Conceptualizing patient-reported outcome measures for use within two Danish psychiatric clinical registries: description of an iterative co-creation process between patients and healthcare professionals.

BACKGROUND: Denmark has national clinical indicator programs for adult patients diagnosed with depression and schizophrenia, respectively. Within each program, the responsible steering group (SG) decided to add some indicators based upon patient-reported outcome measures (PROMs). AIMS: The primary aim was to describe the process of selecting PROMs and defining a national measurement concept for use in clinical practice and for indicator monitoring and the secondary aim s to collect patient recommendations for implementation. METHODS: An interdisciplinary SG of healthcare professionals and a Patient Peer Board (PPB) representing both patient groups co-created the output in an iterative process. The work included literature search, PPB workshops, SG meetings, ratings of PROM topics and items, and a pilot. The PPB discussed the following: item relevance, mode of data collection, graphical format of the online PROMs, and display of results. Finally, requirements for PROM patient information were identified. Based upon input from the PPB, the SG selected the items and specified the measurement concept. RESULTS: The PPB prioritized 20 of 53 suitable items and suggested alternative wording and answer categories. A pilot was performed and 19 items covering well-being, lack of well-being, impairment of functioning, and overall health were selected for clinical testing. The patients recommended concrete, unambiguous, easily understandable information and procedures for data collection and display of results. CONCLUSIONS: The iterative co-creation process based upon a high degree of patient involvement resulted in a set of PROMs, a national measurement concept, and patient recommendations for implementation. The cooperation between patients and professionals was successful.

Evidence for Systemic Infection by Puccinia horiana, Causal Agent of Chrysanthemum White Rust, in Chrysanthemum.

Puccinia horiana, causal agent of the disease commonly known as chrysanthemum white rust (CWR), is a quarantine-significant fungal pathogen of chrysanthemum in the United States and indigenous to Asia. The pathogen was believed to have been eradicated in the United States but recently reappeared on several occasions in northeastern United States. The objective of the study presented here was to determine whether P. horiana could systemically infect chrysanthemum plants, thus providing a means of survival through winters. Scanning and transmission electron microscopy revealed the development of P. horiana on the surface and within leaves, stems, or crowns of inoculated chrysanthemum plants artificially exposed to northeastern U.S. winter temperatures. P. horiana penetrated leaves directly through the cuticle and then colonized the mesophyll tissue both inter- and intracellularly. An electron-dense material formed at the interface between fungal and host mesophyll cells, suggesting that the pathogen adhered to the plant cells. P. horiana appeared to penetrate mesophyll cell walls by enzymatic digestion, as indicated by the absence of deformation lines in host cell walls at penetration sites. The fungus was common in vascular tissue within the infected crown, often nearly replacing the entire contents of tracheid cell walls. P. horiana frequently passed from one tracheid cell to an adjacent tracheid cell by penetration either through pit pairs or nonpitted areas of the cell walls. Individual, presumed, fungal cells in mature tracheid cells of the crown and stems arising from infected crowns suggested that the pathogen might have been moving at least partially by means of the transpiration stream. The demonstration that chrysanthemum plants can be systemically infected by P. horiana suggests that additional disease control measures are required to effectively control CWR.

Effects of frequency of "extreme" temperature highs on development of soybean rust.

Previously, we hypothesized that summer "extreme" diurnal temperature highs in the southeastern United States were responsible for the yearly absence or delay of soybean rust development until fall. Utilizing temperature-controlled growth chambers, a diurnal temperature pattern of 33°C high and 20°C low reduced urediniospore production by 81%. However, that study did not consider the influence of frequency of extreme temperatures on soybean rust. We now report that a temperature high of 35°C for 1 h on three consecutive days, initiated 15 days after inoculation, when lesions had formed, reduced urediniospore production by 50% and required 9 to 12 days for sporulation to resume once the extreme temperature highs ceased. Furthermore, three consecutive days in which the temperature high was 37°C, beginning immediately after inoculation and subsequent dew period, reduced lesion numbers by 60%. The combined effects of reduced numbers of lesions and urediniospores per lesion caused by extreme temperature highs can account for observed absence or delay of soybean rust development in the southeastern United States until fall. A comparison of frequency of extreme temperature highs with numbers of counties reporting presence of soybean rust from 2005 to 2012 verified that extreme temperature highs may be largely responsible for absence or delay of soybean rust development. This is the first report showing the effect of frequency of extreme temperature highs on development of soybean rust. Because the south-to-north progression of soybean rust is required for the disease to occur in the major soybean-production regions of the United States, temperatures in the southeastern United States have a major effect on the entire U.S. soybean industry.

