic conditions of heavy dew or light rain, it appears that flag leaf and the boot sheath may be essential for normal infection (Aujla et al., 1986 and Kumar and Nagarajan, 1998). Dhaliwal et al., (1983), reported that after the primary infection, spread to adjacent florets had been occurred as late as the dough stage. The results of various studies on teliospore germination representing the wide range of environmental conditions conducive for teliospore germination indicate that it is not a limiting factor for disease development. The production of allantoids sporidia, which are the infective agents of the disease, seemed to be the primary factor in epidemiology. Bedi et al., (1990) reported that the optimum temperature and pH for germination and production of sporidia were 20oC and 8.0 respectively. He further described that pre- incubation storage of teliospores at 4oC for one week significantly enhanced their capacity to produce sporidia where as storage at 40oC or more were not conducive. White fluorescent light enhanced germination and no sporidia were formed under complete darkness. Disease-prediction models have been developed in the Karnal bunt disease affected countries that integrate the climatic factors that influence significantly production of sporidia including temperature, humidity, solar radiation, and rainfall (Jhorar et al., 1993; Jhorar et al., 1992 and Mavi et al., 1992). Germinating teliospores produces sporidia possessing thin cell walls that are considered to be short-lived and transitory structures sensitive to drought (Aujla et al., 1985; Nagarajan et al., 1997 and Smilanick et al., 1989). According to Smilanick et al., 1989, sporadia of T. indica at 95% relative humidity, survived no longer than 14 h. However, according to the experiments conducted by Goates (2006), T. indica, T. horrida, T. walkeri, and T. caries sporidia were remarkably durable. Sporadia collected from natural discharge in air dried conditions were viable for 30 days at 10–20% RH at 20◦–22◦C and after 60 days at 40%–50% RH at 18◦C, and newly formed sporidia were commonly observed germinating within 18 h of rehydration. It was observed that sporadia could survive in common field even, after several diurnal periods containing temperature above 38 oC associated with relative humidity below 10% and then rapidly produced hyphae under humid rainy conditions to infect host plants. Similar results were obtained in field experiments over 46 days at temperatures often exceeding 40oC and relative humidity as low as 10% (Lori et al., 2006). It was concluded by these results that teliospore germination prior to a vulnerable stage of host plant. The inoculum remains viable in dry field conditions, which can regenerate rapidly during humid conditions normally associated with disease. According to Sansford, (1998) weather extremes (extremely hot, extremely dry, or very humid and cold) conditions are not favorable for development of Karnal bunt disease and are considered to be limited to moderately cool climates (Jhorar et al., 1992; Sansford, 1998; and Diekmann1998). However, T. indica had never been reported outside of its’ allocation in the southwestern US and Mexico in spite of the historic movement of wheat grains throughout the North America. This relatively limited distribution of Karnal bunt could be attributed to specific weather conditions affecting on the life cycle of the fungus (Diekmann, 1998). Krishna and Singh, (1982) reported the optimum environmental conditions for germination of teliospores of T. indica and concluded that 15-200C as adequate temperature under alternate light and dark regime. Singh, (1994) suggested that successful infection was dependent upon the suitable weather conditions during flowering stage (inflorescence) of wheat plants, which is considered the most vulnerable stage to infection, and optimum temperature range for teliospors germination is 15 to 250C. Smilanick et al., 1985a, reported that the optimum temperature after 3 week incubation in continuous light was 15 to 20oC, over a pH range of 6.0 to 9.5. Moisture was a critical factor in determining weather in disease development (Singh, 1994; Smilanick et al., 1985a and Gill et al., 1993a). Singh, (1994) reported that 82 % relative humidity and preferably free water was required for the teliospores germination. He further added that with high humidity and rainy weather during 2-3 week window at flowering, infection of wheat kernels and individual seed grains increases. In the laboratory tests, survival of teliospores was noted after freezing over several months, but with delayed or reduced germination, (Chahal and Mathur, 1992; Zhang et al., 1984). According to Diekmann, (1993) moisture at the time of flowering may be the most critical element for the establishment of Karnal bunt disease in U.S.A. Prediction made from environmental data and prerequisite of infection by pathogen suggest that, under current climatic conditions, Karnal bunt would never cause major crop losses in the U.S.A. Generally, teliospores at or very near the soil surface germinate in optimum conditions, and consequently release secondary sporidia that become air-borne and infect the host plant at flowering. However, the exact details of many steps of the disease development are still poorly understood and are considered a matter of opinion or conjecture. Krishna and Singh, (1983) reported that teliospores of T. indica survived longer on the soil surface than in soil, and teliospore survival decreased with increased soil depth. No teliospores survived after 27 months at a depth of 15 cm in Pantnagar, India. But in the experimental studies of Babadoost, et al., (2004) it was indicated that teliospores survived for more than 32 months at all three depths (2, 10, and 25 cm) in Montana. There was non significant influence of soil depth on teliospore recovery from soil. Even after a period of twenty months, germination of teliospores recovered from various soil samples buried at 25 cm was significantly higher than those of samples buried in the soil at depth of 2 and 10 cm. The differences between the report by Krishna and Singh, (1983) and the results from studies of Babadoost et al., (2004) may be consequently attributed to the effects of soil moisture and temperature on survival, viability and germination of teliospores, as reported by Rattan and Aujla, 1992. When Babadoost et al., (2004) stored infested soil samples with the teliospores of Karnal bunt fungus in the laboratory in Bozeman, teliospore revival decreased as long as the soil samples were wet. It was also pointed out that temperatures at depths of 2 and 10 cm were higher than those at 25 cm during summer. The Pest Risk Analysis Panel of the North American Plant Protection Organization (NAPPO) estimated the risk of Karnal bunt disease, establishment in commercial wheat growing regions of North America during October 2001. Critical factor of environments conducive to induction of disease development were acknowledged in coincidence with time of occurrence, in phonological terms, with potential disease severity to categorize areas at risk of disease establishment. Data were stratified by the phonological window matching to the critical stage of wheat anthesis which was considered the vulnerable period for each country across North America. Data were analyzed, characterized, and related to disease triangle including to production features, like yield and cropping constituency of wheat, climatology, known disease distribution (survey results), relevant wheat production technology, cultivation practices, chronological disease dynamics and environmental conditions leading to epidemics of disease. The risk model confirmed that majority of the wheat growing areas in North America were not highly susceptible to the establishment of the disease most of the time. Limited area had been acknowledged where risk may be occurred medium or high. By the analysis of existing climatic conditions and planting patterns during winter and spring wheat it was concluded that vulnerable period did not generally coincide with environmental factors condusive to Karnal bunt disease at the greater part of North America. The majority of wheat production territories in Canada and the United States keep up a correspondence to the lowest risk category for the disease. This was true for both winter and spring cultivated regions of both countries. (Anon. 2001). Various models to estimate risk of establishment of Karnal bunt in different countries have been developed (Kehlenbeck et al., 1997; Diekmann 1998; Murray and Brennan 1998; Sansford 1998; Baker et al., 2000). The Humid Thermal Index (HTI) model as a tool for determining potential disease distribution On the basis of prevailing climatic conditions, there are examples for determination, prediction and mapping the potential distribution of diseases. An example of mapping the potential distribution of disease based on relevant environmental indices is provided by studies of Karnal bunt disease of wheat. The disease has been established in regions of southern USA. A number of pest risk areas (PRAs) had been selected to determine whether the disease could be established other areas through grain shipments. The HTI is a disease forecasting model developed by (Jhorar et al., 1992). In this model he found a high correlation between a humid thermal index (HTI) and Karnal bunt disease development over a period of 19 years in the central areas of Punjab, in India. The HTI can be defined as the relative humidity (RH) in mid-afternoon divided by the maximum daily temperature, calculated at the time between first awn visible to half inflorescence emergence. This model was used in the European Union (EU) sponsored to Karnal bunt programme as a device to determine potential distribution within Europe (Sansford et al., 2006). The HTI model has also been as a source to predict potential induction of Karnal bunt in Australia (Murray & Brennan, 1998; Stansbury & McKirdy, 2002) and South Africa (Stansbury & Pretorius, 2001). The model was devised when it was considered that there was a strong positive relationship between disease intensity and both average daily temperature and relative humidity during the month of anthesis of wheat crops (Jhorar et al., 1992; Mavi et al., 1992). An HTI between 2.2 and 3.3 was shown to be particularely favourable for the disease. The HTI has been used to determine prevailing environmental conditions prior to anthesis of European wheat crops for infection and this finding has been extrapolated to predict that disease could establish on susceptible wheat varieties in many areas of Europe (Baker et al., 2005; Sansford et al., 2006). After the analysis of long-term, average data, the HTI model predicts that a number of places in Pakistan, India, Iran, Arizona and South Africa had unfavorable climatic conditions for Karnal bunt despite the disease being present. This contradiction is justified by the reasons that local temperature and relative humidity levels are modified by irrigation (Stansbury & McKirday, 2002). Secondly in some parts of the Punjab wet and humid conditions favor teliospores germination, consequently inoculum is depleted at the stage of crop development when the wheat is not susceptible to infection (Sharma & Nanda, 2003). Teliospores at or near the soil surface possess a level of hypersensitivity to environmental conditions and soil moisture and temperatures of around 5–20oC can stimulate germination (Sansford, 1998). According to Smilanick et al., (1985b) only the teliospores that germinate within the 2 mm of the soil surface will release sporidia that are capable of spreading to canopy of host plants. However if favourable conditions are present for the germination of teliospores other than at the vulnerable period of crops for infection, teliospores undergo ‘suicidal germination’ and the teliospore population will decrease without inducing disease. In contrast, if suboptimal climatic conditions for teliospores germination remain dominant for most of the growing season of crop, teliospores are more likely to survive longer with the potential to become an inoculums’ source for next crop infection (Bonde et al., 2004). A long dry season in hot arid and semiarid climate may be an important suboptimal weather element or device to inhibit ‘suicidal germination’. The dark- coloured, hardy nature, thick-walled teliospores endure harsh, dry summer conditions during the post-harvest period (Singh, 2005). Dormancy accompanied by dry period ensured that large numbers of teliospores remained ungerminated until water stimulates germination during the next growing season. Seasonal rains or irrigation water are required for wheat production in dry regions and, because teliospores act as a reservoir of inoculum by releasing sporidia in areas where the disease is prevalent. However, the situation is different in environments in Europe where rain is more evenly distributed throughout the year than in warmer regions. Consequently, persistent moisture may also stimulate microbial antagonism and degradation that may affect viability and reduce the longevity of teliospores. Therefore, in spite of favourable weather circumstances, prior to anthesis of wheat crops there may be no great reservoir of inoculum capable of sustaining the disease because of a gradual depletion of teliospore reserves. Even if infection did occur under favourable low teliospore population levels would result in reduced disease incidence and increase the possibility of pathogen extinction (Bonde et al., 2004; Garrett & Bowden, 2002). Diekmann, (1993) reported three temperature related parameters as sufficient for discriminating environmental indices where the disease had established and those where it had not. They were (1) the difference between mean daily maximum and minimum temperature in the month of planting, (2) the mean daily maximum temperature in the month of flowering and (3) the mean daily minimum temperature in the coldest month of the year. Due to some drawback this model it was criticized by (Sansford et al., 2006) as it was not based on temperature parameters only, neither rainfall nor soil moisture was included as an essential parameter. Jones, 2007 concluded that the climate throughout the year is important and not just climatic conditions prior to antheis. There is a range of sometimes conflicting information available on how abiotic factors during rest of the year influence the survival of pathogen in establishment of Karnal bunt disease of wheat as summarized by Warham, (1986). With respect to the suitability of the climate in the Pest Risk Analysis (PRA) area, Warham, (1986) came to conclusion that low temperature and high humidity were essential at wheat anthesis for the infection to occur, while dry weather, high temperature and bright sunlight were not favorable for infection process. Rainfall is necessary but rain on its’ own at flowering was not sufficient to cause infection, suggesting that a specific combination of environmental factors was required. Crop irrigation is an additional factor favouring disease. Jhorare et al., (1992) devised a model for the prediction of Karnal bunt disease in the central Punjab, India, by an empirical method. A study of the associations between incidence of Karnal bunt disease and meteorological factors, using chronological meteorological data and data pertaining to disease intensity for Ludhiana district, in the central plains of the Punjab, was made for the reproductive stage of wheat crop. The period studied corresponded to flag leaf emergence (starting 12 February in Ludhiana) and subsequent stages of host plant ending on 18 March. This corresponded to the most important period for the pathogen, when teliospors that germinated at the time could lead to the production of infective sporidia that survived to infect the host and for the Karnal bunt to develop. The first meteorological model used to assess the risk of Karnal bunt infection was developed by Jhorar et al., (1992). They showed that there were non- significant relationships between the Karnal bunt disease development and maximum daily temperature and sunshine duration during the vulnerable stages leading up to anthesis for wheat (for India this was the 9th-11th standard meteorological weeks (SMW). “The two most important factors were determined to be mean maximum daily temperature (r =0.88) and 3 pm RH (r =0.93). The sunshine duration (r = -0.73) was negatively related, while number of rainy days (r = 0.71) were positively related. Regression analysis showed that evening relative humidity and maximum temperature can be put in a disease model as independent variables in simple regression equations. The data were then used to develop a HTI forecasting suitability for disease establishment and spread. The HTI was the monthly average 3 pm RH, divided by the average monthly maximum temperature, for the month leading up to anthesis of wheat crop. The most significant relationship was found between disease intensity and evening relative humidity (A), maximum temperature (B), and the “Humid Thermal Index” (A/B). A best-fit model was developed for forecasting the severity of Karnal bunt in the central Punjab thus: (1) DI = -0.8 + 1.5 HTI (2) HTI = ERH ÷ TMX Where DI = Disease index, HTI = Humid Thermal Index, ERH = evening relative humidity recorded at 14.30 hrs (average of the third, fourth, and fifth reproductive weeks), TMX = maximum temperature (average of third, fourth and fifth reproductive weeks). Jhorar et al., (1992) concluded that the HTI during this part of growing period varied between 1 and 5; the lowest values occurring in extremely dry and warm environment, and highest values representing extremely humid and cold weather, neither of which favoured the disease. An HTI of 2.2-3.3 throughout the third and fourth week of the study period favoured the disease. These conditions resulted from frequent cloudiness and irregular showers which could be predicted, thus permitting a disease forecast to be made. The second meteorological model for assessment of optimum conditions for the Karnal bunt disease was developed for the Pacific Northwest, USA by Smiley, (1997) who noted that temperature and relative humidity were essential for the region, but stressed the significance of specific weather factors such as suitable rainfall and related humidity levels that were essential for teliospore germination, secondary sporidial reproduction, its penetration and infection. Smiley, (1997) reported that utility of these events being synchronized with three to four week vulnerable period leading upto wheat anthesis. Suitable rain and humidity events were defined as: “1. Measurable rain (>3 mm) occurring on each of two or more successive days; 2. At least 10 mm being collected within 2-day interval; 3. Average daily RH must also exceed 70% during both rainy days”. In order to predict the likely risk the pathogen establishing Pest Risk Analysis (PRA) area, the 1996 UK PRA applied the HTI used climatic data from individual meteorological stations in UK and showed that conditions during the “heading” period (broadly speaking May and June) were favourable for infection and disease development, i. e. the majority of the calculated HTI values fell within 2.2 and 3.3. Kehlenbeck et al., (1997) also calculated the HTI for the wheat growing areas of Germany and found that some of the southern areas had HTI values which fell within optimum range for disease development. Murray and Brennan, (1998) also used this methology for Australia. They assessed the locations for potential development of Karnal bunt in Australia. Data for average monthly maximum and minimum temperatures and 3 pm relative humidity were obtained from the Bureau of Meteorology (http://www.bom.gov.au) for 122 wheat growing regions in Australia. The data were compiled up to the end of 1996. Times of sowing and stage for anthesis development were collected from agronomists and wheat breeders of concerned region. The humid thermal index (HTI) was computed for each month coinciding anthesis occurrence at each location, as the ratio of the monthly average 3 pm relative humidity to the monthly average maximum temperature. It was noted that out of 122 sites within Australian wheat belt, 67 had HTIs condusive for the Karnal bunt disease establishment. The location was estimated for potential development of disease as follows: “(i) if HTI < 2.2, locality is too hot or too dry (ii) if 2.2 £ HTI £ 3.3, site is appropriate for Karnal bunt disease (iii) if HTI > 3.3, site is too cold or too wet of the 122 localities, 46 were of category (i), 67 were of category (ii) and 9 were of category (iii). The 67 “reasonable” sites were through the southern wheat growing regions from Western Australia to central New South Wales. Category (i) sites or localities were in Queensland, northern New South Wales, the Victorian and South Australian Mallee, and the northern inland wheat belt of Western Australia. The cool and wet localities, category (iii), were outside the main wheat producing areas, on the south coast of Western Australia, southern Victoria and the north coast of Tasmania”. As the sowing time and growth development stages of wheat crop vary from country to country, region to region and even locality to locality with in a country therefore, life-cycle of the pathogen differ with time between Asian and European countries. The Australian, United Kingdom, and German studies were conducted using individual data. For the improvement of this work further, Baker et al., (2000) undertook provisional meteorological mapping using interpolated environmental data (1961-90) with adjustments in the calculation to allow for the lack of mid-afternoon relative humidity measurements. The result showed that for June, an area covering much of central and southern England had HTIs falling between 2.2 and 3.3 which was therefore, considered using this factor alone, favourable for Karnal bunt development in the crops phenologicially susceptible at that time. There is an existence of strong relationships between environmental conditions at anthesis and establishment of Karnal bunt disease of wheat for sites in India. Mavi et al. (1992) developed a model based on the average maximum temperature during mid to late anthesis (-ve correlation), the “evening relative humidity” (presumably 2:30 pm, +ve corr.) and sunshine duration (-ve) during early to late anthesis, and the number of rainy days in early anthesis (+ve). Although this model has R2 of 0.89, it is likely to be location specific due to the inclusion of sunshine hours and therefore not directly applicable to Australian conditions. Diekmann, (1993) launched “geophytopathology” methology to develop a correlation between Karnal bunt likelihood and (i) the distinction between the mean maximum and minimum temperature in the month of crop sowing; (ii) the average daily minimum temperature in the coldest month of the year; and (iii) the average daily maximum temperature at anthesis of wheat crop. Diekmann compared localities in the world where T. indica did and did not occur for the development of the model. Stansbury & McKirdy, (2002) compiled two previously established meteorological modelling techniques while determining areas in Western Australia (WA) where circumstances were condusive for infection of wheat crop by Karnal bunt pathogen. A strong correlation (r = 0.83) was found between the rainfall model, and the Humid Thermal Index (HTI) model, which used average-monthly data. Rain fall model was developed and based on the per cent opportunity of at least three Suitable Rain Events (SRE) during the vulnerable stages (August to October) of wheat crop. It was concluded that northern wheat growing belts are too hot and dry (HTI < 2.2, chance of SRE 15– 27%), southern regions are unimportant due to too cold and/or wet (HTI > 3, chance of SRE 68–97%), eastern regions are marginal to too hot and dry (HTI around 2.2, chance of SRE 24–50%), and western wheat growing sites are appropriate (HTI between 2.2 and 3.3, chance of SRE 41–78%). Analysis indicated that between and within year infection due to T. indica was more likely to establish if anthesis occurred in northern areas in August, in October in southern regions, in September in eastern belts, and in August, September, and October in western and south-eastern wheat growing localities. Stansbury and Pretorius, 2001, used the meteorological modeling techniques to determine suitable condition for the development of infection, and influence of irrigation schedules in South African wheat growing belt for the favourable environment for Karnal bunt pathogen. Only rainfed spring wheat in the western and southern wheat production areas of the Western Cape experienced climatic suitability to Tilletia indica development. Humid Thermal Indexes (HTI) experienced an appropriate range of 2.2 to 3.3 (HTI range 2.09-3.20, mean 2.58) and most regions indicated at least one Suitable Rain Event (SRE) (range 0.78-2.44) during the vulnerable period of crop. In distinction to central and eastern wheat production belts in South Africa were dry and warm under natural circumstances (HTI range 0.61-1.67, mean 1.06) possessing less than one SRE (range 0- 0.63) for early and mid-sowing wheat crop in these regions. It was concluded that the late sowing of wheat in irrigated areas experienced less condusive climatic conditions for the prevalence of Karnal bunt pathogen. This may not be applied to wheat growing regions where crops are irrigated very 24 h, as minimum critical relative humidity (RH) for the security and the chances of survival of T. indica spores would potentially be increased. A linear disease prediction model was devised by Nagarajan, (1991) for the climatic suitability of Tilletia indica infection by using average weekly weather had a R2 value of 0.89 indicating an appropriate degree of fitness. For the condition of North West India, Y= 0.4381+2.97a-2.77b-0.09c=0.13d; where Y= is the predicted Karnal bunt severity on bread wheat and the variable a to d, represent rainfall duration between 15-20 February, rainfall during 2-28 February, amount of rain days between 5-20 February, amount of rain between 22-28 February, respectively. When an analogous exercise was carried out for Mexico where Karnal bunt disease developed in Sinaloa and Sonora states, the R2 value was 0.91 and the model when validated was found to indicate a reliable forecasting Y= -0.71 +1.6A + 0.95B + 1.07C + 0.20D; where A to D stand for the number of rainy days between 1-7 February, 8-14 February, and 22-28 February; and total magnitude of rainfall between 21-28 February, respectively. Though there are minor differences between both the models for fractional coefficient values, the common denominator is that the number of rainy days during a particular period of February when the ear head emerges. Sources of genetic resistance against the disease. The most successful, efficient and economic method of disease control is through host plant resistance. Screening is carried out by creating artificial epiphytotic circumstances at boot leaf stage. Studies with Tilletia indica aimed at developing trustworthy and practical methods of inoculation for screening germplasm have demonstrated the highest rate of infection occurred by hypodermically injecting a suspension of sporidia into the wheat boot at awns-emerging stage, or slightly before (Aujla et al., 1982, Aujla et al., 1986; Royer and Rytter, 1988; Bains, 1994;). Krishna and Singh, (1982a) adopted injection technique at several phonologic stages of wheat growth from panicle initiation to early milky stage using sporidial suspension and concluded that the most susceptible stage for inoculation was when awns were just emerging. During the artificial inoculations in wheat crop in the field, use of more precise propagules of the allantoids secondary sporadia had required, in order to attain reliable high level of infection (Singh et al., 1988; Fuentes-Davila et al., 1993). The susceptibility of bread wheat to Karnal bunt fungus had been well acknowledged by the work of Fuentes-Davila, et al., (1992) achieving infection levels greater than 50 % under artificial conditions; therefore it is significant to continue evaluating new advanced lines and cultivars, as a measure to overcome the problems, and guidelines for the release of commercial use. However, bread wheat cultivars that had shown constantly low levels of infection were identified to occur (Fuentes-Davila and Rajaram, 1994). According to Bedi et al., (1949) and Fuentes-Davila et al., (1992) sources of resistance against the Karnal bunt disease were also present in durum wheat and triticale germplasm under natural and artificial inoculated conditions. Villareal et al., (1994) during an evaluation with three crop cycle under artificial conditions, concluded that 49 % synthetic hexapolids (SH) were immune to the disease. Villareal et al., 1996) identified 4 SHs as immune sources during a work to evaluate elite bread wheat lines, synthetic hexaploid wheat derivatives, commercial varieties and candidates to release for resistance against Karnal bunt disease of wheat under artificial inoculation. Mujeeb, et al., (2006) evolved immune synthetic hexaploid wheat by crossing (hybridizing) randomly high yielding durum wheat cultivars with several Aegilops tauschii accessions. The F1 combinations were advanced by conventional breeding protocols to F8. Crosses between Triticum aestivam and Aegilops tauschii accessions resulted in F1 hybrids with 2n=3X=21, ABD plants. These hybrid seedlings after colchicines treatment led to hexaploid C-0 (2n=6X=42, AABBDD) seed formation. Stable plants with 42 chromosomes called synthetic hexaploids were screened against biotic stress. An elite SH group of 95 advanced lines was prepared from the initial 420 SH wheats. These 95 germplasm lines were distributed by CIMMYTs’ germplasm bank. Supplementary repositories were at the Kansas, State University Wheat Genetic Resource Faculty, Manhattan, Kansas, USA and at National Agricultural Research Center, Islamabad, Pakistan. High level of resistance in the elite SH wheats were maintained during the Karnal bunt stress screening. The durum cultivars involved in these SH combinations were generally susceptible under the severe greenhouse inoculation tests. The corresponding SH wheats showed immunity under similar conditions. Singh et al., (2003) in his studies for mapping a resistance gene effective against Karnal bunt of wheat reported that most sources of genetic resistance to KB were traced in China, India and Brazil, Gill et al., 1993, Fuentes-Davila et al., (1995) and Singh et al., (1995b) also had same conclusions. In India Indu-Sharma et al., 2005, reported the genetics of Karnal bunt (KB) resistance in populations derived from crosses of four resistant stocks (HD 29, W 485, ALDAN 'S'/IAS 58, H 567.71/3*PAR) and a highly susceptible cultivar, WH 542. The plant materials screened for KB response consisted of F2, BC1 and RILs from all 'Resistant' x 'Susceptible' crosses and RILs from the six possible 'Resistant' x 'Resistant' crosses as well as the parents and F1s. The screening was performed under optimal conditions for disease development with a mixture of isolates from North Western Plains of India using the widely followed syringe method of inoculation. The KB scores of the F1 from the four 'Resistant' x 'Susceptible' crosses indicated partial dominance of resistance. Genetic analysis revealed that HD 29, W485 and ALDAN 'S'/IAS 58 each carried two resistance genes whereas 3 genes were indicated in H 567.71/3*PAR. The six 'Resistant' x 'Resistant' RIL sets showed that the genes in the four resistant stocks were different and that there may be as many as nine genes governing KB resistance in the four parents. Aujla et al., (1989) devised rating scale for identifying wheat cultivars resistant to Karnal bunt disease of wheat. They categorized wheat varieties on the basis of severity and response to T. indica into five classes ranging from highly resistant to highly susceptible and expained the procedure for calculating the coefficient of infection with the help of grades of infection and numerical values of infected grains. Dhaliwal et al., (1986) concluded that germplasm of wild wheat, and Aegiolops agropyron possess the resistance to various diseases including Karnal bunt disease of wheat. Triticum urartua was resistant to T. indica and some Aegiolops. Warham, (1988b) conducted screening against Karnal bunt for resistance in wheat, triticale, rye and barley. None of the bread wheat lines were immune to Karnal bunt disease. Gartan, et al., (2004) during detection of multiple resistance sources concluded that among 100 advanced breeding lines of wheat (T. aestivum) and triticale (Secale cereale), the genotypes HS450, HS455, HPW232, VL861, PW731, PW733, PW738 and PW739 had multiple resistance against a number of wheat diseases including yellow (Puccinia striiformis var. striiformis), brown rusts (Puccinia recondita), powdery mildew (Erysiphe graminis) and Karnal bunt (Tilletia indica). The evaluation of these genotypes may be used in crossing programme with other agronomically superior varieties to incorporate the genes of desirable characters to obtain high yielding, disease resistant varieties. Souza et al., (2005) reported a resistant germplasm line IDO602 (Reg. no. GP- 776, PI 620628), resulting from a first backcross of the hard white spring wheat Borloaug M95 onto the hard red spring wheat Westbred 926, in Idaho, USA, and delivered it in February 2003 for use in research and crop improvement programmes. It is semi-dwarf wheat with resistance to Karnal bunt (T. indica) and stripe rust (Puccinia striiformis). Sharma et al., (2004) obtained a Karnal bunt resistant wheat stock ('KBRL22') germplasm from a cross of two resistant lines ('HD29' and 'W485'). They used it as a donor for introgression in KB-free trait into 'PBW343' (an 'Attila' sib), the most widely grown wheat variety in India. The numbers of KB-free and KB-affected plants in BC1, BC2, BC3 and BC4 as well as the F2 were obtained after artificial inoculations. The segregation pattern in these generations clearly indicated two independently segregating, dominant genes which jointly confer the KB-free attribute. Kaur and Nanda, (2002) evaluated wheat genotypes WL 711, WL 1562, HD 2329, PBW 343, PBW 396 and WH 542 along with resistant genotype HD 29 for susceptibility to Karnal bunt disease (T. indica) during 1995-2002 by artificial inoculation. Results showed that WL-711 was the most susceptible to disease. Singh et al., (2003) obtained resistant sources of wheat from Advanced Varietals Trials and other sources like CIMMYT and were tested at hot spot multilocations under artificially inoculated conditions for Karnal bunt (T. indica) establishment during 1990/91-1993/94 in Indian Punjab, Himachal Pradesh, Haryana, New Delhi and Uttar Pradesh, India. Of the 66 entries, 18 were resistant lines, namely HD 29, HD 30, HD 2385, RAJ 2296, WL 1786, WL 6975, WL 7247, HW 502, PBW 34, PBW 225, W 285, W 382, W 388, W 485, DWL 5010, ND 589, ND 602 and HP 1531. PBW 34 and PBW 225 were released varieties. Nineteen lines received from CIMMYT were also resistant to Karnal bunt. HD 29 and HD 30 had already been registered by the National Bureau of Plant Genetic Resources, New Delhi, as INGR 99012 and 99011, respectively. Indu-Sharma, (2001) demonstrated that out of 43, 680 germplasm lines of bread wheat (Triticum aestivum L. emend. Fiore & Paol.) tested against Karnal bunt disease of wheat, 744 lines expressed stability for resistance. Out of 188 strains possessed multiple resistances to Karnal bunt, brown (Puccinia recondita Rox.ex. Desm.) and yellow (P. striiformis) rusts and were agronomically superior. Six Karnal bunt-free ('KBRL 10', 'KBRL 13', 'KBRL 15', 'KBRL 18', 'KBRL 22', 'KBRL 24') and 3 high- yielding Karnal bunt-resistant wheats ('W 7952', 'W 8086', 'W 8618') were developed by pyramiding of Karnal bunt-resistant genes and pedigree method of breeding respectively. Jafari et al., (2000) inoculated spikes of 26 wheat advanced cultivars/lines by injection of a suspension of secondary sporidia of T. indica at booting stage. Coefficient of infection and percentage of infected grains for each entry were estimated, they were ranked into four groups for their responses to the disease. None of the entries was completely resistant the Karnal bunt disease and most of them were vulnerable. Cultivars Pastour, N-75-3 and N-75-5 were partially resistant. Darab-2 with 68.1% of infected grains and a coefficient of infection of 44.1, was the most susceptible cultivar, Atrak and Niknejad very susceptible and Falat susceptible. A significant correlation was found between coefficient of infection and percentage of infected grain for each entry and the regression model was calculated. Beniwal et al., (1999) studied the effect of three different sowing dates on Karnal bunt development was studied in ten commercially grown wheat varieties in India during 1995-96. The coefficient of infection was greater for crops sown on 16 November than for crops sown on 16 and 31 December. The varieties C.06 (31.96%), WH 147 (31.16%), HD 2009 (26.93%) and HD 2329 (20.93%) had a greater mean coefficient of infection compared to WH 283, HD 2285 and WH 896. Weather parameters including mean temperature (19.5 oC), relative humidity (62.6%) and rainfall (66.9 mm) spread over 6 rainy days from the date of inoculation to harvesting for crops sown on 16 November made them more susceptible to infection than those sown on 31 December, which had a mean temperature of 23oC, relative humidity of 53.7%, and 31.6 of mm rain (3 rainy days only). In Pakistani wheat germplasm generally there is scarcity of resistance against Karnal bunt disease of wheat. Out of 141 wheat varieties/lines evaluated by Ahmad et al, (1999) at the Wheat Research Institute, Ayub Agricultural Research Institute, Faisalabad, Pakistan, during 1993-94, four lines, T-91731, T-911734, T-91736 and T-91740, remained free from karnal bunt infection caused by N. indica (T. indica). T-92733, T- 91729 and D-91690 were moderately resistant while 51 showed a moderately susceptible response. Thirty nine varieties/lines were susceptible and 44 highly susceptible. Iftikhar et al., (1988) during screening of wheat germplasm against T. indica concluded that out of eighty cultivars evaluated only three entries T.C. L.83740, V-86354 and V-86257 were totally free from every kind of infection and thus they were declared to be immune. However, none of the entries was found to fall in resistant class. Eight germplasm lines, i, e. V-85003, V-85028,V-85255, V-85409, V-86231, V-86326, V-86369 and V-86371 exhibited moderately resistant, 10 moderately susceptible (V-83134, V-83156-3, V- 83035, V-84021, V-85195, V-85165, V-85060, V-83152-1,V-85283 and V-86357), 21 susceptible (Dirk,Mexi-Pak-65, SA-75, B. Silver, Lyp.-73,Pb.85, C-591, C-518, C-271, C-217, C-273, Yaccura, Suitleg-86, Sandal, V-85096, V-84658, V-86115, V-86061, V- 852992,V-86184,V-87240) and 38 were highly susceptible (Arz, Pavan, LU.26, Lu.26 S, LU.26 S-1, WL-711, Indus-79, Pak-81, Pb.81, BWp-79, K.Noor-83, Morrocco, Potowar, Barani-70,Chenab-79, SA-42, Pb.76, C-228, Fsd-85, Wandanak, Pari-75, Chakwal-86, Barani-83, V-83171, V-84140, V- 85078, V-85205, V-85072-1, V-84133, V-85054, V- 86215, V-86299, V-85405, V-86303, V-86124, V-86124, V-86240 and V-87239. Chemical control of the disease Karnal bunt is very difficult to control when it is present in wheat area. Seed treatments that are used to control other bunt and smut diseases of wheat are generally ineffective because these only protect the wheat crop at seedling stage. The fungus T. indica, rather infecting the host plant or seedling as in the case with other smuts, infects the seed in the head as it emerges. Several attributes of the etiology of Karnal bunt and the teliospores of T. indica make control a very complex problem. Cultural practices that reduce the Karnal bunt incidence, such as delay in sowing date, reduced nitrogen fertilization or reduced planting density, only affect modest reduction in karnal bunt incidences and may themselves reduce yields (Gill et al., 1993a; Singh, 1994 and Warham and Flores, 1988 Rivera-Castaneda et al., 2001). Due to the hardy nature teliospors, are sturdy, long-lived and very resistant to chemical and physical treatments (Smilaninick et al., 1988 and Warham 1988a). They are seed borne and protected by the sorus and remainder of the partially bunted seeds typical of disease. According to Smilanick et al., (1989) teliospores masked in the soil persist longer than those of present on soil surface and are more sheltered from harsh climatic conditions and chemical treatments. Seed and soil treatments applied for the control of Karnal bunt have been only partially successful. Seeds treatments with fungicides do not kill the teliospores but inhibit their germination (Hoffmann 1986 and Warham et al., 1989). They have reported that fungicides applied to soil at seedling had not reduced the disease, probably because the infection originated from airborne infectious sporidia from the teliospores that germinated outside the test plots. According to Agarwal et al., (1993) numerous chemical compounds have actively against this pathogen by reducing the germination of teliospores. However, Anonymous (1997) proposed that the chemical seed treatments have proved ineffective in killing teliospores with the exception of mercurial compounds which are banned in most countries. Seed treatment does little to eliminate soil-borne inoculum. So in case of chemical control the only valid and the most effective option is the application of foliar fungicides at anthesis stage. According to Smilanick et al., (1987) seed treatment fungicides do not protect wheat plants from infection when seed are planted in teliospore-infested soil, and do not persist long enough within the plant to inhibit the infection of florets. During the evaluation of fungicides they estimated that out of eight seed-applied and 16 foliar- applied fungicides against the Karnal bunt of wheat more than 80 % control was obtained with two applications of either Propiconazole or etaconazole or four application of mancozeb or copper hydroxide when these fungicides were applied to wheat crop when the spikes were still enclosed (feeks’ growth stage 9), with the awns emerged about 1cm. Best control of propiconazole and etaconazole was obtained when they were applied 72 hours after inoculation rather than before inoculation. Foliar application of the fungicide has given control of Karnal bunt disease of wheat to some extent. Two or more application of propiconazol at or after spike emergence reduced the incidence of disease by 95 % (Aujla et al., 1989; Singh; 1994). Sharma et al., (2005) evaluated new fungicides in greenhouse experiments to determine efficacy against the karnal bunt disease of wheat and durum wheat, caused by T. indica. The treatments comprised: 0.05, 0.10, 0.20, 0.40 and 0.80% Folicur (tebuconazole); 0.05, 0.10 and 0.20% Contaf (hexaconazole); 0.05, 0.10 and 0.20% Tilt (propiconazole); 50, 100 and 200 g a.i. thifluzamide/ha; and the control, applied at 48 h after inoculation of sporidial suspension. Infected and healthy grains were counted in the inoculated ear heads and the percent infection was calculated. Folicur at 0.20%, Contaf at 0.10%, Tilt at 0.10% and 100 g a.i. thifluzamide/ha resulted in more than 90% Karnal bunt control, while Folicur at 0.40% and 0.80%, and Contaf at 0.20% resulted in 100% bunt control.. Rivera-Castaneda et al., (2001) during in vitro studies have reported the effectiveness of few commercial fungicides in inhibiting teliospore germination of T. indica. A number of native plants extracts from Sonora, Mexico, were tested to determine their antifungal activity against T. indica. Dichloromethane (DCM) and methanol (MeOH) extracts were incubated with the fungus to measure inhibition of mycelial growth. DCM extracts from Chenopodium ambrosiodes and Encelia farinosa reduced mycelial colony growth of the fungus, but total inhibition was obtained with 500 mg/ml of the DCM extract from Larrea tridentata. Teliospores subjected to this treatment showed no viability when transferred to fresh culture media. None of the evaluated extracts stimulated fungal growth. The extract from Larrea tridentata illustrated potential as control agent for T. indica. A study was carried out by Singh et al., (2000) from 1996 to 1999 wheat cropping season to evaluate fungicides for the control of Karnal bunt (T. indica) through foliar spray under field conditions to ensure healthy wheat seed production. Using a knapsack sprayer the highly susceptible cultivar HD 2329 was sprayed with following fungicides: propiconazole, hexaconazole, tricyclazole, flusilazole, thiophanate-methyl, cymoxanil and carbendazim at 0.05 and 0.1% dosage level. Among the fungicides, the maximum disease control (99.8%) was achieved by two sprays of propiconazole (0.1%) whereas a single spray controlled (97.68%) disease, followed by hexaconazole (94.40%) in the post-inoculation treatment. In the pre-inoculated spray treatments, maximum disease control (99.78%) was achieved with two sprays of propiconazole, while a single spray provided 96.46% control followed by hexaconazole (92.87%). None of the fungicides assured in a complete control of the disease. Flusilazole controlled the disease by 70.25-83.76%, but was phytotoxic to the wheat crop. Tricyclazole, cyamoxanil, carbendazim and thiophanate-methyl resulted non-significant in disease control. Two sprays of propiconazole (0.1%) at 15 days interval reduced the disease from 19.83 to 0.02 and 18.66 to 0.04% in post-inoculation and pre-inoculation treatments, respectively, which is below the level of international seed certification standard for foundation seed (0.05%). Goel et al., (2000) reported that a foliar spray of propiconazole (Tilt 25 EC) at 250 and 500 ml/ha, applied at the boot leaf stage, decreased Karnal bunt disease in wheat up to 78 and 87% respectively, in multilocation trials conducted during 1988-93 at New Delhi, Ludhiana and Gurdaspur (Punjab). Consequently it increased grain yield and was therefore, efficient for the management of this disease in the field. According to Salazar et al., (1997) out of 11 fungicides applied in varying combinations and concentrations to control T. indica infection of wheat in Mexico, propiconazole resulted the best disease control. However, based on costs and quarantine regulations in northwest Mexico, the use of propiconazole would not be economically profitable. Agarwal et al., (1993) found that a range of fungicides that gave significant control of sporidia on wheat crop if applied at the early heading stage. These included mancozeb, carbendazim, fentin hydroxide, bitertanol and propiconazole. Findings from current EU (European Union) research project (Anon. 2004b) reported that some active ingredients, particularly azoxystrobin as well as propiconazole were effective against both pre and post-infection by T. indica when applied as a single spray treatment at flag leaf ligule just visible (first awn visible) or caryopsis watery ripe stage. Krishna and Singh, (1982) evaluated the fungicides for the contol of Karnal bunt of wheat. In a glass-house test against T. indica Plantavax (oxycarboxin), Vitavax (Carboxin), Bayletan (triadimefon) and Bavistan (carbendazim) gave 82-87.55 % control when sprayed on the ear heads two days before inoculation of a sporidial suspension into the boot leaf. Krishna and Singh (1983) reported the chemical that enhanced the germination of teliospores of T. indica. Out of 16 treatments tested for obtaining abundant and early germination of teliospores from the diseased grains of wheat, the best were citrus and tomato juice, ether and hexan. Krishna and Singh, (1986) studied the effect of some organic compounds on teliospores germination and screening of fungicides against the Karnal bunt pathogen. Butyric acid and Lactic acid at 500 ppm. increased percent germination. Among the alcohols and solvents, methanol, ethanol n- butanol, ether and toluence induced early germination and xylene, hexane andchloroform also increased germination. Phenol 2, 4-dihydroxy cinnamic acid, Chlorogenic acid had stimulatory affect on germination. Mancozeb and Metalaxyl are considered broad spectrum fungicides, and exhibited a strong antisporolant activity against three major divisions of the fungi, Oomycota, Ascomycota and basidiomycota when applied before inoculation rather than post infection (curative applicative application). Metalaxyl (260 µg/ml) also provide complete control of disease when used in protectant mode. (Ammerman et al., 1992., Baldwin et al., 1996., Gold et al., 1996 and Mitsuhiro et al., 1999). Chapter 3 MATERIALS AND METHODS

