Internal Transcribed Spacer Sequence-Based Phylogeny and Polymerase Chain Reaction-Restriction Fragment Length Polymorphism Differentiation of Tilletia walkeri and T. indica

ABSTRACT A polymerase chain reaction-restriction fragment length polymorphism assay to distinguish Tilleita walkeri, a rye grass bunt fungus that occurs in the southeastern United States and Oregon, from T. indica, the Karnal bunt fungus, is described. The internal transcribed spacer (ITS) region of the ribosomal DNA repeat unit was amplified and sequenced for isolates of T. indica, T. walkeri, T. horrida, and a number of other taxa in the genus Tilletia. A unique restriction digest site in the ITS1 region of T. walkeri was identified that distinguishes it from the other taxa in the genus. Phylogenetic analysis of the taxa based on ITS sequence data revealed a close relationship between T. indica and T. walkeri, but more distant relationships between these two species and other morphologically similar taxa.     

小麦印度腥黑穗病菌PCR检测

应用PCR方法对小麦印度腥黑穗病菌及其近似种或相关种包括黑麦草腥黑粉菌、狼尾草腥黑粉菌、水稻腥黑粉菌等10种腥黑粉菌共14个菌株进行了检测研究。根据线粒体DNA的序列分别设计了扩增小麦印度腥黑穗病菌的特异性引物和扩增黑麦草腥黑粉菌的特异性引物，根据核糖体内转录区（ITS）DNA片段设计了扩增腥黑粉菌属真菌的引物，应用PCR方法能将小麦印度腥黑穗病菌与黑麦草腥黑粉菌及其它近似种或相关种加以区分。本方法稳定、可靠、重复性强，已分别在不同实验室的不同型号PCR仪上得到验证。     

Susceptibility of European bread and durum wheat cultivars to Tilletia indica

Representative European wheat cultivars were tested under quarantine containment for their susceptibility to Tilletia indica, the cause of Karnal bunt of wheat. Fifteen winter and 15 spring wheat ( Triticum aestivum ) and 11 durum wheat ( Triticum durum ) cultivars were inoculated by boot injection just prior to ear emergence to test their physiological susceptibility. Selected cultivars were then re-tested by spray inoculation after ear emergence to determine their morphological susceptibility, which is a better predictor of field susceptibility. At maturity, the ears and seeds were assessed for incidence and severity of disease. For the physiological susceptibility tests, 13/15 winter wheat cultivars were infected and the percentage of infected seeds ranged from 1 to 32%. For spring cultivars, 13/15 cultivars were infected and the percentage of infected seeds ranged from 1 to 48%. For the durum cultivars, 9/11 were infected and the percentage of infected seeds ranged from 2 to 95%. Across all cultivars, 35/41 were infected. Based on historical Karnal bunt susceptibility categories using coefficients of infection, one cultivar was classed as highly susceptible, three as susceptible, 11 as moderately susceptible, 20 as resistant and only six as highly resistant. The spray-inoculation morphological susceptibility tests broadly confirmed the physiological susceptibility results, although lower levels of infection were observed. Overall, the range of susceptibility was similar to that found in cultivars grown in Karnal bunt affected countries. The results demonstrate that European wheat cultivars are susceptible to T. indica and thus could potentially support the establishment of T. indica if introduced into Europe.     

基于MaxEnt模型的小麦印度腥黑穗病在中国的适生性分析

本文用MaxEnt软件对小麦印度腥黑穗病(Karnalbunt)在我国的潜在分布进行了预测。结果表明,小麦印度腥黑穗病在中国适生范围广。根据适生值的大小,中风险区和高风险区主要分布在我国华北、黄淮海、西南、西北、长江中下游和东北地区的小麦主产区,加强这些区域的调查监测和检疫对保护我国的小麦生产有重要意义。     

Test validation for the detection of Tilletia indica Mitra by multiplex real‐time PCR