Effects of daily temperature highs on development of Phakopsora pachyrhizi on soybean.

Although considerable information exists regarding the importance of moisture in the development of soybean rust, little is known about the influence of temperature. The purpose of our study was to determine whether temperature might be a significant limiting factor in the development of soybean rust in the southeastern United States. Soybean plants infected with Phakopsora pachyrhizi were incubated in temperature-controlled growth chambers simulating day and night diurnal temperature patterns representative of the southeastern United States during the growing season. At 3-day intervals beginning 12 days after inoculation, urediniospores were collected from each plant and counted. The highest numbers of urediniospores were produced when day temperatures peaked at 21 or 25°C and night temperatures dipped to 8 or 12°C. When day temperatures peaked at 29, 33, or 37°C for a minimum of 1 h/day, urediniospore production was reduced to 36, 19, and 0%, respectively, compared with urediniospore production at the optimum diurnal temperature conditions. Essentially, no lesions developed when the daily temperature high was 37°C or above. Temperature data obtained from the National Climatic Data Center showed that temperature highs during July and August in several southeastern states were too high for significant urediniospore production on 55 to 77% of days. The inhibition of temperature highs on soybean rust development in southeastern states not only limits disease locally but also has implications pertaining to spread of soybean rust into and development of disease in the major soybean-producing regions of the Midwestern and northern states. We concluded from our results that temperature highs common to southeastern states are a factor in the delay or absence of soybean rust in much of the United States.

Penetration and establishment of Phakopsora pachyrhizi in soybean leaves as observed by transmission electron microscopy.

For over 30 years, it has been known that Phakopsora pachyrhizi is unusual in that it penetrates from urediniospores directly through the leaf cuticle without entering stomates. This unusual mode of penetration suggests that disease resistance mechanisms might exist for soybean rust that do not exist for most rust diseases. As a result, we decided to conduct a histological study using transmission electron microscopy to further elucidate the mechanisms of penetration and early establishment of P. pachyrhizi in soybean leaves. Based on our study, it was concluded that P. pachyrhizi utilizes primarily mechanical force, perhaps with the aid of digestive enzymes, to penetrate the cuticle on the leaf surface. However, the lack of deformation lines in micrographs indicated that digestive enzymes, without mechanical force, are used by the penetration hypha to penetrate the outer and inner epidermal cell walls. Digestive enzymes, again indicated by the lack of deformation lines, are used by haustorial mother cells to breach the walls of mesophyll cells to form haustoria. The possibility exists for eventual determination of the precise roles of pressure and digestive enzymes in the development of soybean rust and elucidation of some of the determinants of resistance and susceptibility to this important plant disease.

Characterizing Resistance to Phakopsora pachyrhizi in Soybean.

Resistance in soybean to Phakopsora pachyrhizi, the cause of soybean rust, is characterized by either reddish-brown (RB) lesions or an immune response. The RB type of resistance can be incomplete, as evidenced by the presence of sporulating uredinia within lesions. Susceptibility, on the other hand, is exemplified by tan-colored (TAN) lesions, and can be expressed in gradations of susceptibility or partial resistance that are less well defined. This study evaluated traits associated with incomplete or partial resistance to P. pachyrhizi in soybean by comparing 34 soybean accessions inoculated with four P. pachyrhizi isolates. Six accessions produced RB lesions to all four isolates, while 19 accessions produced TAN lesions, including plant introduction (PI) 200492 (Rpp1) and the susceptible check 'Williams'. Williams had among the largest area under the disease progress curve (AUDPC) values and area under the sporulating uredinia progress curve (AUSUPC) values, while eight accessions had lower AUSUPC values. Of the known sources of single-gene resistance, only PI 230970 (Rpp2), PI 459025B (Rpp4), and PI 594538A (Rpp1b) had lower AUDPC and AUSUPC values than Williams. PI 594538A and PI 561356 had RB lesions and had the lowest AUDPC and AUSUPC values. Of the known sources of single-gene resistance, only PI 230970 (Rpp2) and PI 594538A (Rpp1b) produced fewer and smaller-diameter uredinia than Williams. This study characterized reactions to P. pachyrhizi in 34 accessions based on lesion type and sporulation, and defined incomplete resistance and partial resistance in the soybean-P. pachyrhizi interaction.