Survey of Karnal bunt disease of wheat Three types of surveys were conducted in various wheat growing areas of the Punjab. 1). Survey of hot spot areas. 2). Survey of grain markets /storage in various Districts and Towns. 3). Survey of various Research Stations / Research Institutes Seed Corporation and Model Farms etc. (i) Field survey: With the collaboration of agricultural extension workers, farmer’s fields were visited in main wheat growing areas of the Punjab. Survey was conducted in the month of April-June, (2005-2006), which are the harvesting and threshing months of wheat in the Punjab, Pakistan. Farmer’s report about complaints of wheat disease in the fields proved to be the most helpful method in collecting the data about Karnal bunt disease in small towns’ villages and cities. On complaint report, farmer’s fields were visited on the spots during harvesting, threshing and transportation of wheat before putting the grains into storage godowns. A random sample from the edges of each infected field was taken in a replicated manner in the form of at least 20 heads from one acre rendering a fair representative of the entire field. The samples were packed in brown envelops before placing them into a collection bag. They were labeled with following conventions: 1) Unique field number, sampler’s name, growers name, date and place. 2) Identification code, accompanying sample worksheet, name of the district, name of extension worker and other necessary information required for fungus identification and data analysis in the laboratory. 3) Name of wheat cultivars/ line with its pedigree. The fields were located through cooperation of extension workers and close coordination with wheat growers and farmers. Special protocol for sampling was taken into consideration prior to carry over in disease diagnostic laboratory, to maintain sanitation without cross-contamination. To determine the variation in incidence of the disease in the various hot spot areas of the Punjab, 12 main wheat growing districrts were selected. The samples were taken from five various localities in each of the districts, and submitted to disease diagnostic laboratory of plant pathology section, Ayub Agricultural Research Table: 1. Rating scale used to calculate the severity of Karnal bunt of wheat. Infection Symptoms Coefficient category of infection 0 Healthy 0 1* Well developed point infection c*. 25 % seed bunted. 0.25 2 Infection spreading along the groove c. 50 % seed bunted. 0.50 3 Three-quarters of the seed converted to sorus. c. 75 % seed bunted. 0.75 4 Seed completely converted to sorus. c. 100 % seed bunted. 1.00 *categories combined for use in calculations of coefficients of infection Table: 2. An example of the calculation of coefficient of infection (from Aujla et al., 1989) Grade of infection 0 1 2 3 4 Numerical values 0 0.25 0.50 0.75 1.0 Number of grains 200 75 50 25 10 Multiplication with 0x200 0.25x75 0.50x50 0.75x25 1.0x10 numerical values Value after 0 18.75 25 18.75 10 multiplication Gross total 0.00 + 18.75 + 25 + 18.75 + 10 = 72.25 Total grains 200 + 75 + 50 + 25 + 10 = 360 Coefficient of infection 72.25x 100 ÷ 360 = 20.14

