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Hot water and sodium hypochlorite (NaOCl) were evaluated to eradicate teliospores of the Karnal bunt fungus, Tilletia indica, for the purpose of decontaminating grain storage and handling equipment. The germinability of free teliospores and teliospores within the sori of infected wheat was assessed. Temperatures of 25, 60, and 80°C, NaOCl concentrations (wt/vol, pH 11.5) of 0, 0.53, and 1.60%, and immersion periods of 1, 5, 15, and 30 min were evaluated. In other tests, the influence of pH on NaOCl potency and of a delay between treatment and water rinsing were evaluated. Immersion at 80°C in water alone or with NaOCl killed both free teliospores and those within the sori of infected seeds within 1 min. NaOCl at 1.60% at 25°C killed teliospores suspended in water within 15 min, but some teliospores inside sori survived 30 min of this treatment. NaOCl adjusted to pH 8 before use was superior to NaOCl at pH 11.5. An application of 1.60% NaOCl at 25°C for 5 min followed by a 10-min delay before the seeds were rinsed in fresh water killed free teliospores but not all teliospores within sori. This treatment was more effective than the 5-min treatment alone but inferior to the 15-min treatment with NaOCl at a concentration of 1.60%. Because teliospores within the sori of infected seeds are partially protected and much more resistant to NaOCl, we recommend the removal and disposal of seeds from equipment before the treatments are applied. NaOCl radically altered the appearance of the teliospores, leaving a persistent visual indication that they had been treated, while hot water treatment alone did not. Therefore, it is beneficial to add NaOCl to hot water, although the improvement in the sporicidal efficacy was often small.

Tilletia indica, the causal agent of Karnal bunt of wheat, was first detected and reported in the United States in 1996. Karnal bunt occurred in the southwestern United States as early as 1992. Wheat contaminated with teliospores of T. indica is likely to have been transported from the Southwest to other regions, including the Pacific Northwest, before presence of the pathogen was discovered. Teliospore and sporidial germination and infection are highly dependent on climatic conditions. The potential for T. indica to infect wheat in the Pacific Northwest has not been reported. The objective of this study was to use published information on environmental factors favorable for infection and historical climate data for the Pacific Northwest to analyze the environmental risk for Karnal bunt to occur if wheat fields in the Pacific Northwest become contaminated by T. indica. Conditions during the past four decades appeared favorable for infection in nonirrigated wheat during 1 of every 3 years at two (Corvallis, OR, and Spokane, WA) of 13 Idaho, Oregon, and Washington locations examined, and every year at all locations where wheat is irrigated. If introduced to the area, it appears possible for T. indica to become established in selected regions of the Pacific Northwest.

Removal of the flag leaf of wheat, earlier hypothesized to act as a site where secondary sporidia of Tilletia indica multiply and conjugate, reduced the severity of Karnal bunt. Evidence reported here suggests that sporidia reach the flag leaf from the soil by splashing, multiply on the surface of the flag leaf, and move with rain water or dew into the boot of the flag leaf to cause infection of the spikelets. Plants in growth stage 49 (first awns visible) were more vulnerable to infection by secondary sporidia than were plants at other growth stages. At growth stage 49, the flag leaf lamina was fully opened, compared with its preceding stages, which had tubular and closed lamina. Also, leaf posture of the flag leaf at growth stage 49 is erect and thus may facilitate movement of sporidial inoculum into the boot. Upon completion of stage 49, the flag leaf becomes droopy, leading to lesser inoculum movement into the emerging spike. Thus, maximum success can be achieved in artificial creation of Karnal bunt by inoculating with secondary sporidia before onset of growth stage 49. Genotypes whose maturity avoids coincidence of this critical stage of spike emergence with periods of weather favorable for infection by T. indica may be selected for resistance breeding.