Comparative Susceptibility of Kudzu Accessions from the Southeastern United States to Infection by Phakopsora pachyrhizi.

Soybean rust, caused by Phakopsora pachyrhizi, was first discovered in the continental United States in the fall of 2004. The potential for economic loss in the United States hinges largely on whether or not the pathogen can survive winters in the absence of soybean. Kudzu (Pueraria lobata) is known to be a host for P. pachyrhizi in Asia and South America and is widely distributed in the southern United States. This study examined reactions of kudzu collected from several areas of the southeastern United States to three isolates of P. pachyrhizi, one each from Alabama, Louisiana, and Brazil. Susceptible tan (TAN) lesions, resistant reddish-brown (RB) lesions, and immune (IM) response, previously described on soybean, were produced on kudzu based on the evaluation of 125 plants. However, in contrast to soybean, the RB response on kudzu was common, with approximately 50% frequency. IM responses to at least one isolate were observed on five individual plants, and two plants were immune to all three pathogen isolates used in the test. TAN lesions averaged 3.2 uredinia per lesion with an average diameter per uredinium of 121 µm. In contrast, RB lesions had an average of 0.3 uredinia per lesion with an average uredinial diameter of 77 µm. In 25 of 39 (64%) instances in which multiple plants were tested from a site, each reacted the same to the individual pathogen isolates. This suggested a tendency for plants at specific sites to be genetically identical with respect to rust reaction. Only 19 of 125 (15%) individual plants produced a different reaction to one isolate than to the other two isolates. When four kudzu plants previously shown to produce only TAN lesions to P. pachyrhizi isolates Alabama 04-1, Brazil 01-1, and Louisiana 04-1 were inoculated with eight additional isolates from several areas of the world, all 11 isolates produced only TAN lesions. Likewise, when five other plants previously shown to produce only RB lesions when inoculated with the three isolates were inoculated with the 11 isolates, all produced only RB lesions. These results suggest that susceptibility or resistance to P. pachyrhizi in individual kudzu plants often is broad, extending over a wide range of P. pachyrhizi isolates.

Does hut climate matter for piglet survival in organic production?

Piglet mortality in outdoor production systems varies across the year, and a reason for this variation could be fluctuations in hut climate, as ambient temperature might influence piglet survival, both directly and indirectly. Therefore, the aim of the current study was to investigate the impact of farrowing hut climate and year variation on stillbirth and liveborn mortality. A large-scale observational study was conducted at five commercial organic pig-producing herds in Denmark from June 2015 to August 2016. Both year variation (F 3,635=4.40, P=0.004) and farrowing hut temperature (F 2,511=6.46, P=0.002) affected the rate of stillbirths. The risk of stillborn piglets was lowest in winter and during this season larger changes in hut temperature between day 1 prepartum and the day of farrowing increased the risk of stillbirths (F 1,99=6.39, P=0.013). In addition, during the warm part of the year stillbirth rate increased at temperatures ⩾27°C. Year variation also affected liveborn mortality (F 3,561=3.86, P=0.009) with a lower rate of liveborn deaths in spring. However, the hut climate did not influence liveborn deaths. Consequently, other factors than hut climate may explain the influence of year variation on liveborn mortality. These could be light differences causing seasonality in reproduction and lactation.

New Legume Hosts of Phakopsora pachyrhizi Based on Greenhouse Evaluations.

Phakopsora pachyrhizi, the causal organism of soybean rust, was first found in the continental United States in 2004 and has been found on soybean, kudzu, Florida beggarweed, and three Phaseolus species in the field. The pathogen has been reported to occur on more than 90 legume species worldwide and it is likely to infect native and introduced legume species in the United States. The objective of this study was to determine if 176 species representing 57 genera of legumes, the majority of which are either native or naturalized to soybean-growing areas of the United States, could be hosts of P. pachyrhizi. Between one and three accessions of each species, a total of 264 accessions, were inoculated with a mixture of four isolates of P. pachyrhizi. Severity and sporulation were rated on a 1-to-5 scale at 14 and 28 days after inoculation. P. pachyrhizi was confirmed by the presence of sporulating uredinia and/or immunological assay on 65 new species in 25 genera; 12 of these genera have not been reported previously as hosts. Many of the newly identified hosts grow in the southern United States, and like kudzu, could serve as overwintering hosts for P. pachyrhizi.