Institute, Faisalabad for confirmatory test through diagnostic scheme recommended by European and Mediterranean plant protection organization (EPPO) Anonnymous (2004c). To determine the severity of the disease a rating scale was used for Karnal bunt (T. indica), based on Aujla et al., (1989) and Bonde et al., (1996). (ii) Survey of grain markets Survey of grain markets in towns and big cities was conducted to assess the Karnal bunt disease in the territory. After counseling with dealers about the source (local or abroad) and history of the wheat grains, only the local sources of wheat of the territory were taken into consideration. Mostly the small and medium type dealers were selected for sample collection. Big dealers were neglected due to the apprehension of abroad source of wheat grains and mixture of varieties. To determine the precise estimate and analysis of variance for the incidence of disease in grain markets of the Punjab a second survey was conducted after an interval of ten days. During the survey 5 localities were selected with four replications (samples) in each of 24 districts. Sampling Seed lots were sampled according to International Seed Testing Association (ISTA) rules (EPPO, 2004). As the grains for feed or processing were typically more difficult to separate because consignments were usually very large, and transported or stored as large loose bulks. Therefore, for monitoring purposes, grains were sampled in an appropriate fashion to produce a 1–2 Kg thoroughly mixed sample representative of the consignment. The samples were taken from one year old and new lots. The most efficient and rapid wash test method for detecting teliospores in a sample, (size-selective sieving and centrifugation technique of Peterson et al., (2000) was adopted. Direct visual examinations for bunted kernels or teliospores contaminating seed surfaces were not considered reliable methods. However, Karnal bunt teliospores were also detected by visual examination with the naked eye and for the confirmatory test low power microscopy (10– 70X magnifications) was used. To help visualize symptoms, seeds were soaked in 0.2% NaOH for 24 h at 20 °C. This was especially useful for chemically treated seed lots where coloured dyes obscure symptoms (Mathur & Cunfer, 1993; Agarwal & Mathur, 1992). With severe contamination, teliospores had been seen on the surface of seeds (Mathur & Cunfer, 1993). Teliospores were soaked in water; quickly surface sterilized and then germinated on water agar plates. After 20-26 days they were shifted on PDA (potato dextrose agar) media. Identification was mainly based on morphological characteristics to avoid the possible confusion with similar species like Tilletia wlakri (ryegrass bunt) and Tilletia horrida (rice smut) (Durain and Ficher, 1961; Duran, 1987). After completing the entire possible requirement in diagnostic scheme i.e. isolation, detection, germination and confirmation of the culture, a number of replicated 50 g sub-samples were taken to evaluate % infection from all the positive samples. (iii) Survey of Agricultural Farms For the collection of entries and for the assessment of resistance/ susceptibility in various germplasm lines and varieties against the disease, experimental areas of various research institutes, agricultural research station, model farms, and seed corporations were visited. Survey was continued at an interval of 10 days from sowing to threshing of wheat during two consecutive growing seasons (2005-2006) and (2006-2007). With the collaboration and support of research workers from Plant Pathology section Ayub Agricultural Research Institute Faisalabad, germplasm lines/ varieties with all possible desirable characteristics (short duration, early maturing, high yielding, drought resistant, and cultivars possessing a degree of resistance to highly susceptibility to Karnal bunt disease) were selected for screening and further epidemiological studies. Most of the germplsm advanced lines/ cultivars were obtained from Wheat Research Institute Faisalabad, Tareen Model Farm Lodhran, Punjab Seed Corporation Khanewal, Jalindher Seed Corporation Sahiwal and Department of Plant Breeding and Genetics University of Agriculture Faisalabad. Screening of wheat germplasm for the source of resistance (i) Isolation and preparation of mass culturing of Tilletia indica During the survey one year old bunted grains of wheat infected with T. indica, were obtained from storage godowns of Tareen model farm at Lodhran, where wheat cultivar AS 2002 was badly affected by Karnal bunt disease, along with some other bunted old varieties, during the crop years 2004-2005 and 2005-2006. The disease had been appearing here for the last few years on old varieties. Free teliospores were obtained by gently crushing severely affected seeds, classified to category 3 to 5 (Warham, et al., 1986) with a mortar and

Fig. 1: After centrifugation teliospores sedimented into pellet in conical centrifuge test tubes.

