Karnal Bunt of Wheat (Triticum Aestivum Wheat Fields in Arizona Because of the Arizona (Fig

Morris R. Bonde, Gary L. Peterson, and Norman W. Schaad USDA-ARS-FDWSRU, Frederick, Maryland

Joseph L. Smilanick USDA-ARS, Horticultural Crops Research Laboratory, Fresno, California

a precaution. On 21 March 1996, the Sec- retary of Agriculture announced a “Dec- laration of Extraordinary Emergency” to deal with the disease and set into motion +ARNAL"UNT the mechanism to compensate growers and handlers for losses due to quarantine ac- tions (54). On 25 March, a federal quarantine for Karnal bunt was placed on the state of Arizona and parts of Texas and New Mexico where the Karnal bunt–contami- OF7HEAT nated wheat from Arizona had been planted (25). Later, the discovery of Karnal bunt–infected wheat in California extended the quarantine to portions of that state (Fig. 1), and by late summer a national Karnal bunt survey was underway. The efforts of hundreds of state and federal personnel in Karnal bunt of wheat (Triticum aestivum wheat fields in Arizona because of the Arizona (Fig. 2) and California, and of L.), caused by the smut fungus Tilletia discovery of bunted seeds and the confir- many more workers in other states, and indica Mitra (=Neovossia indica (Mitra) mation of T. indica infection by poly- thousands of pages devoted to Karnal bunt Mundkur), was first discovered in 1930 at merase chain reaction (PCR) (17,48). on the Internet underscore the impact of the Botanical Research Station, Karnal, Bunted seeds also were found in remnant the recent discovery of Karnal bunt in the Haryana, in northwest India (29), and now samples of Arizona wheat seed remaining United States. is considered common in the Punjab region in Arizona after a portion of the lots had The main effect of extensive Karnal of India. The disease has been reported been planted in Arizona, Texas, and New bunt is to reduce yield (4) and impart a from Pakistan, Iraq, and Nepal, and is Mexico. Fields in Texas and New Mexico fishy odor and taste to wheat flour, thus found in wheat from Afghanistan (6). It planted with the seed were deep plowed as reducing the quality of the flour (2). Yield was first reported in Mexico in 1972 (16), and since then it has occurred sporadically in localized areas in the states of Sonora and Sinaloa, northwest Mexico. Because the disease was not known in major wheat- producing countries, trade of Karnal bunt– infested wheat grain became highly regulated internationally, and the Mexican gov- ernment in 1984 placed an internal quarantine on Karnal bunt to prevent disease spread within the country (27). On 8 March 1996, the U.S. Department of Agriculture (USDA) and the Arizona Department of Agriculture announced the discovery of Karnal bunt in Arizona (Release No. 0115.96, Ag News FAX; 56). Efforts were initiated to quarantine suspect

Dr. Bonde’s address is: USDA ARS FDWSRU, 1301 Ditto Avenue, Fort Detrick, MD 21702-5023 E-mail: [email protected]

Product names are necessary to report factually on available data; however, the USDA neither guar- antees nor warrants the standard of the product, and the use of the name by USDA implies no approval of the product to the exclusion of others that may also be suitable.

Publication no. D-1997-1024-01F This article is in the public domain and not copy- rightable. It may be freely reprinted with custom- ary crediting of the source. The American Phyto- Fig. 1. Map of the continental United States showing Karnal bunt–regulated areas in pathological Society, 1997. August 1997.

1370 Plant Disease / Vol. 81 No. 12 and quality losses are considered by many the Department of Defense (DOD) at Fort disease. Only a few kernels of some wheat smut pathologists to be minor (11,51). Detrick (28). The facility is a 12.5 × 53.7 heads are infected, and usually only a por- However, since Karnal bunt is the subject m brick and concrete building with five tion of an infected kernel is replaced with of strict quarantines by several wheat-im- attached 7.6 × 18.3 m glasshouses under fungal sorus (Fig. 3). T. indica is a porting countries, T. indica can profoundly negative air pressure. All waste water is basidiomycetous pathogen belonging to the affect international trade of commercial decontaminated by DOD upon exiting the order Ustilaginales. Black, dusty-appearing grain and the movement of wheat germ- facility. Each glasshouse has double-lay- teliospores give this group of organisms plasm (51). In 1983, the Animal and Plant ered glass panels supported by a steel su- the name “smut.” The life cycle of T. in- Health Inspection Service (APHIS) placed perstructure. With permission of state and dica is depicted in Figure 4. The telio- restrictions on wheat coming from coun- federal regulatory officials, plant patho- spores (Fig. 5) of T. indica are diploid tries with Karnal bunt, recognizing that gens and diseases from anywhere in the (2N), thick walled, globose to subglobose, establishment of Karnal bunt in the United world can be investigated at Bldg. 374 (28). and average 35 µm in diameter (range 22 to States could have major economic ramifi- ARS initiated Karnal bunt research at 49 µm) when mature (27). They are very cations on U.S. wheat exports. The spread Frederick in 1982, after Karnal bunt ap- resistant to adverse environmental condi- of Karnal bunt to the United States and its peared in northwestern Mexico. The Kar- tions, remaining viable for 2 to 5 years in establishment therein could have placed nal bunt research program at Frederick was contaminated soil (27). The pathogen is the country at a marked disadvantage in the the first initiated in the United States and seedborne but is not transmitted directly international wheat market as the first ma- has continued for 15 years. from seed into plant (27). Teliospores of T. jor wheat-exporting country to have Karnal In 1983, ARS initiated cooperative re- indica are considered dormant immediately bunt. As shown by responses following the search projects on Karnal bunt in India and after formation and have poor germination discovery in March 1996 of Karnal bunt in Mexico, and established and maintained a up to approximately 9 months (36). Arizona, and later in California, the disease containment laboratory in Logan, Utah, After a period of dormancy and in the has generated considerable concern and under the direction of James A. Hoffmann. presence of moisture, teliospores at the soil debate both within and outside the country. He and his staff cooperated closely with J. surface germinate (45). During the germi- The American Phytopathological Society Michael Prescott of the International nation process, the nucleus undergoes took the position that Karnal bunt is of Maize and Wheat Improvement Center meiosis followed by several mitotic divi- little agronomic significance and should (CIMMYT), who managed an extensive sions. A promycelium (basidium) grows not be regulated (1). Karnal bunt research program in Mexico. out from the spore, and as many as 180 Since 1972, research on specific foreign This presentation is an overview of Kar- haploid (1N) basidiospores (also known as plant pathogens of major threat to U.S. nal bunt, its importance in international primary sporidia) are produced at the tip agriculture has been a primary objective of agriculture, and past and present research (18,20,36) (Fig. 6). Normally, teliospores the USDA, Agricultural Research Service to better understand and control the disease germinating under 2 mm of soil are inca- (ARS), Foreign Disease-Weed Science and make rational decisions. Recent re- pable of reaching the surface (45). How- Research Unit at Fort Detrick, Frederick, views include Gill et al. (20), Mathur and ever, it is not known whether teliospores Maryland. In 1992, the major objective of Cunfer (27), and Singh (36). A very com- beneath the soil surface germinate. The this program became the development of prehensive literature review was published primary sporidia mean lengths and widths rapid molecular means of detecting and by Warham (51) in 1986. among isolates range from 64 to 79 µm and making accurate, timely identifications of from 1.6 to 1.8 µm, respectively. Sporidia foreign plant pathogens. Because of its Symptoms of Disease germinate to produce mycelia, which in regulatory significance, T. indica was a turn produce large numbers of secondary primary target. and Life Cycle of Pathogen The Frederick unit conducts research on The disease is difficult to detect under foreign pathogens in a plant disease con- fields conditions, and generally only care- tainment facility (Bldg. 374) leased from ful examination will reveal evidence of