Comparative Susceptibilities of Legume Species to Infection by Phakopsora pachyrhizi.

Knowledge of the host range of Phakopsora pachyrhizi is important to agriculture in the United States because of the distinct possibility that economic losses could occur to crops other than soybean. Furthermore, it is possible that alternative hosts could provide a means of overwintering of the pathogen, providing inoculum to initiate epidemics in future years. To clarify the potential importance of soybean rust on nonsoybean legumes and their role in overwintering of the disease, multiple accessions of clover, cowpea, pea, kudzu, lima bean, snap bean, and single accessions of coffee senna, Florida beggarweed, hemp sesbania, hyacinth bean, partridge pea, and showy crotalaria were inoculated under greenhouse conditions with urediniospores of P. pachyrhizi; infected soybean plants served as a control. The four criteria used to assess susceptibility were lesion density, proportion of lesions with sporulating uredinia, average number of uredinia per lesion, and average uredinia diameter, each determined 2 weeks following inoculation. Based on lesion densities, percentage of lesions with sporulation, and average numbers of uredinia per lesion, soybean, kudzu, and pea were the most susceptible species, followed by snap bean. However, because infected pea plants defoliated rapidly, urediniospore production presumably was limited, lessening the potential for epidemics on pea. Cultivars of snap bean produced numerous brown to reddish-brown lesions, many of which sporulated, but numbers of uredinia per lesion were lower than on soybean, kudzu, or pea. The presence of both tan (susceptible) and reddish-brown (resistant) lesions on kudzu demonstrated physiological differentiation on that host. Some kudzu plants appeared to be potentially excellent hosts for overwintering of the disease. The average number of uredinia per lesion appeared to be a valid measurement with which to compare host susceptibilities, and may have epidemiological significance. High susceptibility of a host was characterized by numerous uredinia with a wide range of sizes within individual lesions. In contrast, low susceptibility to rust was characterized by no or a few small uredinia.

Effects of temperature on urediniospore germination, germ tube growth, and initiation of infection in soybean by phakopsora isolates.

ABSTRACT Temperature is a critical factor in plant disease development. As part of a research program to determine how specific environmental variables affect soybean rust, we determined temperature effects on urediniospore germination and germ tube growth of four isolates of Phakopsora pachyrhizi, one each from Brazil, Hawaii, Taiwan, and Zimbabwe, and an isolate of P. meibomiae from Puerto Rico, collected over a 25-year period. Also compared were the effects of temperature during a night dew period on initiation of disease by the P. pachyrhizi isolates. All variables were fit to a nonlinear beta function with temperature as the independent variable. Minimum, maximum, and optimum temperatures, along with shape parameters of the beta function for each variable, were statistically analyzed. All Phakopsora isolates behaved similarly as to how temperature affected urediniospore germination, germ tube growth, and initiation of disease. The results suggest that P. pachyrhizi has changed little in the past few decades with respect to how it responds to temperature and that previously collected research data continues to be valid, simplifying the development of soybean rust disease models.

Evaluation of Virulence of Phakopsora pachyrhizi and P. meibomiae Isolates.

Asian soybean rust (ASR), caused by Phakopsora pachyrhizi and recently discovered for the first time in continental United States, has been of concern to the U.S. agricultural industry for more than 30 years. Since little soybean rust resistance is known, and resistance is often difficult to detect or quantitate, we initiated a project to develop a better, more quantitative, method. The methodology determined the average numbers and diameters of uredinia in lesions that developed on leaves of inoculated plants 14 days after inoculation. It was used to compare virulence of P. pachyrhizi isolates from Asia and Australia and P. meibomiae from Puerto Rico and Brazil, collected as many as 30 years earlier, with isolates of P. pachyrhizi recently collected from Africa or South America. Susceptible reactions to P. pachyrhizi resulted in tan-colored lesions containing 1 to 14 uredinia varying greatly in size within individual lesions. In contrast, on these same genotypes at the same time of year, resistance to other P. pachyrhizi isolates was typified by 0 to 6 small uredinia in reddish-brown to dark-brown lesions. Using appropriate rust resistant and rust susceptible genotypes as standards, examination of uredinia 14 days after inoculation allowed quantitative comparisons of sporulation capacities, one measure of susceptibility or resistance to soybean rust. The study verified the presence and ability to detect all known major genes for resistance to soybean rust in the original sources of resistance. It demonstrated that soybean lines derived from the original PI sources, and presumed to possess the resistance genes, in actuality may lack the gene or express an intermediate reaction to the rust pathogen. We suggest that a determination of numbers and sizes of uredinia will detect both major gene and partial resistance to soybean rust.