Fig. 2: Disinfested teliospores by centrifugation with 5 % chlorox in capped test tubes.

pestle. For each infected samples a suspension of suitable magnitude of teliospores in 200 ml of sterilized water was made. Teliospores suspended in water were filtered through 120 mesh sieve, which retained the larger debris on the sieve. The filtrate was centrifuged at 3000 rmp for one minute which sedimented the spores into a pellet in 40 ml of the test solution in capped pre-sterilized, 50-ml-capacity conical centrifuges test tubes (Fig 1). Excess of water was decanted out. The teliospores were disinfected with commercial bleach (5% chlorx) in capped test tubes by centrifugation. The teliospores were again collected in the sort of pellet (Fig. 2), and chlorox was decanted out. The teliospores were rinsed twice in sterile water, each time recovering after centrifugation. Whole of the process was repeated three times to avoid the chances of contamination. A spore suspension was prepared by adding sterile water in the test tubes containing disinfected teliospores. One ml of the spore suspension with the help of macro-pipette was distributed to each of several Petri- plates containing plain agar medium (Fig. 3). The composition of plain agar medium was 20 gm agar dissolved in one litter of water. Inoculated Petri-plates were incubated at 15-20 0C with a regime of 14 hours light and 10 hours darkness. After 25-30 days of incubation, teliospores germinated and primary sporidia were visible on the surface of plain agar medium in the Petri-plates in the form of thread like or star like structures (Fig 4 & 5). Using this primary sporidial culture (Fig. 6), secondary sporidia were produced on PDA. For this purpose about 100 ml of autoclaved PDA was taken into each 250 ml flasks, and the media were solidified in slanting position in the flasks. The slants were then inoculated with a primary sporidial culture by gently placing an inverted culture block size of about 3 x 3 mm (making angle) on the upper edges of the slants. The inverted block of the culture facilitated the sporidia, frequently shedding on the medium (Fig.7, 8A & 8B). The culture slants in flasks were incubated at 18-20 0C. Within 5 to 6 days secondary sporidia started to discharge forcibly from primary sporidial culture in the form of brittle, crustaceous, unibonate colonies with dendric margins (Fig. 9, A & B). Thousand of the sickle shaped and allantoids types secondary sporidia were observed under the microscope. Field evaluation of wheat germplasm lines/ varieties for the source of resistance against Karnal bunt disease under artificial inoculation. Two hundred one advanced lines and forty three commercial cultivars or local varieties received from Wheat Research Institute Faisalabad, were sown in a single row sub.

Fig.3. Germinating teliospores after 10 days on plain agar petri-plates.

Fig.4 (Germination of teliospores after 20 days) in petri-plates containing plain agar.

Fig.5 (Germination status of tetiospores after 30 days) Germinating teliospores having star like structures of primary sporidia on plain agar.

Fig. 6 Germinating teliospores with long slenderical primary sporadia growing from the tip of promycelium (basidium) under power microscope. (100x 60)

Fig :7 Pattern of spore shedding (secondary sporadia) on PDA slants in conical flasks after second day of inoculation. A B

Fig. 8: (A and B) Pattern of sporadia shedding on PDA slants after third day of inoculation.

A

B

Fig. 9 (A & B): production of secondary sporidia on PDA slants after 6 days of inoculation.

Fig.10: Screening of wheat germplsm lines/ cultivars for the source of resistance against Karnal bunt disease under artificial inoculation condition and field evaluation of fungicides.

plots of 3 meter each (Fig. 10). There was 30 cm row to row spacing and 15 cm plant to plant distance. The test entries were boot inoculated by injecting 4 ml of the sporidial suspension of Tilletia indica with a hypodermic syringe starting from second week of February to mid March. Boot inoculation was carried following the procedure of Singh and Krishna, (1983a) and Aujla et al., (1983). Ten heads of each test line were boot inoculated. The inoculated plants of each line/ variety were tagged and labeled. To maintain the vigor of plants, nitrogenous fertilizers were used in a well balanced quantity, with a suitable interval of irrigation to lower the temperature and increase the relative humidity. All the agronomic practices were homogenously applied for all the entries. Inoculated heads were harvested at maturity between 20 to 30th of April. Heads of each line/ variety were hand threshed and the total numbers of healthy grains as well as bunted grains of the inoculated heads were counted and disease incidences were thus calculated. The levels of resistance/ susceptibility of the test cultivars were assessed by using the following modified disease rating scale of Aujla et al., (1989).

Table: 3. Rating scale used to determine level of resistance/ susceptibility.

Level of resistance or Disease rating scale % Grain infection susceptiblity

0 No infection on (panical) Highly resistant

1 1 % or less grains bunted Resistant

3 1.1-2 % of grains bunted Moderately resistant

5 2.1-5 % of grains bunted Moderately susceptible

7 5.1-10 % of grains bunted Susceptible

9 More than 10 % of the grains bunted Highly susceptible

Epidemiological studies (i) Cultivation of susceptible germplasm lines/ varieties for epidemiological studies During the survey for the assessment of Karnal bunt disease, about 66 susceptible germplasm lines/ varieties indicating various degree of infection (from minimum to maximum) were collected from various agricultural research stations for epidemiological studies. These were sown in experimental area of Department of Plant Pathology University of Agriculture Faisalabad during consecutive growing season 2005- 2006 and 2006-2007. The agronomic practices were adopted homogenously in both years, to keep the crop in good condition, producing conducive environment for the development of disease. No fungicide was sprayed so that the entries could get maximum chances for the development of Karnal bunt disease under natural conditions. The entries were early sown in the second week of November in randomized complete block design with four replications. There was 30 cm row spacing and 15 cm plant to plant distance. The data were recorded in both years from February to end of April. At maturity the kernels were picked from each entry and they were hand threshed. The incidence of the disease for each entry was calculated by using the following formula.

Number of bunted grains in 10 kernels Disease incidence = ______X 100 Total number of grain in 10 spikelets

(ii) Development of disease predictive model Monthly environmental data comprising of maximum and minimum air temperature, relative humidity, average rainfall and wind speed were collected from meteorological station, situated at a distance of 100 meter away from the experimental area of Department of Plant Pathology University of Agriculture Faisalabad. All the above mentioned parameters of environment were collected from February to end of the April during 2006 and 2007, respectively. The data of 66 entries and environmental variables were subjected to analysis of variance. Differences in the environmental parameters and disease incidence were determined by Least Significant Difference test (LSD) at (p<0.05). The influence of these environmental parameters on the development of disease was determined by correlation analysis (Steel et al., 1996). All the data were subjected to stepwise regression analysis and the most favourable environmental parameters were determined by significance level. The coefficient (R2), Mallows Cp and Mean Square Error were used to select the best model (Myers, 1990). Chemotherapy of Karnal bunt disease of wheat (i) In vitro evaluation of fungicides at four dosage rates In vitro evaluations of ten fungicides at 40 ppm, 60 ppm, 80 ppm, and 100 ppm against the colony growth of T. indica, were carried out by inhibition zone technique (Khan and Ilyas, 2007). A weighed quantity of each of ten fungicides (Table 4) was amended into the sterilized water to obtain each of the 40, 60, 80 and 100 µg/mg (ppm) aqueous concentration. Twenty ml of sterilized potato dextrose agar medium (dextrose 20 g, starch 20 g, and 20 g agar agar dissolved in water to make the volume 1000 ml) was poured in equal quantity into replicated sets of 90 mm diameter petri-plates and allowed to solidify. A secondary sporidial culture of T. indica (grown on PDA slants in 250 ml conical flasks) was scratched with a sterilized scalpel, removed and stirred thoroughly in 200ml sterilized water taken in a 1000 ml sterilized beaker. This sporidial suspension was filtered aseptically through sterilized muslin cloth and the suspension was diluted further to get about 10,000 sporidia/ ml of water. One ml of this sporidial suspension was transferred to each of 90 mm diameter PDA plates and spread uniformly on the surface of PDA with a sterilized bended (L-shaped) glass rod. With the help of a sterilized 6mm diameter cork borer, a well was made in the center of each PDA Petri- plate with sporidial suspension spread on its’ surface. The wells of the four PDA Petri- plates were filled with aqueous solution of each of four concentrations of the each test fungicide. The four PDA Petri-plates with wells were filled with sterilized water which served as control. The Petri-plates were labeled with the fungicides and its’ dosage rates. All the Petri-plates with wells either with fungicide solution or sterilized water were put at 50C in a refrigerator for 24 hours to allow the diffusion of fungicide solution into the medium of the Petri-plates. These Plates were then incubated at 200C. After 6 days of incubation the diameter of inhibition zone of T. indica colony around the well for each of the concentration of the test fungicides was measured (Fig.11, A-F). (ii) In vivo control of Karnal bunt disease of wheat by protective and eradicative spray of fungicides. A. Protective spray Wheat grains of highly susceptible cultivar AS-2002 were sown in sub-plots of 1.53 x 0.92 meter size, with three replications, with sub-plot to sub plot distance 60 cm, repeat to repeat distance 90 cm, row to row distance in sub-plot 30 cm and plant to plant distance in a row 15 cm. At boot stage, the sub plots were sprayed with each of ten fungicides at their recommended dosage rates (Table 4). The sub-plots sprayed with tap water served as control. Each treatment was replicated four times. The layout of the experiment was in RCBD. After 48 hours of fungicidal spray, each of the twenty heads in each sub-plot was boot inoculated with 3 ml of sporidial suspension of Tilletia indica (10,000 spores/ml). The sub-plots inoculated with sterilized water served as control. Inoculated plants were tagged and labeled. The field was irrigated to lower the temperature. Agronomic practices were homogenously applied in all the replications. At maturity plants

Table: 4. Fungicides evaluated against in vitro colony growth of Tilletia indica and in vivo control of Karnal bunt disease of wheat grains by foliar spray S. Common Chemical name Dose Formulation Manufacturer No. name & % composition Rate Thiophanate Methyl (52.50%) Protocol 500g/ 100 lit 1 + 65 W.P. Agrolet Co. Precombi water Diethofen carb (12.5%) Diamethomorph (6%) 2 Agrohit 50 W.P. 350g/ acre Agrolet Co. + Mancozeb (44%) Metalaxyl (15%) M/S Pak. China 3 Dolomite + 58 W.P. 250g/ acre Chemicals Mancozeb (65%) Fosetyl aluminium (40%) 4 Alert plus 70 W.P. 350g/ acre Agrolet Co. + Mancozeb (30%) M/S Pak. China 5 Crest Carbendazim 50 W.P. 100g/ acre Chemicals Propineb (61.25%) 250g/ 100 lit 6 Anthacal + 75 W.P. Agrolet Co. water Provali Carb (5.5%) M/S Pak. China 7 Shelter Mancozeb (80%) 80 W.P. 600g/ acre Chemicals Thiophaneti methyle M/S Pak. China 8 Thiomil 70 W.P. 200g/ acre (70%) Chemicals Chlorothalonil (30%) w/w 330g/ acre + or M/S Pak. China 9 Reconil-M 70 W.P. Mancozeb 150g/100 lit Chemicals (40%) w/w water + other ingredients (30%) w/w Aluninium ethyl Rhoune-poulenc- 10 Aliette 80 W.P. 2.5g/lit water phosphate Agrochimie

were harvested and hand threshed. The data of inoculated heads were recorded on percent grain infection The data were analyzed statistically by subjecting it to analysis of variance and Least Significance Difference Test (LSD) Steel and Torrie, (1996) to visualize the difference between the effects of various fungicidal spray treatments. B. Curative spray evaluation In another experiment the variety (AS-2002) was sown in various sub-plots and the experiment was designed similar to that of protective application of fungicides. When crop reached to boot leaf stage each of the twenty heads in each replication were boot inoculated with 3 ml of sporidial suspension of T. indica (10,000 spores/ml). Sterilized water sprayed in sub-plots served as control. After 48 hours of artificial inoculation, each of the test fungicides was sprayed at their recommended dosage rates (Table 4) following RCBD. Inoculated plants were tagged and labeled and field was irrigated. At maturity, the inoculated heads were harvested, hand threshed and percent seed infections for each spray treatment was determined. The data were analyzed statistically by subjecting it to ANOVA and LSD test (Steel and Torrie, 1996) to visualize the difference between various fungicidal spray treatments.