Bonde, M. R., Nester, S. E., Khayat, A., Smilanick, J. L., Frederick, R. D., and Schaad, N. W. 1999. Comparison of effects of acidic electrolyzed water and NaOCl on Tilletia indica teliospore germination. Plant Dis. 83:627-632. Definitive identification of free teliospores of Tilletia indica, causal agent of Karnal bunt of wheat, requires polymerase chain reaction (PCR)-based diagnostic tests. Since direct PCR amplification from teliospores has not been reliable, teliospores first must be germinated in order to obtain adequate DNA. We have routinely surface-sterilized teliospores for 2 min with 0.4% (vol/vol) sodium hypochlorite (NaOCl) to stimulate germination and produce axenic cultures. However, we observed that some spores were killed even with a 2-min NaOCl treatment, the shortest feasible duration. Decreasing the NaOCl concentration in our study from 0.4% to 0.3 and 0.2%, respectively, increased teliospore germination, but treatment times longer than 2 min still progressively reduced the germination percentages. In testing alternative met hods, we found “acidic electrolyzed water” (AEW), generated by electrolysis of a weak solution of sodium chloride, also surface-sterilized and increased the rate of T. indica teliospore germination. In a representative experiment comparing the two methods, NaOCl (0.4%) for 2 min and AEW for 30 min increased germination from 19% (control) to 41 and 54%, respectively, by 7 days after treatment. Because teliospores can be treated with AEW for up to 2 h with little, if any, loss of viability, compared with 1 to 2 min for NaOCl, treatment with AEW has certain advantages over NaOCl for surface sterilizing and increasing germination of teliospores of suspect T. indica.

A sucrose-centrifugation method was developed to extract teliospores of Tilletia indica, T. con-troversa, and T. barclayana from soil. Six soil types were artificially infested with teliospores of each of the three fungi separately to produce 102, 103, 104, or 105 teliospores per 10 g. Each 10 g of infested soil was suspended in 200 ml of water with one drop of Tween 20 and shaken for 30 s. The soil suspension was first passed through a 117-μm sieve and then through a 53-μm mesh filter, and the filtrate was collected. The filtrate was then passed through a 20-μm mesh filter, and materials caught on the mesh were washed into two 50-ml centrifuge tubes and spun for 3 min (1,200 × g). The pellet was suspended in 1.6 M sucrose solution and centrifuged for 40 s (200 × g). The supernatant was passed through a 20-μm mesh filter. The materials caught on the 20-μm mesh were collected, and the number of teliospores was determined. This procedure was initially used to extract teliospores of T. indica in soil. For extraction of teliospores of T. contro-versa and T. barclayana, 1.0 M and 1.3 M sucrose solutions, respectively, were used, and the 20-μm mesh was replaced with a 13-μm mesh filter. Teliospores of T. indica, T. controversa, T. barclayana, and T. indica-like fungus on rye grass were successfully extracted from naturally infested soils. The relationships between number of teliospores recovered from the soil and number of teliospores incorporated into the soil were Ŷ= -0.60 + 1.28(X) - 0.04(X2),Ŷ = -1.25 + 1.56(X) - 0.07(X 2), and Ŷ = -0.71 + 1.33( X) - 0.04(X2) for T. indica, T. controversa, and T. barclayana, respectively, where Ŷ = log10 of the number of teliospores recovered from soil and X = log10 of the actual number of teliospores in soil.