Measurement of bone degradation products in serum using antibodies reactive with an isomerized form of an 8 amino acid sequence of the C-telopeptide of type I collagen.

An enzyme-linked immunosorbent assay for measuring type I collagen degradation products in serum (S-ELISA) was developed. The assay uses a high affinity polyclonal antibody which reacts with an isomerized form of an 8 amino acid sequence of the C-telopeptides of type I collagen (EKAHD-beta-GGR). Cross-reactivity to a nonisomerized synthetic peptide form of the 8 amino acid sequence is less than 0.2%. Values obtained in a group of premenopausal women (age, 33.3 +/- 3.11 years) were 69 +/- 24 ng/ml(n = 22). In a group of early postmenopausal women (age, 51.8 +/- 1.88 years) values obtained were 125 +/- 43 ng/ml (n = 46), which represents an increase of 81% (p < 0.001). Values found in untreated patients with Paget's disease were 234 +/- 95 ng/ml (n = 15), and for primary hyperparathyroidism we found 335 +/- 82 ng/ml (n = 10). Intravenous administration of a bisphosphonate (Pamidronate) to Paget's disease patients for 3 days was reflected in the S-ELISA by a decrease in the values of 55% when compared with values before treatment (n = 15). Following treatment with another bisphosphonate (Alendronate) for 6 months, values were decreased to 48 +/- 19 ng/ml (n = 12), which corresponds to a 62% decrease. Clinical results presented in this context support that the assay is a sensitive and specific index of bone resorption. It may, therefore, prove useful in the follow up of treatment of patients with metabolic bone diseases and in the clinical investigation of osteoporosis.

Quantification of the collagenolytic activity of isolated osteoclasts by enzyme-linked immunosorbent assay.

Difficulties in the geometrical definition and measurement of resorption pits is a major problem for the quantitative analysis of bone resorption by isolated osteoclasts cultured on bone or dentin substrates. In this study we developed an enzyme-linked immunosorbent assay (ELISA) for quantification of bone resorption in vitro, which specifically quantifies type I collagen fragments released into the culture medium by the resorptive action of bone cells cultured on slices of bone or dentin. A consistently high correlation between the formation of resorption pits and the release of antigenic collagen fragments was observed for isolated rabbit osteoclasts seeded at various densities and cultured for various periods on bovine, elephant, and human substrates. In a further support of the osteoclastic nature of the collagenolytic effects, a high consistency between pit formation and collagenolysis was also observed when the rabbit bone cells were cultured in the presence of very differently acting but typical inhibitors of pit formation, i.e., the carbonic anhydrase inhibitor acetazolamide, the cysteine proteinase inhibitor epoxysuccinyl-L-leucylamido-(4-guanodino)butane (E-64), the phosphatidyl-inositol 3-kinase inhibitor wortmannin, and the bisphosphonate ibandronate (BM 21.0955). In conclusion, the ELISA represents a simple, precise, and objective way to dynamically monitor bone resorption in vitro through quantification of the collagenolytic activity of isolated osteoclasts.

Applications of an enzyme immunoassay for a new marker of bone resorption (CrossLaps): follow-up on hormone replacement therapy and osteoporosis risk assessment.

An enzyme-linked immunosorbent immunoassay (ELISA) for a new marker of bone resorption (CrossLaps) was evaluated. The ELISA procedure determines degradation products of type I collagen in urine. Values obtained in the ELISA and in pyridinoline by high pressure liquid chromatography were correlated after a correction for creatinine. A high correlation was found (r = 0.77; n = 81). A group of postmenopausal women (n = 180) showed an increase of more than 70% compared to values in premenopausal women (n = 104). Hydroxyproline was increased by 23%, osteocalcin by 52%, pyridinoline by 31%, and deoxypyridinoline by 50%. A highly significant decrease (60.7%) in the CrossLaps values was seen after 12 months in samples from patients receiving hormone replacement therapy compared to a placebo group. The spontaneous bone loss in an untreated group of women was determined by repeated forearm bone mass measurement over 24 months. Baseline values obtained in the CrossLaps ELISA were correlated to the rate of loss, yielding a highly significant r value of -0.61, indicating that CrossLaps might be a useful parameter for assessment of the risk of osteoporosis in postmenopausal women.