Chapter 4

RESULTS Survey of Karnal bunt disease of wheat Analysis of variance (Table 5) revealed that there was a distinction in the percentage of bunted seeds among the grain markets of 24 districts (Table 6). Significant difference was obtained even in the samples drawn from various locations of a district. Similar results were exhibited by the evaluation of samples collected from hot spot areas (fields) and localities of 12 districts (Table 8). However, results of varieties were non- significant indicating that most of the cultivars in the disease affected regions were susceptible. Except varieties and the interaction of varieties with districts, all the possible two way and three way interactions were significant (Table 8). Table 5. Analysis of variance for the assessment of bunted seeds in grain markets of various districts and locations of the Punjab. S. O. V. DF SS MS F P District 23 90102 3917.48 86.67 0.00* Location 4 3028 756.94 16.75 0.004* Replication 3 592 197.48 Dist.x Locat. 92 79380 862.83 19.09 0.00* Error 357 16137 45.20 Total 479 189240 Grand mean 28.41, CV 23.89 *Significant at 0.05 A comprehensive survey for the incidence of Karnal bunt disease of wheat in 17 districts (Table7) of the Punjab assessed in the crop of 2006 revealed that out of 624 samples collected from grain markets and storage godown of various cities and towns, 208 samples were found infected by Karnal bunt fungus. In other words, 33.3 percent of the total collected samples harbored infection by T. indica. The number of infected samples ranged from 6.97 percent in Muzaffargarh district to 60.97 percent in Toba Tek Singh district. Among 17 districts surveyed in the Punjab, three districts such as Muzaffargarh, Dera Ghazi Khan and Lodhran had 6.97, 8.00 and 17.85 percent infected samples respectively (below 20 percent). Seven districts such as Sheikhupura, Bahawalpur, Chakwal, Jhang, Sargodha, Table: 6 All pairwise comparisons test for Karnal bunt disease of wheat for districts in grain markets of the Punjab. Name of district Mean Homogenous group 1-Sialkot 57.74 A 2-R. Y. Khan 49.15 B 3-Gujranwala 44.78 C 4-Faisalabad 42.87 CD 5-D. G. Khan 40.63 CD 6-Bahawalpur 39.37 DE 7-Multan 36.05 EF 8-Khanewal 35.73 EF 9-Sahiwal 34.65 F 10-Bhakkar 33.22 F

11-Nankana Sahib 31.96 FG

12-Chakwal 28.29 GH

13-Kasur 25.29 HI

14-Sargodha 24.93 HI

15-Jhang 24.92 HI 16-Shekhupura 23.03 IJ 17-Layyah 22.21 IJ 18-Ludhran 19.64 JK 19-Muzaffargarh 17.44 K 20-Gujrat 12.94 L 21-Rawalpindi 10.64 LM 22-Mianwali 7.25 MN 23-Vehari 7.14 MN

24-Rajanpur 5.41 N Alpha 0.05, Standard Error for Comparison 2.0692, Critical T Value 1.969, Critical Value for Comparison 4.0733, Error term used: Distt*location*sample 276, DF. There are 15 groups (A, B, etc.) in which the means are not significantly different from one another.

Nankana Sahib and Multan had 22.58, 23.68, 23.07, 23.68, 24.32, 25.00 and 32.60 % infected samples respectively (i.e. below 40 percent), while the remaining seven districts such as Sialkot, Gujranwala, Faisalabad, Sahiwal, Khanewal, Vehari and Toba Tek Singh had 40.54, 48.57, 40.57, 42.85, 45.16, 49.05 and 60.97 percent Karnal bunt infected samples respectively (i.e. above 40 percent). The low number (below 20%) of wheat samples infected by Karnal bunt fungus (T. indica) in districts of Muzaffargarh, Dera Ghazi Khan and Lodhran (Southern Punjab) can be attributed to relatively dry environmental and soil conditions of these districts as compared to the districts of the Eastern Punjab where above 40 percent of the samples were found infected due to existing of relatively moist environmental and soil conditions which probably favour the germination of teliospores of Table 7: Percent wheat sample infection, range of infection and average infection in grain markets and storage godowns of 17 districts of the Punjab by Karnal bunt disease during 2006. Total percent Average Sr no. of -ve +ve sample infection Grade/Rang no Districts samples sampl sampl s percentag e of infection . collecte e e infecte e d d 1 Bahawal pur 38 29 9 23.68 0.57-3.51 1.75 2 Multan 46 31 15 32.60 0.75-4.59 1.75 3 Muzaffargar 43 40 3 6.976 0.50-2.15 1.46 h 4 D.G.khan 25 23 2 8.00 0.65-2.91 1.78 5 Khanewal 31 17 14 45.16 1.50-3.65 1.95 6 Vehari 53 27 26 49.05 1.57-3.74 1.85 7 Gujranwala 37 22 15 48.57 1.85-4.43 2.97 8 Sialkot 35 18 17 40.54 1.75-4.95 2.75 9 Sheikupura 31 24 7 22.55 0.75-2.75 1.05 10 Ludhran 28 23 5 17.85 0.55-3.38 0.95 11 Sahiwal 35 20 15 42.85 1.05-3.68 1.57 12 Faisalabad 69 41 28 40.57 0.95-4.75 2.45 13 Jhang 38 29 9 23.68 0.75-2.24 1.25 14 Toba-Tak 41 16 25 60.97 0.85-4.96 02.45 Singh 15 Sarghodha 37 28 9 24.32 0.45-2.48 0.78 16 Chakwal 13 10 3 23.07 0.39-2.67 1.27 17 Nankana- 24 18 6 25.00 0.75-4.54 2.35 sahib

the Karnal bunt fungus and multiplication of its sporidia which could infect developing grains in the heads of wheat crop. The range of wheat grain infection and average grain infection varied greatly among infected samples of the 17 districts (Table 7). The maximum range of grain infection of 0.75 to 4.59, 1.85 to 4.43, 1.75 to 4.95, 0.95 to 4.75, 0.85 to 4.96 and 0.75 to 4.54 was found in the infected samples of Multan, Gujranwala, Sialkot, Faisalabad, Toba Tek Singh and Nankana Sahib district, respectively with average grain infection of 1.75, 2.97, 2.75, 2.45, 2.45 and 2.35 percent, respectively. The minimum range of grain infection of 0.50-2.15, 0.65-2.91, 0.75-2.75, 0.75-2.24, 0.45-2.48, and 0.39-2.67 was found in infected samples of Muzaffargah, D.G. Khan, Sheikhpura, Jhang, Sargodha and Chakwal districts, respectively with average grain infection of 1.46, 1.78, 1.05, 1.25, 0.78 and 1.27 percent, respectively. The intermediate range of wheat grain infection of 0.57- 3.51, 1.50-3.65, 1.57-3.74, 0.55-3.38, 1.05-3.68 was found in the infected samples of Bahawalpur, Khanewal, Vehari, Lodhran and Sahiwal, respectively with average wheat grain infection of 1.57, 1.95, 1.85, 0.95, 1.57 percent, respectively. Again the maximum and minimum range of grain infection in various districts can be attributed to relatively wet or dry conditions of the atmosphere and soil of the districts surveyed. Table: 8. Analysis of variance for the incidence of Karnal bunt disease of wheat under natural condition in various hot spot areas of the Punjab during field survey 2007. S.O.V. DF SS MS F P Replication 2 9.62 4.81 (Samples) Varieties 2 2.91 1.45 2.72 0.61ns Locations 4 97.17 24.29 45.33 0.00* Districts 11 313832.3 28530.2153241.03 0.00* Dist.x Loc. 44 2034.243 46.23 86.27 0.00* Dist.x Var. 22 73.23 3.32 6.21 0.34ns Loc.x Var 8 106.25 13.28 24.78 0.00* Dist.x Loc.x Var. 88 1156.35 13.14 24.52 0.00* Error 358 191.84 0.53 Total 539 317503.9 Grand mean 30.68, CV 5.67 *Significant at 0.05 ns= non-significant

The survey of kanral bunt disease of wheat crop of 2007 in various hot spots of farmer’s field falling in 13 districts of the Punjab (Table 10) revealed that the hot spots of Bahawalpur, Vehari, Toba Tek Singh, Sahiwal, Multan and Bhakkar had maximum range of wheat grain infection of 0.97-6.5, 1.05-6.5, 1.5-6.5, 0.75-5.9, 0.57-5.5 and 0.75- 5.5, percent respectively with average grain infection of 4.9, 4.50, 4.25, 2.25, 2.25 and 3.00 percent, respectively. The hot spots of districts D.G. Khan and Muzaffargarh had minimum range of grain infection of 0.75-2.0 and 0.87-2.7 percent, respectively with average grain infection of 1.25 and 1.60 percent respectively while the hot spots of district Rawalpindi, Chakwal, Layyah, Jhang and Faisalabad had intermediate range of wheat grain infection of 0.5-3.7, 0.96-3.10, 0.55-3.50, 0.95-3.5 and 0.95-3.50 percent respectively with average grain infection of 1.50, 1.50, 2.00, 2.50 and 1.25 percent, respectively (Table 10).

Table: 9. All pairwise comparisons test for Karnal bunt disease of wheat for districts recorded in hot spot areas (field). Name of district Mean Homogenous group 1-Bahawalpur 80.09 A 2-Vehari 65.19 B 3-T. T. Singh 51.95 C

4-Sialkot 43.15 D 5-Sahiwal 33.16 E

6-Chakwal 32.82 E

7-Khanewal 20.44 F

8-Faisalabad 14.30 G

9-Multan 11.61 H 10-Muzaffargarh 9.16 I

11-D. G. Khan 5.19 J 12-R. Y. Khan 1.13 K

Alpha 0.05, Standard Error for Comparison 0.3665, Critical T Value 1.965. Critical Value for Comparison 0.7204, Error term used: Samples*Varieties*Locations*Distt, 446 DF. There are 11 groups (A, B, etc.) in which the means are not significantly different from one another.

While conducting the survey of hot spot areas to observe variation in disease incidences of 12 districts (Table 9) of the Punjab, there was a non significant difference in varieties indicating that cultivars were either susceptible or resistant to Karnal bunt disease of wheat (Table 8). Regarding the severity of the disease in the hot spots of areas, variation was found among the districts and locations even in the same district, as indicated by the significant interactions of districts, locations and varieties (Table 8). Table: 10. Survey of Karnal bunt disease of wheat under natural condition in different hot spot areas of the Punjab during the crop year 2007. Sr no. Percent infection

Districts Location Varieties Range Average 1 Parwaz-94, Pasban- Rawalpindi Pindi Gujran 90 and MH-97 0.5—3.7 1.50

2 Chakwal 97,Pb-85 Chakwal Karyala 0.96—3.10 1.50 and Yecora 3 Parwaz-94, MH-97 Sahiwal Arifwala and unknown 0.75—5.9 2.25

4 Muzaffar – AS-2002 and Seetpur, Ali pur 0.87—2.7 1.60 garh unknown 5 Sher shah, Bun SH-2003 and Mutltan 0.57—5.5 2.25 bosan unknown 6 Bahawal- Ahmad pur, Inqalab-91 and 0.97—6.5 4.90 pur Khan pur Unknown 7 D. G. khan Shadan Unknown 0.75—2.0 1.25

8 AS2002 and Layyah Karor lalesan 0.55—3.5 2.00 Faisalabad-85 9 Sarai muhajir, AS2002 and Bhakkar 0.75—5.5 3.00 Jahan khan unknown 10 Mulwana Pasban-90 and Jhang morh,Hassu 0.95—3.5 2.50 MH-97 balail 11 Unknown and Vehari Burewala, Luden 1.05—6.5 4.50 Kohinoor-83 12 Chak 387 J.B, Toba Tak- AS-2002 and Chak 1.5—6.5 4.25 Sing Inqabal-91 384,J.B. 13 Watan, and Faisalabad Amin pur Bangla 0.95—3.50 1.25 Unknown

The survey/ assessment of Karnal bunt disease of various hot spots naturally developed at research stations/institutes, model farms and seed corporation farms (Table 11) revealed that hot spots of Jalindhar Seed Corporation Farm Arifwala (Sahiwal) and Barani Agri. Research Institute, Chakwal developed 2.5 and 3.5 percent grain infection with a range of infection of 1-5 and 1-7 percent, respectively while the hot spots at Punjab Seed Corporation (Peeruwal) Khanewal and at Agronomic Research Institute (Karor Laleason) Layyah developed 4.5 and 6.5 percent grain infection with a range of 1-15 and 5-10 percent infection, respectively, while the hot spots of Tareen Model Farm Lodhran and Experimental Farm of the Department of Plant Breeding & Genetics, University of Agriculture, Faisalabad, developed 7.5 and 10.5 percent grain infection with a range of infection 5-15 and 5-45 percent, respectively.

Table: 11. Rang of infection and percent wheat grains infection in hot spots of various research institutes/ research stations, model farms and seed corporation farms during wheat crop of 2007. Percent Percent Coefficient Lines/ varieties Name of place/ Venue Range of Average of infection affected infection Infection Tareen Model Farm AS-2002, Shafaq- Lodhran 06 and KC050 5-15 7.5 11.87 Punjab seed corporation (Peeruwal) Khanewal AS-2002 and 1-15 4.5 10.43 KC050 Jalindher seed AS2002, KC050, corporation Iqbal-91 and 1-5 2.5 7.98 (Arifwala) Sahiwal Pasban -90

Agronomic Research AS-2002 Station (Karore lalesan) 5-10 6.5 13.24 Layyah Experimental area of Department of Plant Breeding and Genetics 75 germplasm lines 5-45 10.5 15.56 University of Agriculture Faisalabad Barani Agricultural Research Instt. Chakwal 35 germplasm lines 1-7 3.5 6.24

Screening of wheat germplasm for the sources of resistance Regarding the approaches towards breeding for disease resistance, the screening of 201 advanced lines of wheat revealed that 16, 9, 12, 33, 44, and 87 lines were highly resistant (immune), resistant, moderately resistant, moderately susceptible, susceptible and highly susceptible, respectively (Table 12). Sixteen highly resistant lines were V-02192, V-03079, V-04022, V-04178, V-05011, V-05020, V-05023, V-05025, V- 05039, V-05136, V-05144, V-05150, V-06122, V-06126, V-06127, V-06128 while the nine resistant lines were V-01078, V-03138, V-04048, V-04076, V-04171, V-04183, V- 05132, V-05152 and V-08794. The twelve moderately resistant lines were V-02156, V- 04157, V-04189, V-04611, V-5010, V-05097, V-5610, V-06121, V-06123, V-06125, V- 06131 and V-06133. The remaining advanced lines were moderately to highly susceptible. Out of 44 commercial cultivars screened against Karnal bunt disease, none was found to be highly resistant or resistant. However, 07, 07, 14 and 16 cultivars were found to be moderately resistant, moderately susceptible, susceptible and highly susceptible (Table 10). The seven moderately resistant cultivars were BWP-97, SA-42, SH-2002, Farid-2006, Blue Silver, Shafaq-06 and Manthar. The seven moderately susceptible cultivars were Chris, Crow, Fontana, Pavon, Moracco, Dirk and Daman-98. The fourteen susceptible cultivars were Pak-81, Sahar, SA-75, Pb-76, Pb-81, Pb-96, Faisalabad-85, Parwaz-94, Uqab-2002, GA-2002, Inqlab-91, Kohsar-95, Chakwal-96 and Chakwal-97 while the sixteen highly susceptible cultivars were C-271, C-273, C-518, C-591, WL- 711, Chenab-2000, Faisalabad-83, Kohinoor-83, Local White, MH-97, Pb-85, Pasban-90, LH-26, Shalimar-88 and Yecora and Mexi-Pak-65.