Tilletia indica Mitra is a fungal pathogen causing Karnal bunt of wheat. Tilletia indica is a quarantine pest in many countries worldwide. In the European Union, imported wheat grain from countries where the fungus is present must be checked for the presence of T. indica teliospores. The inspection services at the borders need rapid, sensitive and reliable detection tests to identify T. indica spores on wheat grain. In this work, validation was carried out according to EPPO Standard PM 7/98 to evaluate the multiplex real‐time PCR test described in ISPM 27 Diagnostic Protocol for regulated pests (Annex 4 Tilletia indica Mitra) by means of a test performance study with nine participating laboratories, and the performance characteristics of the test were established. The original protocol was modified with regard to the extraction of DNA from the pellet obtained from the ‘washing test’ and the enrichment PCR step in order to increase the amount of template DNA for the real‐time PCR. The optimized test still has five teliospores as the limit of detection for the contaminated pellet but has an increased analytical sensitivity and had positive results with three teliospores in 93% of cases instead of 43% for the original test. The two closest Tilletia species, Tilletia horrida and Tilletia walkeri, were used to evaluate analytical specificity (exclusivity) and no cross‐reactions were obtained. Diagnostic sensitivity, diagnostic specificity, accordance and concordance were also evaluated.     

Ustilospores of Tilletia ehrhartae, a smut of Ehrharta calycina, are common contaminants of Australian wheat grain, and a potential source of confusion with Tilletia indica, the cause of Karnal bunt of wheat

Australian wheat consigned for export from Australian ports was surveyed in March 2004 using a national diagnostic protocol for detection and identification of Tilletia indica . No ustilospores of T. indica were detected, confirming previous surveys which have failed to detect T. indica in Australia. However, the survey detected moderate levels of the common smuts Tilletia caries (syn. Tilletia tritici ), Tilletia laevis and Urocystis agropyri , and very low levels (average fewer than six ustilospores per 150 g sample) of an unidentified dark, tuberculate-spored Tilletia in ≈ 60% of samples tested. Comparison with herbarium specimens enabled identification of the majority of the tuberculate ustilospores as Tilletia ehrhartae , a smut fungus known to infect only Ehrharta calycina (perennial veldt grass) and which is common in southern Australia. A smaller number of tuberculate smut ustilospores were identified as Tilletia walkeri , a smut of Lolium spp. recorded in Australia but apparently uncommon. Both T. ehrhartae and T. walkeri bear sufficient resemblance to T. indica for misidentifications to be possible where only a very few ustilospores are seen, although T. ehrhartae ustilospores are always <25  µ m in diameter. The frequent presence of ustilospores of both T. ehrhartae and T. walkeri as contaminants of Australian wheat grain exports has significance for diagnosticians testing Australian export wheat, as it demonstrates the potential for tuberculate ustilospores of species other than those covered in existing diagnostic protocols to be misidentified as T. indica . This paper describes T. ehrhartae in detail, and provides criteria for its differentiation from T. indica , T. walkeri and some other species.     

Rainfall and temperature distinguish between Karnal bunt positive and negative years in wheat fields in Texas

Karnal bunt of wheat, caused by the fungus Tilletia indica, is an internationally regulated disease. Since its first detection in central Texas in 1997, regions in which the disease was detected have been under strict federal quarantine regulations resulting in significant economic losses. A study was conducted to determine the effect of weather factors on incidence of the disease since its first detection in Texas. Weather variables (temperature and rainfall amount and frequency) were collected and used as predictors in discriminant analysis for classifying bunt-positive and -negative fields using incidence data for 1997 and 2000 to 2003 in San Saba County. Rainfall amount and frequency were obtained from radar (Doppler radar) measurements. The three weather variables correctly classified 100% of the cases into bunt-positive or -negative fields during the specific period overlapping the stage of wheat susceptibility (boot to soft dough) in the region. A linear discriminant-function model then was developed for use in classification of new weather variables into the bunt occurrence groups (+ or -). The model was evaluated using weather data for 2004 to 2006 for San Saba area (central Texas), and data for 2001 and 2002 for Olney area (north-central Texas). The model correctly predicted bunt occurrence in all cases except for the year 2004. The model was also evaluated for site-specific prediction of the disease using radar rainfall data and in most cases provided similar results as the regional level evaluation. The humid thermal index (HTI) model (widely used for assessing risk of Karnal bunt) agreed with our model in all cases in the regional level evaluation, including the year 2004 for the San Saba area, except for the Olney area where it incorrectly predicted weather conditions in 2001 as unfavorable. The current model has a potential to be used in a spray advisory program in regulated wheat fields.     