In December 2000 seed harvested from wheat (Triticum aestivum L.) cultivars SST 876 and SST 825 produced under sprinkler irrigation near Douglas, Northern Cape Province, South Africa, contained a substantial amount of partially bunted kernels. Kernel embryos contained black masses of teliospores, and in many instances the endosperm was partially degraded. Teliospores were brown to dark brown, densely echinulate, 25 to 45 μm in diameter with a short mycelial fragment on some of the spores. Hyaline, smooth-walled sterile cells were also present. Teliospores were soaked in sterile distilled water for 2 days, streaked on 2% water agar plates and incubated at 22°C in the dark. Teliospores germinated after 5 days, producing 50 to 250 filiform, nonconjugating, primary basidiospores and forcibly discharged allantoid, secondary basidiospores. Based on kernel appearance, a rotten fish odor in infected grain, teliospore morphology, and germination characteristics, the pathogen was identified as Tilletia indica Mitra, the cause of Karnal bunt (1). This morphological identification was confirmed at the USDA-ARS Systematic Botany and Mycology Laboratory, Beltsville, MD. Molecular verification of 12 South African isolates was provided by the Foreign Disease-Weed Science Research Unit at Fort Detrick, MD, using real-time polymerase chain reaction with the Tin3/Tin10 T. indica-specific primer set (2). Four additional isolates were confirmed as T. indica using the same primer set as well as ITS rDNA sequencing at the Beltsville laboratory. Reference specimens were deposited at the National Fungal Collection in Pretoria, South Africa (PREM 57214), and at Beltsville (BPI 748170). At present, the mode of introduction of T. indica into South Africa, as well as its precise distribution, is not known. It appears, however, that the pathogen is restricted to the Douglas production area in the Northern Cape where quarantine measures have been taken to contain and possibly eradicate the disease. References: (1) L. A. Castlebury and L. M. Carris. Mycologia 91:121, 1999. (2) R. D. Frederick et al. Phytopathology 90:951, 2000.

Acidic electrolyzed water (AEW) is a germicidal product of electrolysis of a dilute solution (e.g., 0.4% vol/vol) of sodium chloride. This solution can be used to disinfest wheat seed or soil samples being tested for teliospores of Tilletia indica, causal agent of Karnal bunt, without risk of damaging the teliospores. The AEW used in this study had a pH of 2.5 to 2.8 and oxidation-reduction potential of approximately 1,130 mV. In simulations of routine extractions of wheat seed to detect teliospores of T. indica, the effectiveness of a 30-min AEW treatment was compared with a 2-min 0.4% sodium hypochlorite (NaOCl) treatment to eradicate bacteria and nonsmut fungi. Each treatment reduced bacterial and fungal populations in wheat seed extracts by 6 to 7 log10 units when determined on 2% water agar with antibiotics. Reductions of 5 log10 units or more were observed on other media. NaOCl and AEW also were very effective at eliminating bacteria and fungi from soil extracts. In studies to detect and quantitate T. indica teliospores in soil, AEW was nearly 100% effective at eliminating all nonsmut organisms. Free chlorine levels in AEW were very low, suggesting that compounds other than those with chlorine play a significant role in sanitation by AEW. The low pH of AEW was shown to contribute substantially to the effectiveness of AEW to reduce microorganisms. A standardized protocol is described for a 30-min AEW treatment of wheat seed washes or soil extracts to eliminate contaminating microorganisms. A significant advantage of the use of AEW over NaOCl is that, with AEW, teliospore germination is not reduced and usually is stimulated, whereas teliospore germination declines after contact with NaOCl. The protocol facilitates detection and enumeration of viable teliospores of T. indica in wheat seed or soil and the isolation of pure cultures for identification by polymerase chain reaction. The germicidal effects of AEW, as demonstrated in this study, illustrate the potential of AEW as an alternative to presently used seed disinfestants.

Karnal bunt of wheat (caused by Tilletia indica) was first detected in the United States in Arizona in 1996. The seed lots of infected, spring-habit, durum wheat associated with the initial detection were traced to planted fields in California, Arizona, New Mexico, and Texas. However, in the summer of 1997, the disease appeared in unrelated, winter-habit, bread wheat located over 700 km from the nearest potentially contaminated wheat from 1996 (and destroyed prior to reinfection). Here, we examined potential invasion pathways of the fungus associated with the movement of wheat into the United States. We analyzed the USDA/APHIS Port Information Network (PIN) database from 1984 through 2000 to determine likely pathways of introduction based on where, when, and how the disease was intercepted coming into the United States. All interceptions were made on wheat transported from Mexico, with the majority (98.8%) being intercepted at land border crossings. Karnal bunt was not intercepted from any other country over the 17-year period analyzed. Most interceptions were on wheat found in automobiles, trucks, and railway cars. The majority of interceptions were made at Laredo, Brownsville, Eagle Pass, and El Paso, TX, and Nogales, AZ. Karnal bunt was intercepted in all 17 years; however, interceptions peaked in 1986 and 1987. Averaged over all years, more interceptions (19.2%) were made in the month of May than in any other month. Our results indicate that Karnal bunt has probably arrived in the United States on many occasions, at least since 1984. Because of the relatively unaggressive nature of the disease and its reliance on rather exacting weather conditions for infection, we surmised that it is possible this disease has a long period of latent survival between initial arrival and becoming a thriving, established disease.