Epidemiological studies Correlation of environmental conditions with karnal bunt disease: Karnal bunt disease incidences recorded on 66 gremplasm lines/ varieties during two consecutive growing season 2005-06 and 2006-07 revealed significant results (Table 13). The individual effect of years and varieties and their interactive effects were significant. Variation in the disease incidence among the 66 germplasm lines/ varieties during two years was observed. Each of the germplasm line/ variety indicated a different degree of Table: 12. Level of resistance/susceptibility of advanced wheat lines and commercial cultivars against Karnal bunt disease of wheat Grade in the Response of disease test line or rating scale cultivar Advanced wheat lines Commercial wheat cultivars and percent against the grain disease infection 0 = No Highly V-02192, V-03079, V-04022, V-04178, V- - infection at resistant or 05011, V-05020, V-05023, V-05025, V-05039, all immune V-05136, V-05144, V-05150, V-06122, V- 06126, V-06127, V-06128 1 = Less than Resistant V-01078, V-03138, V-04048, V-04076, V- - 1% grain 04171, V-04183, V-05132, V-05152, V-08794 bunted 3 = 1.1-2% Moderately V-02156, V-04157, V-04189, V-04611, V- BWP-97, SA-42, SH- grain bunted resistant 05010, V-05097, V-05610, V-06121, V-06123, 2002, Blue Silver, Farid- V-06125, V-06131, V-06133 2006, Shafaq-06 and Manthar 5 = 2.1-5% Moderately V-03094, V-03285, V-04040, V-04122, V- Chris, Crow, Fontana, grain bunted susceptible 04179, V-04188, V-04306, V-05003, V-05004, Pavon, Moracco, Dirk, V-05009, V-05041, V-05042, V-05043, V- 05044, V-05048, V-05049, V-05050, V-05053, Daman-98 V-05054, v-05055, V-05058, V-05060, V-05064, V-05065, V-05066, V-05067, V-05071, V- 05072, V-05082, V-06069, V-09247, V-00BT08, V-00BT015 7=5.1-10% Susceptible V-02156, V-03007, V-03079, V-03094, V- Pak-81, Sahar, SA-75, bunted grain 03138, V-04009, V-04022, V-04040, V-04048, Pb-76, Pb-81, Pb-96, V-04068, V-04112, V-04157, V-04171, V- 04178, V-04179, V-04181, V-04188, V-04189, Faisalabad-85, Parwas- V-04309, V-04611, V-05006, V-05008, V- 94, Uqab-2002, GA- 05056, V-05062, V-05070, V-05093, V-05132, 2002, Inqlab-91, Kohsar- V-05136, V-05144, V-05150, V-05152, V- 95, Chakwal-96, 05620, V-06124, YR9-SEERI-E45, YR10-E30, YR15-E27, YR18-E23, YRA-E27, YRA- Chakwal-97 E40ANZA, LR-2C, LR-10, LR-13, LR-26, V2KC-050 9=More than Highly D-05603, D-05604, D-05607, D-05608, D- C-271, C-273, C-518, C- 10 percent susceptible 05609, D-05612, D-05623, D-05625, D-05627, 591, WL-711, Chenab- grain bunted D-05630, V-04148, V-05085, V-05086, V- 05087, V-05088, V-05090, V-05094, V-05096, 2000, Faisalabad-83, V-05100, V-05101, V-05104, V-05105, V- Kohinoor-83, Local 05107, V-05113, V-05115, V-05119, V-05121, White, MH-97, Pb-85, V-05122, V-06129, V-06130, V-06134, V- 06135, V-06136,V-06137, V-06138, V-06139, Pasban-90, LU-26, V-06140, V-085205, V-087049, V-087094, LR- Shalimar-88, Yecora, 1, LR-2, LR-2B, LR-3, LR-3KA, LR-3BG, LR-9, MexiPak-65 LR-11, LR-12, LR-14A, LR-14B, LR-15, LR-16, LR-17, LR-18, LR-19, LR-20, LR-21, LR-22A, LR-22B, LR-23, LR-24, LR-25, LR-27, LR-28, LR-29, LR-30, LR-32, LR-33, LR-34, LR-35, LR-36, LR-37, LR-38, YR1-E1, YR1-E18, YR2- E35, YR5-E19, YR5-E25, YR6-APR-E38, RR6- YR7, E37, YR7-E36, YR7-E10, YR8-E20, YR8- E26, YR9-E39

Table: 13. Analysis of variance of Karnal bunt disease incidences recorded on wheat varieties during 2006 and 20007. Source of DF Sum of Mean square F Values Prob>F variance squares Replication 3 12.2 4.06 Varieties 65 11749.3 180.76 105.82 0.000* Year 1 4970.5 4970.45 2909.78 0.000* Varieties vs Year 65 2341.00 36.02 21.08 0.000* Error 393 671.30 1.71 Total 527 19744.30 Grand Mean 14.083 CV 9.28 **Significant at 0.05 infection during its growth stages under natural conditions, although similar production technology, cultural and agronomic practices were adopted during both years. The data were analyzed by correlation to determine the influence of environmental factors. There was a significant correlation of maximum air temperature with 62 entries and minimum air temperature with 58 entries. (Table: 14). After the (min./max.) air temperature rainfall had significant correlation in the development of Karnal bunt disease as Table: 14. Correlation of environmental factors with Karnal bunt disease of wheat on different lines/varieties. Lines/ Temperature C Relative Rainfall Wind varieties humidity (mm) velocity Max. Min. mm) 9013 0.89663* 0.91809* - 0.6946 -0.7885 0.2184 0.0001 0.0098 0.1256 0.0623 0.9672 9092-2 0.89663* 0.91809* 0.6946 0.7885 0.0218 0.0155 0.0098 0.1256 0.0623 0.9672 9141 0.90476* 0.92584* -0.7106 -0.7954 0.4957 0.0132 0.0080 0.1135 0.0585 0.9257 9148 0.85395* 0.87621* -0.6249 -0.7516 -0.0832 0.0304 0.0220 0.1847 0.0849 0.8754 9181 0.92428* 0.79822 -0.7755 -0.8118* 0.1911 0.0084 0.0570 0.0699 0.0498 0.7186 9189 0.91521* 0.93581* -0.73527 0.8044 0.0965 0.0105 0.0060 0.0958 0.0536 0.8556 9191 0.92428* 0.94345* -0.77558 -0.8118* 0.1911 0.0084 0.0047 0.0699 0.0498 0.7168 9233 0.91647* 0.93385* -0.80172 -0.8044 0.2822 0.0102 0.0064 0.0551 0.0536 0.5879 9233-5 0.85395* 0.87621* -0.6249 0.7516 -0.0832 Contd… 0.0304 0.0220 0.1847 0.0948 0.8754 9292 0.90964* 0.93059* -0.72150 -0.7997 0.0696 0.0119 0.0071 0.1055 0.0561 0.8958 9296 0.92259* 0.94240* -0.76068 -0.8106* 0.1525 0.0088 0.0049 0.0791 0.0504 0.7729 9316-1 0.91521* 0.93581* - 0.7352 -0.8044* 0.0965 0.0105 0.0060 0.0958 0.0506 0.8556 9316-2 0.92374* 0.94243* -0.7847 -0.8112 0.2181 0.0085 0.0049 0.0645 0.0536 0.6780 9316-3 0.92207* 0.94196* -0.7581 -0.8102* 0.1464 0.0089 0.0050 0.0807 0.0506 0.7819 9327 0.91799* 0.93557* -0.7997 -0.8058 0.2733 0.0098 0.0061 0.0561 0.0529 0.6001 9332-1 0.92267* 0.94103* -0.7898 -0.8102* 0.2350 0.0087 0.0051 0.0616 0.0506 0.6540 9372 0.92428* 0.79822 -0.7755 -0.8118* 0.1911 0.0084 0.0570 0.0699 0.0498 0.7168 9381 0.92160* 0.93973* -0.7930 -0.8091 0.2465 0.0090 0.0053 0.0598 0.0511 0.6377 9428 0.83627* 0.85869 -0.5995 -0.7363 -0.1167 0.0380 0.0285 0.2084 0.0951 0.8257 9435-2 0.89663* 0.91809* -0.6946 -0.7885* 0.0218 0.0155 0.0098 0.1256 0.0503 0.9672 9436 0.58094 0.60232 -0.2996 -0.5134 -0.4124 0.2266 0.2058 0.5640 0.2975 0.4165 9437 0.86444* 0.88657* -0.6406 -0.7607 -0.0613 0.0263 0.0186 0.1705 0.0790 0.9081 9451 0.92298* 0.9414* -0.7886 -0.81050 0.2308 0.0087 0.0050 0.0623 0.05305 0.6598 9459 0.92428* 0.9434* -0.7755 -0.8118* 0.1911 0.0084 0.0047 0.0699 0.0498 0.7168 9459-1 0.92428* 0.94345* -0.7755 -0.8118* 0.1911 0.0084 0.0047 0.0699 0.0498 0.7168 9466 0.92428* 0.94345* -0.7755 -0.8118 0.1911 0.0084 0.0047 0.0699 0.498 0.7168 9475 0.88515* 0.89998* -0.8137* -0.7763 0.3843 0.0190 0.0145 0.0488 0.0695 0.4519 9475-1 0.88241* 0.90424* -0.6694 -0.7763 -0.0188 0.0199 0.0133 0.1458 0.0695 0.9718 9476 0.85395* 0.87621* -0.6249 -0.7516 -0.0832 0.0304 0.0220 0.1847 0.0849 0.8754 9479 0.89341* 0.91497* -0.6887 -0.7857 0.0118 0.0164 0.0105 0.1303 0.0639 0.9822 9480 0.88241* 0.90424* -0.6694 -0.7763 -0.0188 0.0199 0.0133 0.1458 0.0695 0.9718 9481 0.91521* 0.93581* -0.7352 -0.8044 0.0965 0.0105 0.0060 0.0958 0.0536 Contd…0.8556 9489 0.92428* 0.94345* -0.7755 -0.8118 0.1911 0.0084 0.0047 0.0699 0.05980 0.7168 9492 0.90743* 0.92848* -0.7165 -0.7978 0.0603 0.0125 0.0075 0.1091 0.0572 0.9096 9493-1 0.91293* 0.93368* -0.7293 -0.8025 0.0847 0.0110 0.0065 0.1000 0.0546 0.8732 9496 0.87086* 0.89290* -0.6506 -0.7663* -0.0469 0.0239 0..0166 0.1617 0.05045 0.9296 9507 0.92428* 0.94345* -0.7755 -0.8118* 0.1911 0.0084 0.0047 0.0699 0.0498 0.7168 9508 0.88241* 0.90424* -0.6694 -0.7763 -0.0188 0.0199 0.0133 0.1458 0.0695 0.9718 9517 0.83627* 0.85869* -0.5995 -0.7363 -0.1167 0.0380 0.0285 0.2084 0.0951 0.8257 9519 0.91126* 0.92808* -0.8064 -0.7997 0.3069 0.0115 0.0076 0.0526 0.0561 0.5541 9520 0.86930* 0.89136* -0.6482 -0.7649 -0.0505 0.0245 0.0171 0.1639 0.0764 0.9242 9520-2 0.85395* .87621* -0.6249 -0.7516 -0.0832 0.0304 0.0220 0.1847 0.0849 0.8754 9521 0.58094 0.60232 -0.2996 -0.5134 -0.4124 0.2266 0.2058 0.5640 0.2975 0.4165 9522-1 0.81144* 0.83398* -0.5658 -0.7147 -0.1583 0.0500 0.0391 0.2418 0.1105 0.7645 9527-1 0.92428* 0.94345* -0.7755 -0.8118* 0.1911 0.0084 0.0047 0.0699 0.0498 0.7168 9529-2 0.82797* 0.71872 -0.5880 -0.7290 -0.1312 0.0418 0.1075 0.2196 0.1001 0.8043 9529-3 0.83148* 0.85393* -0.5929 -0.7321 -0.1251 0.0402 0.0304 0.2149 0.0980 0.8132 9536 0.92207* 0.9419* -0.7581 -0.8102* 0.1464 0.0089 0.0050 0.0807 0.0506 0.7819 9541 0.91378* 0.9344* -0.7315 -0.8032 0.0889 0.0108 0.0063 0.0985 0.0543 0.8669 9542-3 0.85395* 0.87621* -0.6249 -0.7516 -0.0832 0.0304 0.0220 0.1847 0.0849 0.8754 9546 0.92428* 0.94345* -0.7755 -0.8118* 0.1911 0.0084 0.0047 0.0699 0.0498 0.7168 9554 0.84494* 0.73305 -0.6118 -0.7438 -0.1007 0.0342 0.0974 0.1968 0.0900 0.8493 9595 0.92207* 0.94196* -0.7581 -0.8102* 0.1464 0.0089 0.0050 0.0807 0.0506 0.7819 9602 0.80795 0.83050* -0.5612 -0.7116* -0.1637 0.0518 0.0407 0.2465 0.01127 0.7565 9610 0.91809* 0.93846* -0.7435 -0.8068 0.1137 0.0098 0.0056 0.0902 0.0523 Contd…0.8301 9618 0.92401* 0.94281* -0.7825 -0.8115* 0.2115 0.0084 0.0048 0.0658 0.0499 0.6874 9622 0.92428* 0.9434* -0.7755 -0.8118* 0.1911 0.0084 0.0047 0.0699 0.0498 0.7168 9629 0.92428* 0.9434* -0.7755 -0.8118* 0.1911 0.0084 0.0047 0.0699 0.0498 0.7168 9677 0.89282* 0.9143* -0.6876 -0.7852 0.0101 0.0166 0.0107 0.1311 0.0642 0.9849 Iqbal-2000 0.58094 0.6023 -0.2996 -0.5134 -0.4124 0.2266 0.2058 0.5640 0.2975 0.4165 Barani-70 0.92428* 0.9434* -0.7755 -0.8118* 0.1911 0.0084 0.0047 0.0699 0.0498 0.7168 Barani-83 0.91144* 0.9322* -0.7257 -0.8012 0.0776 0.0114 0.0067 0.1025 0.0553 0.8837 Anmol-91 0.91903* 0.93931* -0.7465 -0.8076 0.1202 0.0096 0.0054 0.0882 0.0519 0.8205 Faisalabad- 0.91686* 0.93734* -0.7398 -0.8058 0.1060 85 0.0101 0.0058 0.0927 0.0529 0.8416 Pasban -90 0.85395* 0.87621* -0.6249 -0.7516 -0.0832 0.0304 0.0220 0.1847 0.0849 0.8754 Parwaz-94 0.88817* 0.90986* -0.6793 -0.7812 -0.0033 0.0181 0.0118 0.1378 0.0665 0.9950 *Correlation significant at P =0.05 Upper values in a column indicate Pearsons correlation coefficient. Lower values indicate level of significance at 5 % level of probablity.

Table: 15. Comparison of monthly environmental conditions and karnal bunt incidence recorded on wheat varieties during 2006 and 2007. Environmental 2006 2007 LSD parameters Maximum temperature 31.1 a 27.8 a 16.53 (oC) Minimum temperature 16.1 a 14.8 a 10.87 (oC) Relative humidity (%) 38.8 a 49.71 a 34.91 Rainfall (mm) 0.57 a 1.10 a 1.88 Wind speed (km/hr) 4.03 a 2.50 a 1.98 Disease incidence 11.16 b 17.15 a 0.223 Mean values sharing the same letters do not differ significantly as determined by the LSD test (P = 0.05) compared to relative humidity which significantly influenced only one line i. e. 9475. Rainfall exerted significant influence on 21 lines/ cultivars. There was absolutely no correlation between the Karnal bunt disease incidences and wind velocity. Eighteen lines i.e. 9191, 9296, 9316-1 ,9316-3, 9332-1, 9435-2, 9459, 9459-1, 9475, 9496, 9507, 9527- 1, 9536, 9546, 9595, 9618, 9622, 9629 and one variety Barani-70 were influenced significantly by more than 50 % environmental factors in the disease development (Table 14). By the comparision of weather data during the vulnerable stages for the disease incidence recorded on wheat varieties/ lines, (Table 15) it was concluded that during 2006, average maximum temperature was 31.1 oC while the average minimum temperature was 27.8 oC. Relative humidity and rainfall were recorded 38.8 % and 0.57 mm respectively. High rainfall (1.10 mm), relative humidity (49.1 %), low maximum temperature (27.8 oC) and low minimum temperature (14.8 oC) during the year 2007 caused the more disease incidences than year 2006. Disease predictive models Two years combined environmental conditions and Karnal bunt disease incidence data subjected to stepwise regression analysis revealed a model consisting of air temperature (max./ min.) rainfall wind speed and relative humidity explained 84 % of the variability in disease development (Table 16). Thus all the environmental variables were exerting significant influence on Karnal bunt disease development during two years period. When these data were split by year, a model consisting of minimum and maximum air temperature, rainfall, relative humidity and wind speed explained 81 % of the variability in Karnal bunt disease development during 2005-06 (Table 17). However, in the second year relative humidity and rain fall were eliminated by stepwise regression and a model consisting of only three environmental variables (i.e. maximum air temperature, minimum air temperature and wind speed) explained 90 % of the variability in Karnal bunt disease development (Table 18).

