Production of a Mating Inhibitor by Ustilago hordei

ABSTRACT Ustilago hordei, the cause of barley covered smut, was found to produce a factor that inhibited its own mating. The mating inhibition factor (MIF) specifically inhibited mating of U. hordei and other Ustilago spp., but not teliospore germination or sporidial growth. MIF did prevent teliospore germination of Tilletia caries and T. contraversa. MIF was found at low levels in culture supernatants of either mating type of U. hordei grown separately, but at higher levels when both mating types were grown together, in the supernatants of MAT-1 mating type cells transformed with the MAT-1 pheromone gene mfa1 and of MAT-2 cells transformed with either mfa1 or the MAT-1 pheromone receptor gene pra1. Diploid cells produced no detectable inhibitor, nor did MAT-1 cells with a disrupted mating type locus that deleted both mfa1 and pra1. MIF production was restored when mfa1, but not pra1, was added back to the MAT-1Delta cells. MIF activity was altered by protease treatment. Highly purified MIF from MAT-1 cells contained cysteine methyl ester, farnesyl cysteine, farnesyl cysteine methyl ester, and a dodecapeptide with a mass consistent with that of MAT-1 pheromone lacking the terminal cysteine. Since smut fungi must first mate to become pathogenic, mating inhibition has the potential to be an effective method of disease control for these pathogens.     

Polymerase Chain Reaction Detection of Ustilago hordei in Leaves of Susceptible and Resistant Barley Varieties

ABSTRACT Although Ustilago hordei infects barley seedlings, symptoms of the disease covered smut are not visible until heading. Natural or artificial inoculation usually results in inconsistent infection, even in highly susceptible lines. Thus, breeding for resistance to covered smut is time consuming and difficult. The ribosomal DNA internal transcribed spacer (ITS) regions of U. hordei were sequenced and a primer pair was developed for polymerase chain reaction (PCR). These primers amplified a 574-bp fragment from DNA of Ustilago spp., but did not amplify DNA from barley or other common barley pathogens. DNA extracted from as few as four U. hordei sporidia was detected by this method. U. hordei DNA was amplified from leaf tissue of inoculated susceptible and resistant plants at different stages of plant development in differential varieties. Growth of the fungus in different leaves of an individual plant was variable. Several highly resistant varieties were shown to contain U. hordei DNA in the first three to four leaves, but not in later leaves. Thus, although the fungus can infect many resistant plants, the plants remain symptomless. Detection of U. hordei prior to heading should assist efforts for breeding for resistance and provide clues concerning the mechanisms of resistance employed by different resistance genes.     

进境碱茅上腥黑粉菌新记录的初步研究

腥黑粉菌主要侵染禾本科植物。2004年曾从来自美国的碱茅草种中检出一种腥黑粉菌,是碱茅上腥黑粉菌的新记录,迄今国内外尚无其相关研究的报道,本文从冬孢子形态、萌发生理特性及基因序列比较等几方面对该菌进行了初步研究,讨论了该菌的分类地位,根据研究结果,该菌与Tilletia bromi具有较近的亲缘关系,为腥黑粉菌潜在新种。     

公主岭霉素复合剂的研究

两年试验结果:公主岭霉素与粉锈宁混用,可治高粱三种黑穗病和玉米丝黑穗病;与拌种双混用,可治小麦散票穗病和腥黑穗病;与瑞毒霉混用,可治谷子白发病和粒黑穗病。     

Observations on seedling-infecting smuts of barley from Turkey and UK

The identification of smut fungi in smutted barley ears from Turkey and the UK was confirmed. Inoculation of seed showed that certain cultivars from both countries could be attacked by Ustilago hordei from either country and by U. avenae from Turkey. The implications are discussed.     

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.     

不同杀菌剂对稻粒黑粉病菌冬孢子萌发的影响

稻粒黑粉病(Rice kernel smut)是一种真菌病害。在离体条件下,采用孢子萌发测定法,测定了嘧菌酯、灭菌唑、拌种灵、多菌灵、咪酰胺和二硫氰基甲烷抑制稻粒黑粉病菌冬孢子萌发的最低抑制浓度(MIC);同时研究了药剂浸种处理对稻种的安全性以及浸种处理效果。结果表明,嘧菌酯和二硫氰基甲烷对稻粒黑粉病菌冬孢子萌发的抑制活性较高,MIC为0.4μg/mL,多菌灵的抑制活性次之,MIC为0.8μg/mL,其他3种药剂的抑制活性不高,MIC均大于1.6μg/mL。种子安全性试验表明,嘧菌酯在2.0、4.0、8.0μg/mL和多菌灵在4.0、8.0、16.0μg/mL处理3d和7d后,稻种的萌芽率与对照相比无显著性差异;而二硫氰基甲烷在4.0和8.0μg/mL处理下,所选稻种的萌芽率显著低于对照,说明该药剂在浸种处理后会影响所选稻种的萌芽,但其在低剂量处理下对稻种的萌芽率影响较小。关于稻粒黑粉病浸种处理的合适农药剂型仍需要进一步研究。     