To examine the epidemiology of Tilletia indica teliospores in naturally infested soils from wheat fields in both Karnal bunt-regulated regions in Texas, soil was grid sampled from fields that were bunted-kernel positive for Karnal bunt in 1997, 2001, both years, or never. Aliquots of soil from each point were pooled, and teliospores were extracted using a size-selective sieving-sucrose centrifugation method. Teliospores were enumerated microscopically, and low quantities (< 8 per 25 g of soil) were identified in 14 of 15 fields sampled from the regulated regions of Texas, including fields that have never tested positive for bunted kernels, indicating a widespread distribution. No teliospores were isolated from the single field examined outside of the regulated regions. The percent clay was significantly, negatively correlated with the baseline teliospore number and the estimated (extrapolated) number of teliospores per sample, indicating a potential impact of soil composition on teliospore survival. The latter factor was also significantly, positively correlated to the number of times a field had tested positive.

A study was conducted to determine the relationship between soilborne Tilletia indica teliospore density and Karnal bunt incidence in an Arizona durum wheat field in 2005 and 2006. Soil samples were collected from 507 sample points according to a grid marked in a 7.7-ha field. Approximately 500 g of soil from the top 5 cm was collected from each sample point, and teliospores were recovered from 25-g aliquots by a modified size-selective sieving, sucrose centrifugation procedure. Twenty-five and 50 wheat heads were collected from a 1-m2 area around each sample point in May 2005 and June 2006, respectively. Wheat head samples from each sample point were bulked, threshed, and examined for the presence of bunted kernels. Additionally, data for soilborne teliospores and percent bunted kernels from 70 sample points in 2005 and 2006 that corresponded to sample points from a 2004 bunted kernel survey conducted by the USDA and Arizona Department of Agriculture were analyzed. Soilborne teliospore numbers ranged from 6 to 1,000 per 25-g soil sample in the 2-year study. No bunted kernels were recovered in 2005; however, two sample points yielded bunted kernels in 2006. Weather data from three time periods in 2004, 2005, and 2006 were applied to the humid thermal index model and suggested that a conducive environment for disease development existed in 2005. Based on the data from this research, we concluded that even though high numbers of soilborne teliospores were present in the field, and although a conducive environment was present for disease to develop on only one occasion, a direct relationship between soilborne teliospores and disease incidence may not exist.