Table.16. Summery of stepwise procedure for independent variables influencing Karnal bunt disease of wheat during growing season 2005-07 Variables Step Number In Model R2 C (p) F Prob>F entered 1 Min. temp 1 0.48 976.20 373.96 0.0001*

2 Rain fall 2 0.73 326.05 357.84 0.0001*

3 Wind speed 3 0.79 163.04 117.26 0.0001* Relative 4 4 0.81 101.36 51.00 0.0001* humidity

5 Max. temp. 5 0.84 30.41 66.74 0.0001*

* Significant at P = 0.05

Regression equation Y = -53.78+1.81x1* + 0.19x2*+0.08x3 +5.69x4* -1.97x5.

Where x1 indicate maximum temperature, x2 minimum temperature, x3 relative humidity, x4 indicates rain fall and x5 indicates wind speed respectively. *Indicates significant regression at 5 % level of probably Table: 17. Summary of stepwise procedure for independent variable influencing Karnal bunt disease during growing season 2005-06

Variable Number Step Model R2 C (p) F Prob>F entered In

1 Min temp. 1 0.43 410.84 155.07 0.0001*

Relative 2 2 0.72 93.51 219.75 0.0001* humidity

3 Rainfall 3 0.75 71.27 18.09 0.0001*

4 Max. temp. 4 0.76 56.19 13.54 0.0003*

5 Wind speed 5 0.81 3.00 55.66 0.0001*

*Significant at P = 0.05 Regression equation Y = -38.18 + 1.81x1 *+ 0.26x2 + 0.18x3* + 5.69x4* -1.97x5.

Where x1 indicate minimum temperature, x2 maximum temperature, x3 relative humidity, x4 rainfall and x5 wind speed respectively. *Indicates significant regression at 5 % level of probably Table: 18. Summery of stepwise procedure for independent variables influencing karnal bunt disease during growing 2006-07. Variables Step Number In Model R2 C (p) F Prob>F entered

1 Min. temp 1 0.55 702.79 237.61 0.0001*

Wind 2 2 0.85 99.31 400.40 0.0001* speed

Max. 3 3 0.90 3.00 98.31 0.0001* temp.

*Significant at P = 0.05

Regression equation Y = 24.64 + 0.87x1* + 0.18x2 * - 1.08x3. Where x1 indicate minimum temperature, x2 maximum temperature, x3 wind speed respectively. *Indicates significant regression at 5 % level of probably Chemotherapy of Karnal bunt disease of wheat (i) In vitro evaluation of fungicides against the mycelial growth of T. indica An in vitro evaluation of ten fungicides (Table 4), amended into PDA, against colony growth of T. indica revealed that the effectiveness of the fungicides depended on kind of fungicide and its dosage rate evaluated. However, the efficacy of fungicides for inhibiting colony growth of T. indica increased with an increase in their dosage rates (Table 19). Dolomite and Shelter at their 40µg/ ml and 100µg/ ml dosage rates were the most and statistically equally effective fungicide in inhibiting the colony growth of Tilletia indica as these fungicides produced 3.50 and 3.35 mm diameter at 40µg/ ml and 7.77 and 7.75 mm diameter inhibition zones of the fungus at 100µg/ml (Fig. 11). Shelter however at 100 µg/ml dosage rate displayed the same and statistically equal effectiveness as Dolomite at 80 µg/ml. Crest, Antracol Alert plus at 100 µg/ml dosage rate were less effective than Dolomite and Shelter as these fungicides produced 3.77, 3.50, 3.10 mm diameter inhibition zones respectively. However, there was no statistically difference between the effectiveness of Crest and Antracol and between that of Antracol and Alert plus. Alert plus and Antracol at 100 µg/ml dosage rate displayed the same effectiveness as Dolomite and Shelter displayed at 40 µg/ml. Reconil, Agrohit and Thiomil were the least effective fungicides A B

C D

F E

Fig. 11 : Mean inhibition zones of Tilletia indica by action of various fungicides. A = control (water), B = Alert plus at100 ppm, C = Shelter at 80 ppm, D = Antracal at100 ppm, E = Dolomite at100 ppm F = Shelter at100 ppm

against T. indica, while Aliette and Protocol pre-combi were ineffective in inhibiting the growth of the fungus (Table 19). (ii) In vivo control of Karnal bunt disease of wheat by protective and curative (eradicative) spray of fungicides. The in vivo evaluation of effectiveness of the 10 test fungicides, as protective spray (i.e. spray before inoculation) and as eradicative spray (i.e. spray after inoculation) revealed that the protective sprays were more effective than eradicative sprays in controlling Karnal bunt disease of field grown wheat plants (Table 20). The protective spray of Dolomite and Shelter, though statistically equally effective, were the most effective and caused 62 and 64 percent reduction in occurrence of Karnal bunt disease respectively, while the eradicative spray of Dolomite and Shelter reduced 39 and 41 percent disease, respectively on wheat grain and there was no statistical difference between the effectiveness of both fungicides. Table: 19. Mean inhibition zones of colony of Tilletia indica by various fungicides at 4 dosages rates amended in PDA medium. Mean Inhibition zone (mm) at 4 dosage rates Fungicides 40 ppm 60 ppm 80 ppm 100 ppm Crest 2.52 h* 3.40 ef 3.75 e 3.77 e Dolomite 3.50 ef 5.70 c 7.75 a 7.77 a Agrohit 0.25 l 0.50 l 0.5 0 l 0.5 l Alert plus 0.00 l 0.00 l 2.00 j 3.1 fg Shelter 3.35 ef 4.30 d 6.45 b 7.75 a Reconil 0.00 l 1.00 k 1.00 k 1.00 k Aliette 0.00 l 0.00 l 0.00 l 0.00 l Antracol 2.25 ij 2.8 0 gh 3.50 ef 3.5 ef Protocol pre- 0.00 l 0.00 l 0.00 l 0.00 l combi Thiomil 0.25 l 0.25 l 0.5 l 0.5 l Water 0.00 l 0.00 l 0.00 l 0.00 l (control) * Values having the same letters do not differ significantly at 5% level of significance as determined by LSD = 0.44 and EMS = 0.10 The protective sprays of Crest, Agrohit, Alert plus and Antracol were intermediate in their effectiveness and respectively caused 33.79, 34.65, 18.93 and 41.19 percent reduction in Karnal bunt infection of wheat grains. However, there was not significant different between the effectiveness of Crest, Agrohit and Antracol. Table: 20. Percent decrease in wheat kernals infections by Tilletia indica by protective and

Spray of fungicides before Spray of fungicides after Inoculation Inoculation

Percent

Mean percent Percent decrease in Fungicides Mean percent kernel infection decrease in kernel kernel infection kernel infection infection over

over control control

Crest 22.45 efg* 33.79 28.81 bc 7.30 Dolomite 12.73 h 62.45 19.01 fg 38.84 Agrohit 22.16 efg 34.65 28.64 bc 7.82 Alert plus 27.49 cd 18.93 28.89 bc 7.04 Shelter 12.24 h 63.90 18.50 g 40.48 Reconil 33.01 ab 2.71 26.41 cde 14.98 Aliette 30.86 abc 8.99 30.97 abc 0.35 Antracol 19.94 fg 41.19 23.59 def 24.07 Protocol 33.39 ab 1.53 30.10 abc 3.15 pre- combi Thiomil 32.43 ab 4.36 28.87 bc 7.08 Control 33.91 a 0.00 0.00 abc 0.00 eradicative spray of various fungicides. *Values having the same letters do not differ significantly at 5% level of significance as determined by LSD value = 4.91 at alpha = 0.05, EMS = 8.87 .

Antracol being comparatively less affective caused 24.07 percent decrease in kernel infection by its curative spray. The eradicative sprays of Crest, Agrohit and Alert plus were ineffective in controlling wheat kernel infection. The protective as well as curative sprays of Reconil M, Alliette, Protcol Pre-combi and Thiomil were ineffective in the control of wheat kernel infection by T. indica. From the analysis of environmental paramaters for the development of Karnal bunt disease it was concluded that during the vulnerable growth stages of wheat minimum temperature, maximum temperature and rainfall played a vital role. If the average minimum temperature ranges between 11 to 16 oC and max. temperature 27.5 to 31.1 oC in the month of Feb. and March that are commonly considered booting stage (depending upon varietal character and date of sowing) then the spray of fungicides is necessary to avoid the infection. With the deviation of above mentioned ranges of the air temperature numbers of the fungicidal sprays can be adjusted accordingly. Rainfall (at least 1.10 mm) during the early booting stages provides additional critical circumstances for the multiplication and dispersal of sporadia and hence in the progress of the disease.

Chpapter 5

DISCUSSION

1- Survey of Karnal bunt disease During the comprehensive survey it was assessed that Karnal bunt disease was found in all the wheat growing areas of the Punjab. However its’ incidence varied from district to district. Distinction in the incidences of the disease among the districts may be attributed to the differences in the climatic factors, as the specific weather conditions affect on the life cycle of the fungus (Diekmann, 1998) and soil type play an important role in the survival and germination of teliospores (Babadoost, et al., 2004). According to Sansford et al., (2006) effect of T. indica on yeild and quality varied from year to year and location to location. Because the wheat kernels as well as the grains present in them are partially infected therefore, the incidence and severity of the disease differ from location to location; even within a field it may be patchy. Management/ production practices, adoptation of different technologies by farmers, sowing of newly evolved commercial varieties, diversity in local climate especially from flag leaf stage to maturity of the crop and degree of susceptibility of cultivars are the factors which may provide justification to variation in disease incidences in various localities of a district.