Effect of biological seed treatment withCylindrocarpon olidum var.olidum on control of common bunt (Tilletia laevis) of wheat

The goals of the present study were to determine ifCylindrocarpon olidum var.olidum exerts antibiotic activity on teliospore germination ofTilletia laevis in vitro and to evaluate the efficacy of a seed treatment with this antagonist against artificial seedborne infection in the field under natural cropping conditions. Germination of bunt teliospores was completely inhibitedin vitro on water agar medium supplemented with the antagonist’s culture filtrate. In field experiments seed application reduced the incidence of common bunt from 82.9% to 40.4%, and from 81.4% to 42.0% in 1995/1996 and 1997/1998, respectively.C. olidum var.olidum may have a potential againstT. laevis and is worthy of further research on its use as a biocontrol agent for common bunt. http://www.phytoparasitica.org posting July 14, 2005.     

高粱花黑穗病生物学特性及侵染途径研究

 高粱花黑穗病(Ustilago Kenjiana Ito)是我国高粱上发生的一种病害,对产量影响轻微。冬孢子萌发适温是25-30℃,在水滴里萌发率最高,最适pH是6.0-6.5。在不同营养液里以酵母汁及花粉汁里萌发率最高,在人工培养基上病菌生长最适温度是25℃,最适pH是5.9.碳源以葡萄糖、半乳糖、甘露糖和果糖里生长最佳。氮源以蛋白胨和天冬素生长最好.菌瘿黑冬孢子变褐后不经休眠即可萌发。冬孢子在室外可存活一年半,在土壤里经一冬便丧失生活力,室内能存活三年.以冬孢子接种土壤和种子均未发病,属于花器局部侵染,当年侵染当年发病的非系统侵染病害.冬孢子在高粱孕穗或尚未开花的小花护颖与外颖缝隙中侵入子房,而不是在开花期或开花后侵入,也不是从柱头、花柱或其它部位侵入,当年尚能重复侵染.     

Arguments for a low risk of establishment of Karnal bunt disease of wheat in Europe

Tilletia indica, which causes Karnal bunt of wheat, has been perceived as a pathogen that has a high risk of establishment in the UK and other countries in Europe. This viewpoint is challenged with arguments for the risk of establishment being much lower. The present distribution of the pathogen and its likely widespread dissemination with consignments of germplasm and wheat seed from Mexico over a 20 year period without establishment outside the hot arid and semi-arid zones strongly suggests that T. indica is unsuited to environments other than those similar to where it is now found. A critical density of teliospores needs to remain viable and ungerminated between periods of wheat anthesis and then germinate during a narrow window of opportunity to ensure infection and establishment. Despite the results of outdoor teliospore longevity experiments, it is by no means certain that this can occur under European conditions. Generally cooler conditions and more frequent periods of rain in Europe are likely to trigger teliospore germination at times unsuitable for infection making establishment much more unlikely. A model based on year-round soil conditions, especially moisture content, is seen as appropriate to predict teliospore survival and germination potential. It is only after areas where teliospores can survive and germinate in sufficient numbers to guarantee regular infection cycles have been defined can another model, based on temperature, rainfall and/or humidity prior to anthesis, be applied to determine if environmental conditions are suitable for infection. David Jones is a retired employee of the Central Science Laboratory (CSL). Statements and opinions expressed in this paper are those of the author and are not the views of the Central Science Laboratory.     

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.     

红花锈病菌的生物学特性及侵染研究

 本病为害特征为叶片干枯,植株早衰,含油量下降,地下茎感染,使许多病株在营养期萎蔫死亡。初侵染源——冬孢子粘附于种子表面和冠毛上;散落田间时也能侵染。冬孢子人工接种下侵染真叶,但田间自然条件下不起作用。新鲜冬孢子有少量萌发,隔年冬孢子萌发率明显提高。冬孢子萌发温度极限为10至33℃,适温18至28℃,最适温度为25℃。冬孢子水滴上萌发率低,红花幼苗榨出液具激发作用;而夏孢孢子水滴上萌发远高于榨出液。冬孢子干藏可存活两年.夏孢子残存期可延续166天以上。共鉴定87份红花种质抗病性,差异很显著,但无一表现免疫。     

Attempted somatic hybridization of Puccinia striiformis f. sp. tritici and P. striiformis f. sp. hordei

Attempts were made to produce somatic hybrids between isolates of Puccinia striiformis f. sp. tritici and f. sp. hordei. A mixed infection was produced on a common susceptible barley host, Fong Tien, using white-spored isolates of P. striiformis f. sp. tritici and yellow-spored isolates off. sp. hordei. Selection was made for non-parental spore colour on selective wheat and barley hosts, and variants thus isolated were analysed for virulence markers, and for isozyme and double-stranded RNA (dsRNA) markers, all of which clearly differentiated the parental isolates. Two white-spored (non-parental) isolates were found on the selective barley host which otherwise resembled the parental f. sp. hordei isolate in virulence, isozyme and dsRNA markers. The most likely explanation of the origin of these isolates is mutation to white spore colour in the f. sp. hordei isolate.     