Susceptibility of Wheat to Tilletia indica During Stages of Spike Development

ABSTRACT Karnal bunt of wheat is caused by the fungus Tilletia indica, which partially converts kernels into sori filled with teliospores. Despite minor overall yield and quality losses, the disease is of considerable international quarantine concern. Plant development stages reported susceptible to infection vary considerably. A study was designed to better define the susceptibility period by inoculating wheat spikes at different growth stages with naturally liberated secondary sporidia under optimal conditions for disease development. Spikes of a resistant and susceptible cultivar were inoculated at eight growth stages from awns emerging to soft dough. Spikes became susceptible only after emerging from the boot and continued to be susceptible up to soft dough stage at which low levels of disease occurred. Disease severity in both cultivars peaked when spikes were inoculated after complete emergence, but before the onset of anthesis. Disease levels tapered off gradually in spikes inoculated after anthesis. The results broaden the known susceptibility period of wheat to T. indica to include stages long after anthesis, and indicate that infection from airborne inoculum is not possible during boot or awns emerging stages, which are commonly referred to as the most susceptible stages.     

TaqMan-MGB探针在小麦印度腥黑穗病菌和黑麦草腥黑穗病菌鉴定上的应用

根据小麦印度腥黑穗病菌Tilletia indica和黑麦草腥黑穗病菌T.walkeri核糖体ITS序列设计了两对通用引物和两条特异性探针,建立了小麦印腥印度腥黑穗病菌Tilletia indica和黑麦草腥黑穗病菌T.walkeri的实时荧光PCR检测方法,检测的灵敏度为1个冬孢子.这种检测方法可以直接用于样品小麦印腥和黑麦草腥黑穗病菌冬孢子的快速检测,整个检测过程缩短至1天.     

Reaction of selected winter wheat cultivars from Europe and United States to Karnal bunt

A study was conducted to determine the susceptibility of popular United States (U.S.) and European winter wheat cultivars to the fungal pathogen Tilletia indica. Historically, the disease has been limited to autumn-sown spring-habit wheat areas and not associated with winter wheat. In 1997, Karnal bunt was observed on winter wheat in limited regions of Texas. This region marks the southern end of the contiguous U.S. central winter wheat belt, which extends north into Canada. The aim of this study was to assess the levels of disease resistance in winter wheat. Fifty U.S. and European winter wheat cultivars were tested using two different greenhouse inoculation procedures. For each cultivar, 12 spikes in boot were inoculated by boot-injection with a sporidial suspension (1.0 ml/boot, 10,000 spores ml⁻¹), and 12 other emerged spikes were spray-inoculated with the same concentration. The experiment was repeated for three seasons. Among cultivars, mean seed infection ranged from 2.1 to 87.2% and 0 to 15.6% for boot-injected and spray-inoculated treatments, respectively. Results showed that the majority of winter wheat cultivars tested were susceptible to Karnal bunt.     

麦秆中腥黑粉菌冬孢子的鉴定

子鉴定为小麦印度腥黑粉病菌Tilletia indica.     

Image analysis for the identification of the quarantine pest Tilletia indica*

Diagnosis of the teliospores of Tilletia indica (Karnal bunt) detected in wash tests is complicated by the fact that wheat grain can be contaminated by ‘look‐alike’ species, namely Tilletia walkeri (ryegrass bunt) and Tilletia horrida (rice smut). Although morphological diagnosis is possible when there are relatively large numbers of teliospores present, it is difficult with only a few spores as there are significant overlaps in the characteristics of each species. Molecular methods can confirm presumptive identifications, but these take 2–3 weeks. Image analysis offers the potential for more rapid confirmation. An image analysis system was developed for use on bleached spores. Bleaching spores reveals additional morphological characters (spore profile and spore wall layers) that may be used in an image analysis system to discriminate species. The image‐processing software automatically locates spores on a given image and calculates perimeter, surface area, number of spines and spine size, maximum and minimum ray radius, aspect ratio and roundness. Principal components analysis (PCA) is performed on the parameters to obtain a linear separation of spore species. Accuracy of 97% in separating T. indica and T. walkeri has been achieved in preliminary tests using PCA, but further evaluation is required.     