Fig. 3. Typical wheat head with Karnal bunt infection. Under natural field conditions, symptoms are not readily appar- Fig. 2. Arizona Department of Agriculture laboratory in 1997 testing wheat seed sam- ent. Surveying for Karnal bunt infection ples for the presence of Tilletia indica teliospores. In 1996, more than 4,700 wheat requires harvesting the seed and thresh- fields were preharvest and postharvest tested. ing the grain to expose the kernels.

Plant Disease / December 1997 1371 sporidia with mean lengths for different nels. Dhaliwal and Singh (14) presented to higher leaves. In this manner, the fungus isolates of 11.9 to 13.0 µm, and a mean evidence that T. indica may travel in steps travels up the plant to reach developing width of 2.0 µm (31). At the time of flow- from the soil surface to susceptible heads. heads. Here, the sporidia on the glumes ering of the wheat plants, primary and According to them, sporidia from the soil germinate and penetrate the stomates if the secondary sporidia are presumably surface germinate on lower plant leaves, plant is in the 2- to 3-week susceptible splashed and blown onto the surface of colonize the leaf surface, and produce fur- period at or near anthesis. Mycelia grow to glumes enclosing developing wheat ker- ther sporidia which are splashed or blown the base of the glumes and up into the developing kernels (21). The fungus is re- stricted to the pericarp, where it is entirely intercellular (12,35). As the kernels mature, large numbers of teliospores are produced (Fig. 7). At harvest, they are rede- posited on the soil surface to perpetuate the pathogen and disease. Teliospore numbers in a T. indica–contaminated wheat field in the Punjab in India were reported to vary from 2 × 103 to 50.5 × 103 spores per cm3 of soil (20). Epidemiology Karnal bunt initiation and development is dependent on suitable weather conditions during the period wheat plants are flowering and most susceptible to infection (36). According to Singh (36), the optimum temperature range for teliospore germination is 15 to 25°C. Smilanick et al. (45) reported that the optimum after a 3- week incubation in continuous light was 15 to 20°C over a pH range of 6.0 to 9.5. Moisture is a critical element in determining whether there will be a disease out- break (20,36,45). Teliospore germination requires at least 82% relative humidity

Fig. 5. Teliospores of Tilletia indica observed microscopically. Mature spores are dark brown, and immature spores are light brown. Teliospores average 35 µm in diameter and are easy to see in a water wash from contaminated wheat seed.

Fig. 4. Life cycle of Tilletia indica. Teliospores (diploid) germinate at the soil surface and produce haploid primary sporidia (equal basidiospores), which are blown or splashed to the surface of leaves. These germinate to produce mycelia, which produce secondary sporidia. The secondary sporidia are blown or splashed to higher leaves, germinate, and produce more secondary sporidia. In this way, the pathogen moves in steps up to the developing wheat head. On the head, secondary sporidia Fig. 6. Germinated teliospore with long germinate, penetrate the glumes through stomates, and establish infection. Mycelia slender primary sporidia (basidiospores) grow down to the base of glumes and up into the developing kernel. Eventually, growing from the tip of a promycelium diploid teliospores are produced, which are returned to the soil. (basidium).