Identification and Differentiation of Tilletia indica and T. walkeri Using the Polymerase Chain Reaction.

ABSTRACT Karnal bunt of wheat, caused by Tilletia indica, was found in regions of the southwestern United States in 1996. Yield losses due to Karnal bunt are slight, and the greatest threat of Karnal bunt to the U.S. wheat industry is the loss of its export market. Many countries either prohibit or restrict wheat imports from countries with Karnal bunt. In 1997, teliospores morphologically resembling T. indica were isolated from bunted ryegrass seeds and wheat seed washes. Previously developed PCR assays failed to differentiate T. indica from the recently discovered ryegrass pathogen, T. walkeri. The nucleotide sequence of a 2.3 kb region of mitochondrial DNA, previously amplified by PCR only from T. indica, was determined for three isolates of T. indica and three isolates of T. walkeri. There was greater than 99% identity within either the T. indica group or the T. walkeri group of isolates, whereas there was =3% divergence between isolates of these two Tilletia species. Five sets of PCR primers were made specific to T. indica, and three sets were designed specifically for T. walkeri based upon nucleotide differences within the mitochondrial DNA region. In addition, a 212 bp amplicon was developed as a target sequence in a fluorogenic 5' nuclease PCR assay using the TaqMan system for the detection and discrimination of T. indica and T. walkeri.

Selection of monoclonal antibodies for immunoaffinity chromatography: model studies with antibodies against soy bean trypsin inhibitor.

To facilitate selection of monoclonal antibodies for immunoaffinity chromatography, an ELISA screening procedure was developed. The assay is based on the avidin-biotin system and provides a profile of the monoclonal antibody which is based on the binding characteristics of the antigen binding site when exposed to different elution reagents. The elution profiles of 5 monoclonal antibodies to soy bean trypsin inhibitor (SBTI) were determined and for 2 of the antibodies the results obtained in the ELISA were verified using column experiments. The affinity constants were determined for the same 5 monoclonal antibodies and no correlation was seen with the ease of elution. The elution profiles presented here are easily obtained and the results indicate that a general screening procedure for suitable combinations of antibodies and elution conditions can be carried out using an elution ELISA assay when modified as described herein.

Survival of Teliospores of Tilletia indica in Soil.

This study was conducted to assess survival of Tilletia 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 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 25-cm depths in polyvinyl chloride tubes containing the same field soil as the infested bags. Tubes were buried vertically in the ground at Bozeman, MT, in October 1997. Soil samples were assayed for recovery and germination of T. indica teliospores 1 day and 8, 20, and 32 months after incorporation of teliospores into soil. The rates of 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 incorporation 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 begin after breakdown of the sheath-covering teliospore. The results of this study showed that teliospores of T. indica can survive in Montana for more than 32 months and remain viable.

Germinability of Teliospores of Tilletia indica after Hot Water and Sodium Hypochlorite Treatments.

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.

Size-Selective Sieving for Detecting Teliospores of Tilletia indica in Wheat Seed Samples.

A method was developed to isolate teliospores of Tilletia indica from infested grain. The technique was evaluated to determine its sensitivity for detection and quantification of teliospores, the time required to conduct an individual test, and its utility for the detection and identification of the pathogen for phytosanitary regulation and seed certification. A seed wash of a 50-g grain sample was washed through 53-µm and 20-µm pore size nylon screens to remove unwanted debris and to concentrate and isolate teliospores. The material retained in the 20-µm screen was suspended for direct microscopic examination or plated on water agar for teliospore germination and identification by polymerase chain reaction (PCR) utilizing two pairs of T. indica-specific primers. The reliability of detection for both light microscopy and PCR are 100% at an infestation of five teliospores per 50-g sample. The proportion of teliospores recovered from grain samples artificially infested with T. indica was 0, 82, 88, 81, and 82%, respectively, at infestation levels of 0, 1, 2, 5, and 10 teliospores per 50-g wheat sample. Extraction efficiency was comparable to the centrifuge seed-wash method currently used by most seed health laboratories. Sample analysis using size-selective sieving was more than 83% faster than the standard centrifuge seed wash.