Allen, T. W., Workneh, F., Steddom, K. C., Peterson, G. L., and Rush, C. M. 2008. The influence of tillage on dispersal of Tilletia indica teliospores from a concentrated point source. Plant Dis. 92:351-356. A study was conducted to determine the impact of tillage on dispersal of Tilletia indica teliospores from a concentrated point source in Arizona. The infested source was created using a 300-ml teliospore suspension, containing approximately 9.0 × 10 4 teliospores/ml, sprayed onto a 1-by-3-m soil area. Approximately 400 g of soil was collected before tillage treatments, representing the teliospore baseline, and after each of five disk passes, to an approximate depth of 20 cm, through the infestation source (n = 597). Soil samples were collected along three parallel lines extending from the infested area at increments of 1, 3, or 10 m to a total distance of 10, 30, and 50 m, respectively. Teliospores were recovered from soil samples by a combined sizeselective sieving sucrose-centrifugation technique. Immediately following teliospore infestation, an average of 3.6 × 103 teliospores per 25 g of soil sample were recovered from the infestation area. Two different trends in recoverable teliospores occurred at 0- to 10-m sampling distances following five plow passes: either a decrease in the number of teliospores recovered, represented at points 0, 1, and 2 m, or an increase in recoverable teliospores found at points 3 to 10 m. The study was repeated twice for a total of three experiments, and teliospores were recovered to a maximum distance of 24 m. However, the numbers recovered from distances beyond 10 m were sporadic. Based on data from this research, we conclude that teliospores are not distributed in large quantities to great distances by tillage and, therefore, tillage cannot account for the spatial distribution of teliospores in many infested wheat fields. Additional keywords: Karnal bunt

The survival of teliospores of the Karnal bunt of wheat pathogen, Tilletia indica, was determined in field plots in Tucson, AZ. Two methods were used to test viability during a 48-month period in which 21-μm-pore-size polyester mesh bags of teliospore-infested soil were buried in irrigated and nonirrigated field plots at two sites. One method determined the total number of viable teliospores in a soil sample, regardless of whether or not they could be extracted from the soil using a sucrose centrifugation technique. The total number of viable teliospores declined over time in both irrigated and nonirrigated field plots and in the same soils in the laboratory. Based on nonlinear regressions, total number of viable teliospores decreased from 55.7% at time zero to 9.7 and 6.7% for nonirrigated and irrigated field soils, respectively, in 48 months. Total number of viable teliospores in soil in the laboratory decreased from 55.7 to 34.0% after 48 months. The second method determined germination percentages of teliospores extracted from the soil samples by means of a sucrose centrifugation technique. Based on linear regressions of transformed data, germination of teliospores extracted from irrigated and nonirrigated field soils, and control (laboratory) soil, significantly decreased over time. The rate of decrease in germination was significantly greater for teliospores from irrigated field plots than from nonirrigated plots and the laboratory soil. At time zero, 55.7% of teliospores germinated, and by 48 months, average germination of teliospores extracted from soil in nonirrigated plots had decreased to 13.6% compared with 4.4% in irrigated plots and 36.8% for teliospores in the laboratory control. Regression over time of total number of viable teliospores accounted for more of the overall variability than did regression over time of germination percentages of extracted teliospores. Neither field site nor soil depth had any effect on total number of viable teliospores or on teliospore germination percentages.

To determine the potential for Tilletia indica, cause of Karnal bunt of wheat, to survive and become established in new areas, a teliospore longevity study was initiated in Kansas, Maryland, Georgia, and Arizona. Soil from each location was infested with T. indica teliospores and placed in polyester mesh bags. The bags were placed within soil from the same location within 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 ends to allow interaction with the outside environment, however 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 maintained dry in a laboratory. During the first 2 years, viability 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 nearly 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. We hypothesized that soils, irrespective of weather, affect teliospore longevity