1372 Plant Disease / Vol. 81 No. 12 (RH) and preferably, free water (36). If and in their digestive systems (41); how- lections but concluded that there was no high RH and/or several rainy days occur ever, whether these spores result in disease evidence for races. Each collection (two during the 2- to 3-week window at flow- is not documented. from Mexico and one each from India and ering, infection of wheat kernels is likely Pakistan) produced the same disease se- to occur (36). The longer the period of high Pathogen Variation verity rankings on six wheat accessions humidity and rainy weather, the greater the Isozyme analyses have established evi- that differed in susceptibility to T. indica. number of seeds infected and the higher dence of high genetic variation within T. In nature, heterothallism of T. indica most the extent of infection of individual seeds. indica. Bonde et al. (7,8) resolved the pro- likely causes a constant reassortment of Apparently, these conditions are rarely, if tein products of 36 presumed isozyme loci genes, thus leading to the instability of ever, encountered over large land areas, or of 66 monoteliospore cultures of T. indica. races. Teliospore collections can thus be the inoculum levels are not adequate, since Of these 36 isozyme loci, 15 (42%) were expected to be an assortment of spores of Karnal bunt disease losses have never been polymorphic (having allelic variation). The different genetic makeup. reported to be large. relatively low average coefficient of simi- Subsequent to discovery of Karnal bunt For Karnal bunt to establish and per- larity (0.83) among cultures was in marked in Arizona and California in 1996, telio- petuate in most of the major U.S. wheat contrast to that of T. foetida and T. caries, spores of a smut pathogen were found that production regions, teliospores of the causal agents of common bunt of wheat, in tested positive for T. indica using PCR pathogen must be able to survive freezing which intraspecific variation was nearly primers developed at Frederick to differ- conditions. In laboratory tests, teliospores absent. The authors believed the greater entiate T. indica and Tilletia horrida, the survived freezing over several months, variation in T. indica was due primarily to rice kernel smut fungus sometimes referred although some workers reported germina- the high level of outcrossing in T. indica. to as T. barclayana. This smut was deter- tion was delayed (13) or reduced (57) by In contrast, the common bunt pathogens mined to be infecting ryegrass seeds in freezing. It is not known whether Karnal have ineffective sexual cycles, and out- Oregon and the southeastern United States. bunt can survive in northern states such as crossing is rare. Fusion of basidiospores of Extensive sequencing of regions of mito- Montana or North Dakota. However, the T. foetida and T. caries occurs almost to- chondrial and nuclear DNA is being con- Frederick unit has been conducting an tally between basidiospores formed on the ducted by ARS and APHIS scientists to overwintering temperature experiment in same basidium. Forced outcrossing of T. better understand the molecular variation sealed containers in Maryland since 1992, indica probably promotes sexual recombi- within T. indica and to determine the rela- with the appropriate state and APHIS per- nation and genetic variation. tionship of the ryegrass pathogen in Ore- mission. Preliminary results indicate that Variability was also observed in num- gon and the southeastern United States to many Karnal bunt teliospores can survive bers and sizes of chromosomes in T. indica T. indica. Maryland winters in buried sealed contain- (49). At least 11 chromosomes were ob- ers for at least 3 years. Soil temperatures served in T. indica, ranging in size from PCR for Rapid Identification during the period dipped periodically to 2 about 1 to 3.3 megabases. Many isolates of T. indica to 3°C below freezing for a few weeks. contained unique karyotypes. Differences Although there is considerable genetic Although T. indica spores can survive the in karyotypes of teliospores and mono- variability within T. indica, the use of PCR mild winter conditions found in Maryland, sporidial lines derived from the same telio- primers selected from mitochondrial DNA little is known about survival under more spore indicated that karyotype changes sequences has proven extremely valuable extreme conditions further north and the may occur through meiosis. for rapid presumptive identification of T. survival of free spores. Moisture at the The existence of races of T. indica is indica. The technique is especially useful time of flowering may be the most critical controversial. Gill et al. (20) reported the for differentiating free teliospores of T. factor in determining where in the U.S. identification of four races in India based indica from those of T. horrida that are in Karnal bunt could become established. on different levels of aggressiveness. the same size range. By using primers Predictions made from weather data and Bonde et al. (6) recognized differences in TI17M1/M2 and TI57M1/M2 (48), or known requirements for infection by T. aggressiveness among four teliospore col- primers Ti-1/Ti-4 (17), DNA extracted indica suggest that, under current climatic conditions, Karnal bunt will never cause major crop losses in the U.S. (15).

Dissemination of the Pathogen Dry teliospores can survive for many years under laboratory conditions. In fact, spores used in experiments at Fort Detrick have been stored on laboratory shelves for up to 16 years. Long-range teliospore dissemination can occur by transport of infected and/or contaminated seed (36). However, teliospores can also be spread by air currents. Teliospores of T. indica were collected 3,000 m above fields being burned after harvest in Mexico (9). The evidence indicated that the updraft of air from burning fields carried teliospores to great heights and acted as a mechanism of dispersal. Vehicles also transport the pathogen. Boratynski et al. (10) demon- strated that T. indica teliospores were present on rail cars moving from Mexico into California. Animals and birds act as trans- Fig. 7. Bunted kernels removed from infected plants in the field. Most infected kernels porters of viable teliospores on their bodies are only partially infected.