基于ISSR标记开发的一种用于小麦矮腥黑穗病菌的分子鉴定方法

 小麦矮腥黑穗病菌(Tilletia controversa Kühn, 简称TCK)是小麦上的一种重要检疫性真菌。本研究利用内部简单重复序列(Inter-simple sequence repeat, ISSR)技术研究TCK及其近缘种的DNA多态性,开发了一种可靠而简单的方法用于TCK的分子鉴定。用ISSR引物P4从TCK中扩增出一条1 113 bp的特异性条带,据此设计了一对特异性引物TCKF/TCKR,在12个TCK菌株中均能扩增得到一条882 bp的特异性条带,而其他近缘种包括小麦网腥黑穗病菌(T. caries)和小麦光腥黑穗病菌(T. foetida)及相关黑粉菌的14个菌株均无扩增条带。用该特异性引物检测TCK的下限为25 μL反应体系中可检测到1 ng DNA模板。本研究开发的种特异性引物,可将TCK与其形态上相似的近缘种尤其是小麦网腥黑穗病菌准确区分开,本研究基于ISSR标记建立的小麦矮腥黑穗病菌的分子鉴定方法为腥黑粉菌的检疫提供了一种便捷的方法,是对现有分子鉴定方法的一个补充。     

An Allee Effect Reduces the Invasive Potential of Tilletia indica

ABSTRACT The Karnal bunt pathogen, Tilletia indica, is heterothallic and depends on encounters on wheat spikes between airborne secondary sporidia of different mating types for successful infection and reproduction. This life history characteristic results in reduced reproductive success for lower population densities. Such destabilizing density dependence at low population levels has been described for a range of animals and plants and is often termed an Allee effect. Our objective was to characterize how the Allee effect might reduce the invasive potential of this economically important pathogen. We developed a simple population model of T. indica that incorporates an Allee effect by calculating the probability of infection for different numbers of secondary sporidia in the infection court. An Allee effect is predicted to be important at the frontier of an invasion, for establishment of new foci by a small population of teliospores, and when the environment is nonconducive for the production of secondary sporidia. Using estimated model parameter values, we demonstrated a theoretical threshold population size below which populations of T. indica were predicted to decline rather than increase. This threshold will vary from season to season as a function of weather variables and their effect on the reproductive potential of T. indica. Deployment of partial resistance or use of fungicides may be more useful if they push population levels below this threshold.     

应用聚合酶链反应技术鉴定印度腥黑穗病菌

用一对专化于印度腥黑穗病菌的引物Ｔ１１７Ｍ１（５＇－ＴＣＣＣＣＴＴＧ－ＧＡＴＣＡＧＡＡＣＧＴＡ－３＇）和Ｔ１１７Ｍ２（５＇－ＡＧＡＡＧＴＣＴＡＡＣＴＣＣＣＣＣＣＴＣＴ－３＇）可特异地扩增印度腥黑穗病菌产生一段８２５ｂｐ的产物，而稻粒黑粉病菌则不能被扩增。实验还表明，用聚合酶链反应（ＰＣＲ）方法检测灵敏度可达到１００个未萌发的冬孢子，这为进口粮印度腥黑穗病菌的检疫提供了有力工具。     

进境小麦中沙地牧草腥黑粉菌的鉴定

从上海口岸进境的澳大利亚小麦中发现一种类似小麦印度腥黑粉病菌的腥黑粉菌冬孢子，对该菌冬孢子进行了形态学特征和PCR检测，根据结果，将这种冬孢子鉴定为沙地牧草腥黑粉菌Tilletia ehghartaTle；本研究设计了T．ehrhartaea的特异引物Eh2/Eh4，结合引物Till／Til4建立了T．ehdmrtae的套式PCR检测方法。     

Pathogenic and genetic variability in <Emphasis Type="Italic">Tilletia indica</Emphasis> monosporidial culture lines using universal rice primer-PCR