Surveys for Tilletia walkeri on annual ryegrass (Lolium multiflorum) were conducted during 1997 and 1998 in the southeastern United States, where suspect teliospores of the Karnal bunt fungus, Tilletia indica, were found in USDA-APHIS surveys of wheat (Triticum aestivum) seed in 1996. T. walkeri is morphologically similar to T. indica. Annual ryegrass is a common weed in wheat fields in the southeastern United States. Between April and June 1997, ryegrass seed samples were collected from 190 fields of wheat in 47 counties in Georgia and from 26 fields in 17 counties in Alabama and south-central Tennessee. In 1998, 70 samples were collected from 40 counties in the same regions of the three states. The teliospores from these samples were 23 to 45 μm in diameter (average about 33 μm) and ranged from light brown to dark reddish brown. They had coarse, widely spaced cerebriform ridges on the surface and were surrounded by a gelatinous sheath. The ryegrass bunt was identified as the recently described species T. walkeri, occurring on ryegrass seed from Australia and Oregon. In 1997, teliospores of T. walkeri were found in 13 samples from eight counties in central Georgia and from one field in Tennessee. In 1998, more teliospores and bunted seeds were found, possibly due to frequent rain in the region throughout the flowering period for ryegrass. Teliospores were found in 26/70 of the samples, and among these, only a small number of bunted seed were found in 12 of 13/70 samples. In one wheat field in Morgan County, Georgia, about 50% of the ryegrass seed collected was partially bunted, and a small percentage was completely bunted. Fields with teliospores were widely distributed and generally matched the locations where teliospores were found in APHIS wheat seed surveys in 1996 to 1998. T. walkeri occurs at very low levels on ryegrass in the Southeast and is the source of teliospores, initially identified as those of T. indica, associated with wheat seed in APHIS surveys. No bunted wheat seeds or teliospores of T. indica were found in the survey.

Four Tilletia indica teliospore field populations, two from Mexico and one each from India and Pakistan, were tested for virulence on five Karnal bunt-resistant cultivars, one moderately susceptible, and two Karnal bunt highly susceptible wheat cultivars. The five resistant cultivars represented the most Karnal bunt-resistant germ plasm in the breeding programs at the International Maize and Wheat Improvement Center (Centro Internacional de Mejoramiento de Maiz y Trigo [CIMMYT]), Mexico, and the Department of Plant Breeding, Punjab Agricultural University, Ludhiana, India. Plants at the boot stage were inoculated by injecting into the boot 1 ml of a water suspension containing 10,000 allantoid sporidia per ml, incubated in a mist chamber for 3 days, then maintained until maturity in a greenhouse. All inoculated and control wheat spikes were harvested individually, and percentages of T. indica-infected seeds were determined. In addition, infected seeds from 10 randomly selected infected spikes per treatment were examined to estimate the proportion of each infected seed converted to a sorus. On the most resistant wheat cultivar (HD-29), percentage of seeds infected varied from 10 to 30%, depending on pathogen aggressiveness. On the most susceptible cultivar (Bacanora), infection varied from 55 to 84%. Although there were differences in pathogen aggressiveness, there was no evidence of the existence of races among the field populations. Wheat cultivars resistant to the Mexican fungal populations also were resistant to those from Asia, and vice versa; there was a significant correlation (P ≤ 0.05) between percentage of seeds infected and extent of fungal colonization of infected seeds with all but one pathogen population when comparing resistant versus other wheat cultivars.

The effect or Karnal bunt (T.i.) infection on viability, germination and vigor of wheat seed was determined using the topographical tetrazolium viability test, the rolled paper towel germination test, and the accelerated aging vigor test. Infection had very little effect on seed viability, irrespective of the age of the seed, while germination of infected seed appeared to depend upon the wheat cultivar and age of the seed. In contrast, there was a signigicant reduction in vigor of infected seed. Infected seeds would therefore appear to have a lower survival rate in storage compared to healthy seeds of the same seed lot

Sporidia of T.i. were produced in potato-dextrose broth with or without supplemental sucrose after 1 wk on a shaker. Secondary sporidia produced in liquid cultures were characteristically filiform and distinguishable from allantoid secondary sporidia produced on potato-dextrose agar plates. Boot and spray inoculations of bread wheat with filiform secondary sporidia resulted in lower levels of infection than similar inoculations with allantoid secondary sporidia. When screening germ plasm for Karnal bunt resistance, allantoid secondary sporidia produced in solid media should be used as inoculum

Forty-seven chemical products recommended for the control of bunts and smuts were screened for their effectiveness as wheat seed treatments to control Tilletia indica. Chemicals that reduced teliospore germination appeared to be limited in the length of their activity. Most were effective for up to 6 mo but only a few for longer periods of time.(...) There is currently no chemical seed treatment that can guarantee that wheat seed is not carrying viable T.i. teliospores