Plant Disease / December 1997 1373 from mycelia obtained from germinated wheat, caused by T. controversa, consid- enough within the plant to inhibit the in- teliospores washed from wheat seeds can ered by many plant pathologists to be a fection of florets (43). Seed treatment fun- be quickly tested. These primers tested minor disease of the U.S. Pacific North- gicides are of value only when infected or positive with over 78 isolates of T. indica west, with high rates of infection in local- contaminated seed is planted in soil not and negative with 69 isolates of T. horrida ized small areas within individual fields infested with teliospores. Hot water treat- (48). Because the selected primers failed to (26). The environmental requirements of ments can reduce teliospore germinability differentiate between T. indica and isolates the two diseases are different, but disease without killing the seed (30,46), but water of the newly discovered ryegrass pathogen incidence for each is driven by very spe- temperatures that kill all teliospores within discussed above, primers are now being cific weather factors. Whereas dwarf bunt infected seeds reduce seed germination designed from sequences of the cloned occurs only in autumn-sown wheat in areas (42). Nonselective heat treatments, such as 2,300-bp product from amplification with where snow cover maintains temperatures water of 60°C or higher (42), burning primers Ti-1/Ti-4. Also, inter-transcribed near freezing for several weeks at the soil wheat stubble (37), or soil solarization spacer (ITS) regions of nuclear fungal surface (26), Karnal bunt apparently occurs (36,37), dramatically reduce the viability rDNAs have been sequenced by APHIS to only where high moisture, preferably rain, of teliospores. Practical limitations to the gain more information on the relatedness occurs for several days at the time of flow- utilization of these methods include the between the ryegrass and wheat isolates (L. ering of wheat plants. expense of hot water or solarization and, as Levy, R. Meyer, and A. Tschanz; APHIS, previously cited, the dispersal of viable PPQ, Beltsville, MD.; personal communi- teliospores in the air above burning fields cation) and may be useful for identification. Control (9). Fungicides applied to soil at seeding Control of Karnal bunt is desirable at have not reduced the disease (43), probably two levels: (i) to manage the disease where because the infections originated from Crop Losses it occurs so that losses in yield and quality airborne infectious sporidia from telio- In the foothills of Himachal Pradesh, are minimized, and (ii) to contain the dis- spores that germinated outside the test Jammu, and Kashmir states of India, Gill ease or contaminating teliospores for trade plots. Among the fumigants tested to con- et al. (20) reported that the percentage of or regulatory purposes. Control measures trol teliospores, only methyl bromide killed wheat samples that contained at least one suitable for regulatory purposes usually teliospores within the unbroken sori of infected kernel increased from 25% in must be more stringent than those needed infected seeds when used at high doses on 1977–78 to 86% for the 1982–83 crop year. for management purposes. For example, moist soil (47) or on moist grain after har- Percentages of wheat seed samples with treatments applied to comply with insect vest (J. L. Smilanick and M. R. Bonde, bunted seeds were 9, 34, and 17% in Hary- quarantines, so that potentially infested unpublished). Seed fumigation tests with ana during the 1974–75, 1977–78, and products can leave regulated areas and be ethylene oxide were promising, although 1978–79 cropping seasons, respectively, accepted elsewhere, often must meet a both it and methyl bromide reduced seed and 60% of samples had infected kernels in minimum efficacy standard termed probit germination dramatically when applied at 1982 (38). In 1989–90, 1990–91, and 9, which dictates not more than three pests effective doses (46; M. R. Bonde, unpub- 1991–92, 63, 94, and 62% of wheat sam- should survive in a population of 100,000 lished). ples, respectively, had bunted kernels (20). treated individuals (34). Several attributes Foliar fungicide applications have Taken collectively, these figures suggest of the etiology of Karnal bunt and the te- achieved significant control of Karnal that disease levels increased slowly over liospores of T. indica make control a very bunt. Two or more applications of propi- time. However, the actual percentage of challenging problem. Cultural practices conazole at or after spike emergence re- infected seeds per sample is a much better that reduce Karnal bunt incidence, such as duced the incidence of infected seeds by measure of the effect of the disease on crop delays in sowing date, reduced nitrogen 95% (3,37,43,44). In many locations, the yield and quality. fertilization, or reduced planting density, cost of propiconazole use can be partially A detailed analysis of wheat samples only affect modest reductions in Karnal recouped by increases in yield or quality collected from 15 districts of eastern Uttar bunt incidence and may themselves reduce that often occur by the control of diseases Pradesh in 1987, a year of particularly high yields (20,36,52). The teliospores, which other than Karnal bunt. However, when Karnal bunt incidence in India, revealed are long-lived and very resistant to chemi- propiconazole is applied to emerged that an average of 3.79% of seeds per sam- cal and physical treatments (46,53), are spikes, as was done in some reports where ple were infected (32,33). In 1978–79, one borne within and protected by the sorus good control of Karnal bunt occurred, resi- field in Madhya Pradesh had 23% of the and remainder of the partially bunted seeds dues of the fungicide may occur in the seed infected (40). However, data collected typical of the disease. Teliospores are pro- grain and pose a regulatory issue. In con- by survey teams in northwest India showed duced in large numbers; one infected seed trast to results in Mexico (43,44), Singh that even during the worst epidemic years, can contain >100,000 teliospores. Telio- and coworkers (37) reported that an appli- the loss was only 0.2 to 0.5% of total pro- spores within the interior of sori are further cation of propiconazole that preceded spike duction (24). According to Singh (36), the protected by the surrounding mass of telio- emergence by 2 days, eliminating or mini- state of Uttar Pradesh in northeast India spores. Teliospores buried in soil persist mizing grain residues, reduced Karnal bunt experiences less than 1% loss even in the longer than those on the soil surface and incidence by more than 95%. More studies worst years. Yield losses in Mexico from are more protected from physical and of fungicide timing are needed, particularly 1982 to 1989 averaged 0.12% (11). The chemical treatments (47). with natural infection, but the sparse and data from India and Mexico suggest that a Seed and soil treatments applied for the irregular incidence of Karnal bunt makes few fields can have significant levels of control of Karnal bunt have been only this work difficult and time-consuming to disease in some years, but disease severity partially successful. Fungicides applied to conduct. over large areas is never more than a few seeds do not kill the teliospores but inhibit Genetic resistance can provide excellent percent. In general, the percentage of their germination (23,53); some chemicals control of Karnal bunt. Workers in Mexico bunted seeds reported in surveys is consis- with fungistatic activity that will persist and India have identified resistant lines tently low, although the percentage of more than 6 months include carboxin, whose ancestry was traced to China, India, positive samples (where Karnal bunt could thiram, pentachloronitrobenzene, and chlor- or Brazil (19,20,39). Tolerant wheat culti- be detected) can be high after seasons with othalonil (53). Seed treatment fungicides vars, such as WL 1562 in India and particularly conducive weather. do not protect wheat plants from infection Arivechi and Guamuchil in Mexico, have In many ways, the epidemiology of Kar- when seeds are planted in teliospore-in- been released to growers; genes involved nal bunt parallels that of dwarf bunt of fested soil, and they do not persist long in resistance have been identified; and