Tilletia indica Mitra is the causal agent of Karnal bunt of wheat, an important disease prevalent in several countries. The disease is internationally quarantined and the pathogen due to its heterothallic nature shows high variability. In the present study, we compared the pathogenic behaviour of various isolates of T. indica collected from different geographical locations of India and genetically characterized monosporidial (Ms) culture lines raised from these isolates of the pathogen. Pathogenic variability revealed existence of three pathotypes based on aggressiveness on a set of differential host genotypes. Monosporidial culture lines viz., 5 each from KB1, KB2, KB4 and KB5 and three lines of KB3 were established and analyzed genetically using 12 Universal Rice Primers (URPs). Amplification showed 98.44% polymorphism and primer URP 13R produced 100% polymorphic bands. Maximum similarity (83%) was between KB1MsB and KB1MsD as calculated by Jaccard’s similarity coefficient, whereas, minimum similarity was between KB1MsC and KB4MsB; KB1MsE and KB3MsA (46%). Three groups were formed among all Ms culture lines. One major group consisted of 13 lines with approximately 70% similarity, the second group consisted of 7 culture lines showing 55% similarity and the third group consisted of 3 Ms lines. URPs were able to differentiate the Ms culture lines raised from different T. indica isolates and the results indicated heterogeneity in the pathogen population.     

Teliospore Survival and Plant Quarantine Significance of Tilletia Indies (causal agent of Karnal bunt) Particularly in Relation to China

The influence of temperature, light and soil conditions on teliospore germination of Tilletia (Neovossia) Indica was investigated. The teliospores were germinated by incubation in an aqueous suspension in a concavity on a water agar surface and the germination percentage was scored directly under low magnification of the compound microscope. The optimal temperature range for germination was found to be 15 to 22°C. Germination was extremely low at 2°C, but total inhibition was only observed after prolonged exposure to 35®C (in darkness) or to - 18°C under dry conditions. Light was shown to have a stimulating effect on germination (artificial daylight was found to be better that near ultraviolet light). No stimulation was achieved by prolonging the illumination from 12 to 24 h. Teliospores from 5-year-old wheat samples were shown to be viable. The weather conditions of the various wheat- growing areas of China were compared with the results obtained for the survival of teliospores of T. indica. It is concluded that a considerable risk exists for establishment and spread of the Karnal bunt disease if first accidentally introduced into China. Strict quarantine precautions, and policies can minimize or even eliminate this risk.     

套式PCR直接检测印度腥黑穗病菌冬孢子

用印度腥黑穗病菌冬孢子制备模板DNA ,利用印腥特异性引物T3 /T6,T3 /T4和套式PCR(nestPCR)扩增技术直接检测印腥冬孢子 ,检测的灵敏度可达 1个冬孢子。检测时间缩短为 1天。这种简单、快速、灵敏、实用和准确的PCR检测技术适用于口岸印腥检疫的需要 ,解决了常规PCR检测中DNA制备需要萌发冬孢子和检测时间长的难题。     

Survival of Tilletia indica teliospores under European soil conditions

As part of developing a European Pest Risk Analysis (PRA) for Tilletia indica , the causal agent of Karnal bunt of wheat, teliospore survival studies were done outside under quarantine containment at three European sites (Norway, UK, Italy). At each site, experiments were set up in three consecutive years (Experiments 1, 2 & 3) to determine teliospore survival over time (1–3 years) at 5, 10 and 20 cm depths. Experiments were sampled annually and survival assessed in relation to teliospore recovery and to germination at recovery (T0) and 3 months after recovery in case of burial-induced dormancy ( T3 ). Teliospores survived at all three sites at all depths over all the time periods studied. At each site, there was no evidence of a marked decline in teliospore recovery between sampling years, except in one set of years in one Norwegian experiment. There was no consistent effect of depth on recovery. In general there was little evidence for a marked decline in teliospore germination between sampling years. There was some evidence of a decrease in germination with increasing depth in the UK, and for some time-depth interactions. After 3 years' incubation (Experiment 1), mean teliospore recovery and mean germination were: UK: 61% recovery and 31% ( 33% ) germination for T0 (and T3 ); Italy: 30% recovery and 36% ( 29% ) germination; and Norway: 12% recovery and 19% ( 49% ) germination. Germination for laboratory controls ranged from 20–59% (UK), 18–41% (Italy) and 28–59% (Norway). There was no evidence for burial-induced dormancy except in Norway. Teliospores of T. indica can survive for at least three years in European soils. This prolonged period of survival could support establishment of the pathogen if it were introduced into areas of European cereal production.