In the past, Karnal bunt was known to occur in Sialkot district of the Punjab in 1950 in Northern areas of the country (Saleem and Akhtar, 1988) and was considered to be a disease of minor importance as it occurred in traces in foothill districts of Punjab (Munkdur, 1943). According to (Warham 1986, 1992), Karnal bunt affects wheat crop in the northern regions of Pakistan and India. Increasing incidence, severity and distribution of the disease toward the Southern Punjab have provided a great threat and alarming situation for the Plant Pathologists and Plant Breeders as these areas were considered free of this disease due to different climatology. During survey it came to know that seed corporations and seed companies were the main source of spreading the disease, because most of these companies get the infected grains from affected areas at low rates, mix it with normal healthy seeds and sell it at high price during growing season. The survey results were close to that of Mirza, (2005) who pointed out that in Punjab MH 97, Inqilab-91 and Wattan varieties were mostly affected with Karnal bunt disease. Average infection in grain markets and storage godowns of main districts of the Punjab coincided with survey conducted by Mirza, (2005) on farm disease assessment in the Punjab conducted during growing season 2003- 04 by Mirza, (2005). However, the survey results vary year to year as the climatic conditions seldom remain same in every year during the growing season of wheat. Normally the nature of the Karnal bunt disease is sporadic but in epidemic years it causes substantial losses to crop in the Punjab, Sindh and North Western Frontier Province (Ilyas et al., 1989). The disease remained endemic to Sialkot for a considerable time (Hassan, 1971) but with the passage of time it had spread to new sites extending from Sialkot to Mardan in the north and towards Jhang, Khanewal and Muzaffargarh in the south (Bhatti and Ilyas, 1986). During a Karnal bunt survey of the wheat crop carried earlier (Ilyas et al., 1989) the disease was reported to be absent in wheat samples collected from Multan, D.G. Khan and other districts of the southern Punjab but now it is prevalent in these districts. This spread of Karnal bunt in all 17 surveyed districts of the Punjab is of great quarantine concern. This aggravating condition and high wheat grain infection by Karnal bunt disease at farmer’s fields, research stations/institutes, seed corporations and Model Farms may serve as a source of inoculum to other unaffected areas of the Punjab for next year’s crops and may further increase the incidence of Karnal bunt disease in the Punjab, thus aggravating more and more quarantine status of the wheat produce of the Punjab. The biodiversity of the pathogen, its successful establishment and gradual distribution in various agro-ecological zones may further be attributed to physiological races. Aujla et al., (1987) reported the existence of four pathotypes, K1, K2, K3 and K4 among 21 N. indica isolates or field collection from different geographic locations of Punjab and Himachal Pradesh on a set of 17 differentials. As the N. indica is a heterothallic fungus and it survives through sexual reproduction, there is no well- developed race concept based on virulence patterns of different N. indica isolates on different host genotypes and genetics of pathogen (Datta et al., 1999). Singh et al., (1995) and Boned et al., (1996) considered it appropriate to classify the variation in the isolates as aggressiveness. Biological analysis of the N. indica isolates was done by field inoculations to check the level of virulence of the isolates (Datta et al., 1997). A substantial differential reaction was noted between wheat host plants and fungal isolates representing genetic distinction among N. indica isolates. The order of the virulence pattern of N. indica isolates obtained on the basis of susceptible reactions on the host plants for each isolate was as follows – Ni4>Ni5>Ni1>Ni7>Ni8>Ni6>Ni2. An individual isolate showed a differential reaction against two host cultivars and similarly, an individual host cultivar gave different reactions against two N. indica isolates. Thus, Datta et al., (1999) reported that different Karnal bunt resistance genes perhaps existed in these cultivars and that there was genetic variability for pathogenicity in the isolates. However, they also suggested that though the virulence typing explains the pathogenic behaviour of fungal isolates, it is a rough estimate. Further, it was influenced by environmental conditions which made the reproducibility of the results difficult in successive seasons even under controlled conditions. Screening of advanced lines/ cultivars of wheat against the disease Breeding for resistance has been a successful research activity and certainly is the best control method in the long term. However, variation in the pathogen may sometimes pose a threat to wheat production. Therefore, it needs a continuous breeding programme for the source of resistance against the diseases. The susceptibility of bread wheat to Karnal bunt disease of wheat has been well documented (Fuentes-Davila et al., 1992, 1993) reaching infection levels greater than 50% under artificial condition; therefore it is important to continue evaluating new advanced lines and commercial cultivars against the disease in various agro-ecological regions. The identification and tagging of K-B resistant genes is important for further breeding and developing resistant wheat cultivars because of quarantine restrictions on the pathogen and the large effect of the environment on the disease development. The most reliable and sufficient levels of genetic resistance to KB, have been observed among Chinese, Indian and Brazilian wheat cultivars. (Gill et al., 1993; Fuentes-Davila et al., 1995 Singh et al., 1995b). Wheat line HD29 is potentially an important source of genetic resistance to Karnal bunt providing resistance to diverse isolates of T. indica (Gill et al., 1993). A true breeding mapping population (RILs) was developed from the cross of WL711x HD29, because Karnal bunt screening over a number of replications and years to obtain reliable phenotypic data was required to establish reliable markers/traits associations. In experiments during 1998-99, little disease occurred because of warm temperature during flowering, illustrated well the large influence of environment on KB disease even in inoculated nurseries. The distribution of KB disease severity on the RILs was skewed toward that of resistant parent in three experiments, suggesting the segregation of multiple genes with dominant complementary gene action in wheat line HD29. This was consisted with the results of previous studies (Morgunov et al., 1994). Karnal Bunt of wheat is such a disease which is very effective in low quantity. In views of its importance Fuentes-Davila 1996, reported that 1-4 % of kernels are enough to render the wheat grains unacceptable for human consumption. According to Sekhon et al., (1992) the flour from grains with over 3 % bunted kernels imparts an off colour and unpleasant odour. Therefore according to modern requirement a modified rating scale of (Aujla et al., 1989) was used and lines or varieties having 2.1-5 % grain infection were considered as moderately susceptible and lines/ cultivars with more than 10 % infected grains were declared as highly susceptible. The highly resistant advanced lines of wheat identified in the present screening can further be exploited, as resistant sources against Karnal bunt disease, in breeding programmes for the development of disease resistant commercial cultivars after determining their genetics or these advanced lines can be released directly as commercial cultivars if these were found to possess other desirable agronomic characters. The sources of resistance in the wheat germplasm against Karnal bunt disease are not uncommon. Aujla et al., (1980) reported ten wheat lines with only 1-5 percent grain infection among 286 lines screened by them. Earlier Gautam et al., (1977a) screened 96 wheat lines and reported lines with less than 1 percent infection. In a field screening trial of superior genetic stocks consisting of 350 lines, Gautam et al., (1977b) found 160 lines with no infection. Aujla et al., (1985) screened germplasm under artificial epiphytotic conditions against Karnal bunt and reported 26 lines which remained disease free and 58 lines having 0-5 percent infection. The screening of 38 wheat lines, included in NUWYT of 2004-05 at NARC, Islamabad revealed that all the lines were highly susceptible except 5 lines that remained free from grain bunt infection (Anonymous, 2005). As regards the commercial cultivars, there is scarcity of resistance in them except a few moderately resistant cultivars. The scarcity of resistance in the commercial cultivars is a serious threat as regards the quarantine status of wheat of Pakistan. This scarcity of resistance in commercial cultivars against Karnal bunt disease across the border (Krishna and Singh, 1983) and in the country has already been reported (Iftikhar et al., 1988; Anonymous, 2005). Weather conditions and disease predictive models A part from the importance of weather conditions or climatic factors, in creating epidemics and distribution of Karnal bunt disease these are interlinked/ interrelated with each other to certain extant. Three environmental factors i. e. maximum air temperature, minimum air temperature and rain fall were tabulated to get a better scenario in Karnal bunt over all correlations. Maximum air temperature and minimum air temperature play a key role with disease development and influenced significantly almost all the lines/ cultivars. Various models to estimate risk of establishment of Karnal bunt in different countries have been developed (Kehlenbeck et al., 1997; Diekmann 1998; Murray and Brennan 1998; Sansford 1998; Baker et al., 2000). This study was taken into consideration and concentrated only on phenological timing. Although this timing is acknowledged to fluctuate, we used the weather parameters within a sowing region in order to forecast the timing of the reproductive period; wich is the only wheat stage vulnerable to infection by Karnal bunt. As the disease is believed to be a dynamic process and temperature is a key factor in disease tetrahedron in the presence of host followed by a certain period of time. Development of Karnal bunt depends on favourable weather conditions for infection and disease development from heading to flowering (anthesis) of the wheat crop. Moderate temperature, high relative humidity or free moisture, cloudiness and rainfall during anthesis favour disease development (Fuentes-Davila, 1996), which is also reported by our results to some extent as max. and min. air temperature had influenced directly on 62 and 58 lines and cultivars respectively in the month of February and March, the flowering and heading stages of wheat. Rainfall had influenced 21 lines/ varieties. Results of Workneh, et al., (2008), revealed that temperature and rainfall were the main factors affecting the Karnal bunt occurrence in Texas. A weather based model was developed for the prediction of disease to evaluate bunt-negative and bunt-positive fields. The model accurately forecasted the occurrence of disease except for the year 2004, when the climatic conditions were predicted favourable for the development of the disease. However, according to their arguments wet and cool weather during vulnerable period of crop received rainfall seven times during eighteen day period in April 2004. Thus greater frequency with twice amount of rainfall as compared to last years was too wet for the development of disease. The view is supported by remarks of Sharma and Nanda (2003), reported that prolonged wet conditions are unfavourable for Karnal bunt development in India. Jhorar et al., (1992), also made the observation that conditions for the establishment of Karnal bunt disease are unfavourable when HTI values are too high, and the large HTI values are the result from high relative humidity and cool temperature. The excessive rainfall wash off the sporadia from the plants consequently, fungus infection is reduced. One possible explanation as pointed out by Indian situation is that excessive rainfall may have produced conducive weather circumstances for the germination of spores before the onset of vulnerable stage of the host, consequently exhausting the inoculm potential. There is conflicting information available on how abiotic conditions during the rest of year affect the survival of pathogen and development of Karnal bunt, summarized by Warham (1986). For Karnal bunt of wheat to develop, conditions must be suitable for teliospore of T. indica to germinate to produce sporidia between the flag leaf emergence and anthesis stages of the growing wheat plant and for these spores to infect the developing ears (Nagarajan et al., 1997). Relationship exists between condition at anthesis and development of Karnal bunt for sites in India. Mavi et al., (1992) studied the relationship of weather conditions during the reproductive stage of wheat crop to disease intensity over a 19 year period in Ludhiana, in India and developed a model based on the average maximum temperature during mid to late anthesis (-ve correlation), the “evening relative humidity” (presumably 2:30, +ve correlation) and sunshine duration (-ve) during early to late anthesis, and number of rainy days in early anthesis (+ve). As the climatic conditions and the wheat growing season in Indo-Pak. have no big difference, therefore vulnerable stages in main wheat growing regions of both countries are the same (exist mostly in Feb. and March). Therefore, environmental conditions including maximum air temperature, minimum air temperature, rainfall relative humidity and wind speed were employed for the assessment of Karnal bunt disease occurrence. Using the climatological data and stepwise discriminant analyses, Diekmann, (1993) conducted a study to determine areas of the world that were at low risk for Karnal bunt. Although recognizing limited Karnal bunt distribution that could be due to the difficulty of recognizing the disease in the field, Diekmann believed the limited distribution was largely due to the very specific environmental requirements for disease development. During our observations when the data were split by varieties to develop model for each variety it could not be done, because parameter estimates were biased and these data did not fit well to the model. More number of observations were needed to get goodness of fit. These environmental parameters had high degree of multi-colinearity and are sensitive to their order or sequence of entry into the model. However, it is evident that all the five environmental parameters had significant influence on disease development. Air temperature (max. / min.) and wind speed should be studied critically with great detail and care in future to reach towards a sound biological conclusion for Karnal bunt disease prediction. Present studies provide some base line information in this regard. Disease-risk locations were characterized by a high difference between daily maximum and minimum temperatures in the planting month, a relatively low daily maximum temperature in the month of flowering, and mild winter. Jhorar et al., (1992) found a high correlation between a humid thermal index (HTI), defined as the high RH in mid-afternoon divided by maximum daily temperature, calculated at wheat ear emergence, and the disease incidence over a19-year period in the central Punjab in India. Evaluation of fungicides against the Karnal bunt disease of wheat Protection of the wheat from Karnal bunt infection by applying a systemic fungicide to the seed has no value due to two reasons: i) systemic fungicides did not persist long enough to inhibit floret infection and (ii) the inoculum for Karnal bunt is primarily soil-borne and not seed-borne like that of dwarf bunt (Hoffmann, 1982; Brooks and Buckley, 1977). Therefore, an unfortunate consequence of applying systemic fungicides to wheat at late growth stages give rise to the apprehension of high level of residue in the grain at harvest. Contact fungicides, because they do not redistribute systemically within the spike, would probably have insignificant residues, as the residual chemical would be removed when the grain is threshed. In this experiment discovery of Shelter and Dolomite in which mancozeb is main portion of active ingredient, is a ray of hope for controlling the Karnal bunt disease as the mancozeb belong to Ethylenebisdithiocarbamates (EBDCs) group of non-systemic (surface acting) fungicides and could be used on wheat crop without acute toxicity. Karnal bunt disease of wheat has assumed an alarming situation in the Punjab during the previous two to three decades (Anon, 2005) and has been reported to cause, depending upon the cultivar affected, upto 30 percent grain losses (Anon, 1986; Ilyas et al., 1989). This calls for control of disease either by the use of host resistance or through the use of chemotherapy. Since resistance to karnal bunt disease in available commercial wheat cultivars is absent (Anon., 2005), the chemotherapeutic control of the disease can be achieved by foliar application of fungicides at proper plant stage growth (Singh and Prasad, 1980; Singh et al., 1985; Ilays et al., 1989a). Foliar spray of fungicides may protect the plants from infections or eradicate the established infection (Vyas, 1984). There are few contradictory reports on the chemotherapeutic control of Karnal bunt disease of wheat. According to Bedi, (1980) fungicides are not effective against Karnal bunt disease. Although reduction in the disease was achieved on two occasions by the application of carbendazim (Krishna and Singh 1982, Singh and Prasad, 1980). Seed treatment was not preferred on the basis of some sound reasons. Fungicides applied as seed treatment had controlled neither artificial nor natural infection by Karnal bunt pathogen, they reduced the tillering capacity of plants, also delayed heading by about one week. (Smilannick et al., (1987). Secondly the infection in the spike of wheat is initiated by airborne sporadia which may come from other fields of remote areas and the effect of seed dressing fungicide persist no longer upto the heading or booting stage of wheat crop. Fungicides effective in inhibiting the in vitro colony growth of T. indica were also equally effective for in vivo control of the karnal bunt infection of wheat grains. However, the protective applications of these fungicides were comparatively more effective in controlling wheat grain infection than their eradicative applications. Regarding the time of application (Protective or curative spray) our results are very similar to that of Ammerman et al., 1992., Baldwin et al., 1996., Gold et al., 1996 and Mitsuhiro et al., 1999. Application of Mancozeb and Metalaxyl provide the best control against the Downey mildews when applied before inoculation rather than post infection (curative application). They also reported that these fungicides possess a broad spectrum activity against fungal pathogen from all the three major divisions of fungi: Oomycota, Ascomycota and basidiomycota. Dolomite and Shelter which are basically a combination of Mancozeb and Metalaxyl provided the better control when used as a protective spray in the field against the Karnal bunt disease of wheat. Under the present situation of scarcity of resistance to Karnal bunt disease the control of the disease, through chemotherapy is not uncommon (Singh and Prasad, 1980; Singh et al., 1985; Ilyas et al., 1989a; Krishna and Singh, 1982). Krishna and Singh, (1982) reported that Bavistine (Derosal-60) and Bayleton, when sprayed at boot leaf growth stage prior to inoculation (protective spray) controlled the karnal bunt disease. Singh and prasad (1980) found that a single spray of Benomyl or Bevistine or Dithane M-45 at boot leaf growth stage was effective against Karnal bunt disease. Singh et al., (1985) also reported that spray of either Mancozeb or Carbendazim can prevent karnal bunt disease of the wheat plants. Ilays et al., (1989a) reported that Spotless, Tilt or Baycor were the most effective fungicides that significantly reduced the grain infection, the least effective spray were that of Bayleton, Topas C-50 and Dithane M-45 while that of Brassicol (PCNB), Bayfidan and Bavistine were intermediate in their effectiveness for reducing karnal bunt disease over the unsprayed control.

Chapter 6 SUMMARY Karnal bunt (partial bunt) disease caused by Tilletia indica being a floret- infecting is sporadic in nature. For the successful infection, coincidence between conducive weather conditions and susceptible stages of wheat crop is utmost necessary. Vulnerable period for the invasion of fungus remains for a short time about 3-4 weeks. Emergence and existence of vulnerable stages (starting early booting to milky) vary in each germlasm line and cultivar. It is also dependent on date of sowing, production technology and other agronomic practices to some extent. During the survey it was assessed that the disease is extending its broad-spectrum activity from north of the Punjab (Sialkot) toward the south of Punjab (Multan, Bahawalpur and D. G. Khan divisions). This indicates that the hot and dry climatic conditions of south Punjab exert no adverse effect on hardy nature teliospores of the pathogen. These conditions provide security to teliospores from suicidal germination. Consequently during the susceptible period of next growing season teliospores release sporadia to infect the host plant. During the epidemiological studies it was concluded that Karnal bunt disease severity was higher in 2007 as compared to 2006 in about all the entries except with a deviation of few entries. After temperature the other environmental factor that exerted a significant influence is rainfall. More rainfall with comparative low air temperature (min./ max.) during month of Feb. and March 2007 (1.99 & 1.33mm) rendered more disease incidences as compared to (0.52 & 1.19mm) year 2006 respectively. There was a significant correlation of maximum air temperature with 62 entries, minimum air temperature and rain fall exerted a significant influence on 58 and 21 entries respectively. In screening trials a large number of germplsm lines were categorized between moderately resistant to highly susceptible. Only 16 lines were immune or highly resistant and 9 were resistant. None of the cultivars which are being practically sown in the Punjab was found immune or resistant. Only seven varieties (BWP-97, SA-42, SH-2002, Blue silver, Farid 2006, Shafaq-06 and Manthar) were found moderately resistant to Karnal bunt disease of wheat. The protective spray of Dolomite and Shelter were most effective and reduced the disease upto 62.45 and 63.90 % respectively, while the eradicative spray of these fungicides caused 38.84 % and 40.48 % reduction in the disease. Chapter 7

CONCLUSIONS 1. Karnal bunt disease is rapidly spreading from East and North Punjab to southern areas of the Punjab and Sindh. 2. Seed corporations including private seed companies are the main source of spreading the disease, as these companies mix the diseased grains with healthy seeds and sell it during next growing season. 3. At present, a few varieties i.e Blue silver, Menthar, Farid-06 and BWP-97 which were evolved from Regional Research Institute Bahawalpur, and Shafaq-06, SA- 42 and SH-2002 evolved from Wheat Research Institute Faisalabad are avaible. 4. Out of 200 germplasm lines obtained from Wheat Research Institute Faisalabad only 16 lines were highly resistant and 9 were resistant all the other lines fall in the rank of moderately resistant, moderately susceptible, susceptible, and highly susceptible. 5. During the correlation analysis of environmental factors, max. air temperature and min. air temperature had a significant influence on all the line/ varieties. 6. Based upon the environmental conditions of two years data combined stepwise regression indicated that a model combining of air temperature (max. /min.) relative humidity and rainfall expained 84% 0f the variability in disease development. 7. When the data were split by year a model consisting of min./max. air temperature, relative humidity, rainfall and wind speed explained 81 percent of the variablity in disease development during wheat growing season 2005- 06. 8. In the second year (2006-07) a model consisting of min. air temperature wind speed max. air temperature explained 90 percent of the variability in Karnal bunt disease development. 9. Dplomite and Shelter were the best fungicides to control the disease and were staticially equally effective against the disease. 10. Protective spray at early booting stage was more helpful than curative spray.

Chapter 8

RECOMMENDATIONS 1. Farid-06, Menthar, Bwp-97, Shafaq-06 and blue silver must be grown in the affected areas. 2. Seed corporations and private seed companies should not be allowed to sell the seeds until they got the certificate of disease free seed from plant pathologist. 3. Plant protection department must be provided with necessary facilities and options to check the storage godowns freely to get rid of the problems of adulterations. 4. Local quarantine measures should be strictly adopted so that the pathogen could not enter in the disease free areas. 5. Protective spray of Shelter and Dolomite at the recomended doses during the early booting stage of wheat crop provide the best control against the disease. 6. Research workers, extension workers and Agricultural university staff should work in collaboration with each other. At least fortnightly meeting must be held between these personells to exchange views for the solution of farmers, problems. .

Chapter 9 FUTURE STUDIES 1- Assessment of precise number of protrctive and curative foliar sprays to attain 100 % control of Karnal bunt of wheat disease with Dolomite and Shelter fungicides. 2- Survival of teliospores in different soils containing various proportions of sand, silt and clay with various combinations of macro and micronutrient. 3- Germination of teliospores to release the primary and secondary sporadia at various levels of soil moisture and temperature. 4- Survival and longivity of secondary sporadia under dry and wet conditions and its virulence/ aggressiveness with passage of time. 5- As the heterothallism has been reported in the pathogen, there is dire need to study the infection process in detail and histology of infection of wheat by Tilletia indica. 6- Biological control of the Karnal bunt disease of wheat.

Chapter 10

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