1374 Plant Disease / Vol. 81 No. 12 immune selections, based on resistance regulated by oil temperature, auger speed, techniques to detect and identify the telio- originating from goat grass (Triticum and length of tube. The Holo-Flite Thermal spores of T. indica, which are necessary to tauschii), are under development (50). Processor, or similar dry heat processor, is conduct the massive Karnal bunt survey more economical to purchase and operate (4,700 fields were pre- and postharvest Treatment of Karnal Bunt– than methods such as pelletization or ex- tested in Arizona alone) and developing trusion, other methods that potentially feasible methods to decontaminate har- Contaminated Wheat destroy Karnal bunt spores. vested wheat, facilities, and equipment. Steam-flake milling. Following the dis- The study was conducted in a Karnal However, during the first year of the Na- covery of Karnal bunt in Arizona and Cali- bunt–contaminated shed belonging to Ari- tional Karnal Bunt Survey, it became ap- fornia, there was a major effort to test zona Grain, Inc., in Casa Grande, using a parent that other smut pathogens, morpho- wheat in fields, grain elevators, and rail small scale Holo-Flite test model. Clean logically similar to T. indica, were also cars for the presence of teliospores of T. mill feed was artificially infested with present and complicated identifications. indica. As a result of these assays, large teliospores of T. indica. The Holo-Flite During the summer of 1996, T. indica–like quantities of grain testing positive for Kar- was operated at a range of temperatures teliospores were detected as free spores in nal bunt were left in storage facilities and speeds, and three replicated samples wheat in the southeastern United States. In throughout the region. It was recognized were taken at each time/temperature set- the fall of 1996, similar teliospores were by USDA and state officials in Arizona and ting. The temperature of the treated mill detected in ryegrass seed lots in Oregon. California that a method was needed feed was recorded as it left the machine. This was particularly significant because as within a few weeks to treat the contami- Spore viability was determined by extrac- much as 80% of the world’s grass seed is nated grain so it could be moved safely tion of teliospores from the product and produced in Oregon, and as much as 60% from these storage facilities and preferably germination testing. Results indicated that of the ryegrass seed lots tested were con- still maintain some economic value. One teliospores in mill feed can be killed with taminated with a pathogen that could not such method tested, found highly success- dry heat if the product reaches tempera- be reliably distinguished from T. indica at ful and adopted, was steam-flake milling. tures of 84, 101, or 110°C for 12, 5, or 2 that time by either PCR or spore morphol- This milling procedure is used to generate min, respectively (G. L. Peterson, T. Bo- ogy (M. R. Bonde, M. Palm, G. L. Peter- feed for huge livestock feed lot operations, ratynski, D. Harder, and K. Kostas, un- son, L. Levy, and R. Meyer, unpublished). is readily available in the region, and was published). However, despite the fact that ryegrass and shown effective in destroying Karnal bunt wheat are both grown in Oregon (often teliospores with no modification to its Present Problems adjacent to each other), and teliospore standard operational specifications (Fig. 8). numbers were sometimes high in ryegrass In a large cooperative test conducted by and Future Research samples, no spores of T. indica morphol- ARS (G. L. Peterson), APHIS (T. Boratyn- One of the main reasons for the mam- ogy could be detected in wheat samples ski), Arizona Department of Agriculture moth effort to deal with Karnal bunt in the from the same areas. This suggested that in (D. Harder), and California Department of United States is an economic considera- Oregon the pathogen infected ryegrass and Food and Agriculture (K. Kosta), four tion. The United States sells about $5 bil- not wheat, and therefore was an organism truckloads of Karnal bunt–contaminated lion worth of wheat per year in the foreign different from T. indica. durum wheat were treated by steam-flake market. In order to maintain these sales, In January 1997, R. Ykema, Arizona milling. In the process, grain was loaded phytosanitary certificates are required indi- Department of Agriculture, found an in- via closed system conveyer belt into 7.6- cating that the wheat comes from regions fected ryegrass seed in an Oregon seed lot m-high steam cabinet towers and heated 30 where Karnal bunt is not known to occur. (personal communication), and in Febru- min to 109°C, then passed through rollers In the United States, as wheat moves along ary, infected ryegrass seeds were discov- that compressed the steamed grain into the transportation pipeline, much of it ered by G. Peterson in wheat samples from flakes. Grain samples were taken from the making its way to the major ports for ex- trucks prior to treatment, then sampled porting, it is commingled with other wheat every 15 min as grain moved through the lots. As long as foreign customers are con- mill. Subsequent teliospore germination cerned about Karnal bunt and have regula- tests showed that all spores were killed by tions preventing entry of wheat with T. the process, and steam-flake milling was indica teliospores, it is imperative this adopted for large-scale use within the pipeline be kept free of Karnal bunt con- quarantine areas. tamination. For this reason, quarantines Holo-Flite Thermal Processor. It gen- were put into effect in the United States. erally is believed that flour milled from Karnal bunt likely has little direct effect contaminated grain poses no phytosanitary on wheat yield or quality except perhaps risk. However, the untreated mill feed by- for localized small areas of high infection. product does pose a minimal risk because Indeed, the quality effects from these hot of the potential introduction of viable telio- spots can be easily diluted by mixing in- spores into a field via animal waste. In a fected seed with seed lots with no disease. cooperative effort between Bay State However, in spite of the minor direct ef- Milling (R. Hampel, D. Reinig, R. Strews- fects on crop yield and quality, the poten- bury), ARS (G. L. Peterson), and CDFA tial for economic losses to the U.S. wheat (K. Kosta), tests were conducted to evalu- industry is real because of possible export ate the effectiveness of heat treating mill reductions. feed using a system known as a Holo-Flite APHIS, ARS, several state departments Thermal Processor. The system consists of of agriculture, and international organiza- a hollow, jacketed tube containing two tions such as CIMMYT (Mexico) are hollow twin augers. Temperature is regu- working cooperatively to answer questions lated by heated oil, which is pumped pertaining to the Karnal bunt problem. Fig. 8. Steam cabinet towers in Arizona through the hollow augers and jacket, Immediately following discovery of Karnal used for steam-flake milling to decon- evenly transferring the heat into the com- bunt in the United States, research at taminate wheat infected and/or infested modity. Temperature of the product is APHIS and ARS centered on improving with Karnal bunt teliospores.

Plant Disease / December 1997 1375 southeast United States (G. L. Peterson, fruitful discussions and suggestions, and for pro- 18. Fuentes-Davila, G., and Duran, R. 1986. unpublished). viding the map of the 1997 Karnal bunt regulated Tilletia indica: Cytology and teliospore for- area and the photograph of the steam-flake milling mation in-vitro and in immature kernels. Can. In spring 1997, the Arizona Department facility; and Joel Floyd, APHIS, PPQ, for drawing J. Bot. 8:1712-1719. of Agriculture in Phoenix and the USDA, and providing the Karnal bunt life cycle. Thanks 19. Fuentes-Davila, G., Rajaram, S., and Singh, ARS in Frederick independently demon- also are expressed to Roy Gingery, Wilda Mar- G. 1995. Inheritance of resistance to Karnal strated that the “ryegrass pathogen” could tinez, Mary Palm, Arnold Tschanz, and Ron bunt (Tilletia indica Mitra) in bread wheat infect wheat after injections of sporidia Ykema for critical reviews of the manuscript, and (Triticum aestivum L.). Plant Breed. 114:250- to Pat Freaner and Gail Hoover for typing the 252. into the boot cavity under optimum green- manuscript, and Susan Nester for taking photo- 20. Gill, K. S., Sharma, I., and Aujla, S. S. 1993. house conditions and high inoculum con- graphs and preparing figures. Karnal Bunt and Wheat Production. Punjab centrations. However, virulence tests per- Agricultural University, Ludhiana. formed under highly conducive artificial 21. Goates, B. J. 1988. Histology of infection of Literature Cited wheat by Tilletia indica, the Karnal bunt conditions can result in erroneous conclu- 1. American Phytopathological Society. 1996. pathogen. Phytopathology 78:1434-1441. sions. For example, it is well recognized Position statement of: The American Phyto- 22. Hildebrand, D. C., Scroth, M. N., and Sands, that some bacterial pathogens only infect pathological Society: The use of quarantines D. S. 1988. Laboratory Guide for Identifica- for wheat Karnal bunt. APSnet. On-line: Kar- certain plant species under artificial condi- tion of Plant Pathogenic Bacteria, 2nd ed. N. nal Bunt Symposium. W. Schaad, ed. American Phytopathological tions (22). Some fungal pathogens, such as 2. Aujla, S. S., Grewal, A. S., Gill, K. S., and Society, St. Paul, MN. pp. 60-80. Peronosclerospora sorghi and P. sacchari, Sharma, I. 1980. Effect of Karnal bunt on 23. Hoffmann, J. A. 1986. Chemical seed treat- have been shown to infect a broader range chappati making properties of wheat grains. ments for Karnal bunt. Proc. Bien. Smut of plant species when large numbers of Crop Improve. 7:147-149. Worker’s Workshop, 3rd. Ciudad Oregon, 3. Aujla, S. S., Sharma, I., Singh, P., Singh, G., Mexico. spores are used under optimum greenhouse Dhaliwal, H. S., and Gill, K. S. 1989. Propi- 24. Josh, L. M., Singh, D. V., and Srivastava, K. conditions but are not known to infect conazole - a promising fungicide against Kar- D. 1980. Wheat Disease Survey - I. Karnal those same species in the field (5). As nal bunt of wheat. Pesticides 23:35-38. Bunt 1975-1980. Wheat Pathology Series No. pointed out by Whitney (55), the inappro- 4. Bansal, R., Singh, D. V., and Joshi, L. M. 7. Division of Mycology and Plant Pathology, 1984. Effect of Karnal bunt pathogen Indian Agricultural Research Institute, New priate reduction to synonymy of the rice (Neovossia indica [Mitra] Mundkur) on Delhi. smut pathogen, T. horrida, with Neovossia weight and viability of wheat seed. Indian J. 25. Mark, L. 1996. Karnal bunt - A strange- barclayana probably resulted from errone- Agric. Sci. 54:663-666. sounding wheat disease with serious implica- ous conclusions made from infection stud- 5. Bonde, M. R., and Peterson, G. L. 1983. tions. APHIS News Feature. On-line: Press Comparison of host ranges of Peronoscle- ies when two species of Pennisetum were Release, August 8. rospora philippinensis and P. sachari. Phyto- 26. Mathur, S. B., and Cunfer, B. M. 1993. Dwarf inoculated and incorrectly described as pathology 73:875-878. bunt. Pages 23-29 in: Seed-borne Diseases infected by T. horrida. Presently, evidence 6. Bonde, M. R., Peterson, G. L., Fuentes-Dav- and Seed Health Testing of Wheat. Jordbrugs- is not conclusive as to whether T. indica ila, G., Aujla, S. S., Nanda, G. S., and Phil- forlaget, Frederiksberg, Denmark. and the ryegrass pathogen are the same lips, J. G. 1996. Comparison of the virulence 27. Mathur, S. B., and Cunfer, B. M. 1993. Karnal of isolates of Tilletia indica, causal agent of bunt. Pages 31-43 in: Seed-borne Diseases organism. Field studies are being initiated Karnal bunt of wheat, from India, Pakistan, and Seed Health Testing of Wheat. Jord- to determine if the ryegrass pathogen will and Mexico. Plant Dis. 80:1071-1074. brugsforlaget, Frederiksberg, Denmark. infect wheat under natural field conditions. 7. Bonde, M. R., Peterson, G. L., and Matsu- 28. Melching, J. S., Bromfield, K. R., and King- Research is underway on the taxonomy moto, T. T. 1989. The use of isozymes to solver, C. H. 1983. The plant pathogen con- identify teliospores of Tilletia indica. Phyto- tainment facility at Frederick, Maryland. Plant of T. indica and similar species, and on the pathology 79:596-599. Dis. 67:717-722. identity of the ryegrass pathogen. In order 8. Bonde, M. R., Peterson, G. L., and Royer, M. 29. Mitra, M. 1931. A new bunt of wheat in India. to answer some of the most pressing ques- H. 1988. Inheritance of isozymes in the smut Ann. Appl. Biol. 18:178-179. tions about Karnal bunt, an expanded re- pathogen Tilletia indica. Phytopathology 30. Mitra, M. 1937. Studies on the stinking smut 78:1276-1279. search effort is being mounted by the or bunt of wheat in India. J. Agric. Sci. 7:459- 9. Bonde, M. R., Prescott, J. M., Matsumoto, T. 478. USDA and some states in order to better T., and Peterson, G. L. 1987. Possible dis- 31. Peterson, G. L., Bonde, M. R., Dowler, W. M., understand the relationship of T. indica to semination of teliospores of Tilletia indica by and Royer, M. H. 1984. Morphological com- other smut organisms. the practice of burning wheat stubble. (Abstr.) parisons of Tilletia indica Mitra from India In addition to resolving taxonomic ques- Phytopathology 77:639. and Mexico. (Abstr.) Phytopathology 74:757. 10. Boratynski, T. N., Matsumoto, T. T., and 32. Rai, R. C., and Singh, A. 1989. Karnal bunt of tions, further research is required in the Bonde, M. R. 1985. Interceptions of Tilletia wheat. A threat to wheat seed production in areas of ecology and epidemiology, espe- indica at the California-Mexico border in eastern Uttar Pradesh. Narendra Deva J. Ag- cially on the eventual geographical limits Mexican railroad boxcars. (Abstr.) Phytopa- ric. Res. 4:27-31. of the disease caused by environmental thology 75:1339. 33. Rai, R. C., Singh, A., and Singh, R. V. 1988. 11. Butler, L. 1990. Karnal bunt, quarantine and Status of Karnal bunt of wheat in eastern Ut- restrictions. Although present research infor- the international shipment of CIMMYT wheat tar Pradesh. Narendra Deva J. Agric. Res. mation suggests that Karnal bunt is limited seed. Proc. Bien. Workshop Smut Fungi, 7th. 3:183-185. by very specific moisture conditions, further 12. Cashion, N. L., and Luttrell, E. S. 1988. Host- 34. Robertson, J. L., Preisler, H. K., Frampton, E. research is required to develop more parasite relationship in Karnal bunt of wheat. R., and Armstrong, J. W. 1994. Statistical Phytopathology 78:75-84. accurate disease models to predict Karnal analyses to estimate efficacy of disinfestation 13. Chahal, S. S., and Mathur, S. B. 1992. Germi- treatments. Pages 47 to 65 in: Quarantine bunt outbreaks. Additional research also is nation of deep-frozen Tilletia indica and Til- Treatments for Pests of Food Plants. J. L. required to improve detection and identi- letia barclayana teliospores. FAO Plant Prot. Sharp and G. J. Hallman, eds. Westview fication of T. indica; improve decontami- Bull. 40:31-35. Press, San Francisco. 14. Dhaliwal, H. S., and Singh, D. V. 1988. Up- nation of wheat seeds, facilities, and equip- 35. Roberson, R. W., and Luttrell, E. S. 1987. to-date life cycle of Neovossia indica (Mitra) Ultrastructure of teliospore ontogeny in Til- ment; develop Karnal bunt–resistant wheat Mundkur. Curr. Sci. 57:675-677. letia indica. Mycologia 79:753-763. cultivars; and refine chemical control 15. Diekmann, M. 1993. Epidemiology and geo- 36. Singh, A. 1994. Epidemiology and Manage- strategies. phytopathology of selected seed-borne dis- ment of Karnal Bunt Disease of Wheat. Re- Karnal bunt is an important disease because eases. Int. Center Agric. Res. Dry Areas search Bulletin No. 127, Directorate of Ex- (ICARDA). periment Station, G. B. Pant University of of its influence on global trade of grain. A 16. Duran, R. 1972. Further aspects of teliospore Agriculture and Technology, Pantnagar, India. solution to the present situation will require germination in North American smut fungi. 37. Singh, B. B., Aujla, S. S., and Sharma, I. continued cooperative efforts of scientists and Can. J. Bot. 50:2569-2573. 1993. Integrated management of wheat Karnal regulators at an international level. 17. Ferreira, M. A. S. V., Tooley, P. W., Hatzilou- bunt. Int. J. Pest Manage. 39:431-434. kas, E., Castro, C., and Schaad, N. W. 1996. 38. Singh, D.V., Josh, L. M., and Srivastava, K. Isolation of a species-specific mitochondrial D. 1986. Varietal susceptibility and spread of Acknowledgments DNA sequence for identification of Tilletia Karnal bunt of wheat in India. Rachis Barley We thank Robert Nave, USDA, APHIS, Na- indica, the Karnal bunt of wheat fungus. Wheat Newsl. ICARDA 4:10-16. tional Coordinator, Karnal Bunt Program, for Appl. Environ. Microbiol. 62:87-93. 39. Singh, G., Rajaram, S., Fuentes-Davila, G.,

1376 Plant Disease / Vol. 81 No. 12 and Morgunov, A. 1995. Genetic basis of resistance to Karnal bunt in bread wheat. Bien. Workshop Smut Fungi, 9th. CIMMYT, El Batan, Mexico. 40. Singh, S. 1980. Report on Coordinated Ex- periments (Wheat Pathology) 1978-79. Wheat Project Directorate, Indian Agric. Res. Insti- tute, New Delhi. 41. Smilanick, J. L., Dupler, M., Goates, B. J., Hoffmann, J. A., Clark, D., and Dobson, D. 1986. Germination of teliospores of Karnal, dwarf, and common bunt fungi after ingestion by animals. Plant Dis. 70:242-244. 42. Smilanick, J. L., Hershberger, W., Bonde, M. R., and Nester, S. E. 1997. Germinability of teliospores of Tilletia indica after hot water and sodium hypochlorite treatment. Plant Dis. 81:932-935. 43. Smilanick, J. L., Hoffmann, J. A., Cashion, N. L., and Prescott, J. M. 1987. Evaluation of seed and foliar fungicides for the control of Morris R. Bonde Gary L. Peterson Karnal bunt of wheat. Plant Dis. 71:94-96. 44. Smilanick, J. L., Hoffmann, J. A., and Dr. Bonde graduated with a B.S. degree in Mr. Peterson is a biologist who received his Prescott, J. M. 1987. Control of Karnal bunt botany from the University of Maine in 1967 B.S. degree from St. Mary’s College of with foliar fungicides, 1986. Fungic. Nemati- and from Cornell University with M.S. and Maryland in 1977. Since 1978, he has been cide Tests 42:96. Ph.D. degrees in plant pathology in 1969 with the USDA-ARS Foreign Disease-Weed 45. Smilanick, J. L., Hoffmann, J. A., and Royer, and 1974, respectively. Since 1974, he has Science Research Laboratory and currently been employed by the USDA-ARS Foreign M. H. 1985. Effect of temperature, pH, light, is assigned to research on new and Disease-Weed Science Research. He has and desiccation on teliospore germination of emerging plant diseases that affect conducted research to determine the threat international trade. In addition to work on Tilletia indica. Phytopathology 75:1428- of foreign plant pathogens to major U.S. Karnal bunt, he has conducted research on 1431. crops. Among the diseases are Karnal, graminaceous downy mildews, soybean 46. Smilanick, J. L., Hoffmann, J. A., Secrest, L. dwarf, and common bunts of wheat; downy rust, citrus canker, sorghum ergot, and R., and Wiese, K. 1988. Evaluation of chemi- mildews of maize, sorghum, and sugarcane; kernel smut of rice. His research and pro- cal and physical treatments to prevent germi- soybean rust; and sorghum ergot. He began fessional activities since 1982 have focused nation of Tilletia indica teliospores. Plant Dis. his research program on Karnal bunt in on detection, identification, epidemiology, 72:46-51. 1982 and has had extensive cooperative and trade issues associated with Karnal 47. Smilanick, J. L., Prescott, J. M., Hoffmann, J. research projects in India and Mexico. bunt and dwarf bunt of wheat. A., Secrest, L. R., and Wiese, K. 1989. Envi- ronmental effects on survival and growth of secondary sporidia and teliospores of Tilletia indica. Crop Prot. 8:86-90. 48. Smith, O. P., Peterson, G. L., Beck, R. J., Schaad, N. W., and Bonde, M. R. 1996. De- velopment of a PCR-based method for identification of Tilletia indica, causal agent of Karnal bunt of wheat. Phytopathology 86:115-122. 49. Tooley, P. W., Carras, M. M., Beck, R., Peter- son, G., and Bonde, M. R. 1995. Separation of Tilletia indica chromosomes using CHEF gel electrophoresis. Mycologia 81:61-67. 50. Villareal, R. L., Mujeeb-Kazi, A., Davila, G. F., and Rajaram, S. 1996. Registration of four synthetic hexaploid wheat germplasm lines derived from Triticum turgidum × T. tauschii crosses and resistant to Karnal bunt. Crop Sci. 36:218. 51. Warham, E. J. 1986. Karnal bunt disease of wheat: A literature review. Trop. Pest Norman W. Schaad Joseph L. Smilanick Manage. 32:229-242. Dr. Schaad received his B.S., M.S., and 52. Warham, E. J., and Flores, D. 1988. Farmer Dr. Smilanick is a research plant pathologist Ph.D. degrees from the University of at the Horticultural Crops Research surveys on the relation of agronomic practices California, Davis, in 1964, 1966, and 1969, Laboratory of the USDA-ARS facility in to Karnal bunt disease of wheat in the Yaqui respectively. He then studied the character- Fresno. He received a B.S. from the Valley, Mexico. Trop. Pest Manage. 34:373- ization of bacterial ribosomes for 2 years University of California, Davis, in plant 381. under the direction of C. I. Kado. Dr. Schaad science in 1977, an M.S. in plant pathology 53. Warham, E. J., Prescott, J. M., and Griffiths, was a professor of plant pathology at the in 1980 from Colorado State University, and E. 1989. Effectiveness of chemical seed University of Georgia in Griffin from 1971 a Ph.D. in plant pathology in 1984 from the treatments in controlling Karnal bunt disease until 1982, specializing in identification, University of California, Riverside. Prior to of wheat. Plant Dis. 73:585-588. detection, and ecology of bacteria. He taught moving to Fresno in 1986, he studied the 54. Washington Post. 1996. Declaration of ex- seed pathology and headed a research biology and control of Karnal bunt in a traordinary emergency. March 22. p. A23. program in detection and control of cooperative project with CIMMYT as a 55. Whitney, N. G. 1989. Taxonomy of the fun- seedborne bacteria at the University of research associate of James A. Hoffmann of gus causing kernel smut of rice. Mycologia Idaho from 1982 until 1986. In 1986, he took the USDA-ARS in Logan, Utah. Subse- 8:468-471. a position as Director of Biotechnology of quently, he investigated postharvest treat- 56. Ykema, R. E., Floyd, J. P., Palm, M. E., and Harris Moran Seed Company in Gilroy, ments employing biological control, fumi- Peterson, G. L. 1996. First report of Karnal California. Since 1992, he has been Research gants, heat treatments, and fungicides for Leader and Bacteriology CRIS Leader of the bunt of wheat in the United States. Plant Dis. small grains and horticultural crops to USDA-ARS Foreign Disease-Weed Science 80:1207. reduce postharvest decay losses or to affect Research Unit in Frederick, Maryland, containment of pathogens for quarantine 57. Zhang, Z., Lange, L., and Mathur, S. B. 1984. where he specializes in developing im- purposes. His present research activities Teliospore survival and plant quarantine sig- proved molecular techniques for detecting are focused primarily on the use of bio- nificance of Tilletia indica (causal agent of seedborne pathogens. logical control and heat treatments for citrus Karnal bunt) particularly in relation to China. fruit to control postharvest decay. EPPO Bull. 14:119-128.

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