Use of inter-simple sequence repeats and amplified fragment length polymorphisms to analyze genetic relationships among small grain-infecting species of ustilago

ABSTRACT In the smut fungi, few features are available for use as taxonomic criteria (spore size, shape, morphology, germination type, and host range). DNA-based molecular techniques are useful in expanding the traits considered in determining relationships among these fungi. We examined the phylogenetic relationships among seven species of Ustilago (U. avenae, U. bullata, U. hordei, U. kolleri, U. nigra, U. nuda, and U. tritici) using inter-simple sequence repeats (ISSRs) and amplified fragment length polymorphisms (AFLPs) to compare their DNA profiles. Fifty-four isolates of different Ustilago spp. were analyzed using ISSR primers, and 16 isolates of Ustilago were studied using AFLP primers. The variability among isolates within species was low for all species except U. bullata. The isolates of U. bullata, U. nuda, and U. tritici were well separated and our data supports their speciation. U. avenae and U. kolleri isolates did not separate from each other and there was little variability between these species. U. hordei and U. nigra isolates also showed little variability between species, but the isolates from each species grouped together. Our data suggest that U. avenae and U. kolleri are monophyletic and should be considered one species, as should U. hordei and U. nigra.     

Evaluation of screening protocols to determine genetic variability of grain sorghum germplasm to Sporisorium sorghi under field and greenhouse conditions

Covered kernel smut caused by Sporisorium sorghi is a serious problem in grain sorghum (Sorghum bicolor)-growing areas in the world, particularly where seed dressings are unavailable or too expensive. In a screening protocol designed to eliminate the possibility of escapes, seed subjected to a teliospore suspension coupled with a partial vacuum, and seeds directly infested with dried teliospores, were equal in development of smutted sorghum panicles. Inoculation with sporidia from teliospores was ineffective when plants were inoculated at the boot stage of growth and was minimally effective when plants were inoculated at the 10-12 leaf stage of growth. Environmental conditions were more conducive to disease incidence when sorghum was grown under greenhouse conditions or planted late in the field. Sorghum planted in warm (>20C) soil had the greatest incidence of covered kernel smutted panicles.     

土壤湿度对小麦矮腥黑穗病菌(TCK)冬孢子萌发的影响

通过土壤湿度对小麦矮腥黑穗病菌(Tilletiacontronversa Kühn,TCK)萌发率的影响试验研究表明,其冬孢子在土壤质量含水量为1%～28%(相对含水量3.57%～100%)范围内均可萌发,其适宜萌发的土壤质量含水量范围为10%～25%(相对含水量17.85%～89.3%),最佳土壤相对含水量范围在65%～75%之间;不同分离物在相同土壤湿度培养下,多数分离物冬孢子的萌发率之间差异不显著。根据分离菌T_t1和T_t2在不同土壤湿度下培养50d和60d的冬孢子萌发率,建立了TCK冬孢子萌发率与土壤相对含水量的关系模型,此结果为TCK的风险评估提供了基础数据。     

Wheat Diseases in Turkey1

Cereal crop production is one of the most important projects in the agricultural improvement programme for Turkey. Overall, wheat represents 61 % of the cereal crop. While wheat production was almost 4 million tons in 1950, this reached a limit of 10 million tons in 1970. A number of diseases continue to afflict the crop from time to time, causing heavy losses. The most important of these are: bunt ( Tilletia foetida and T. caries ), loose smut ( Ustilago nuda ), rusts ( Puccinia strilformis, P. graminis f. sp. tritici, P. recondita f. sp. tritici ), leaf blotch ( Septoria tritici ), root and foot rots ( Fusarium spp., Drechslera sorokiniana, Pseudocercosporella herpotrichoides, Alternarla alternate, Sclerotium spp. and Rhizoctonia spp.). The diseases appear all over the country. Average losses due to rusts in epidemics are estimated at about 30–35 % in the whole country. Annual losses from covered smut are about 15–20 %, and from loose smut from 0.1 % to almost 20 % depending on the weather conditions of the year, on variety and on locality. Root rots have gained importance in some places, especially in Thrace in the last few years. In addition to the above diseases, several others occur occasionally but are of minor importance: powdery mildew ( Erysiphe graminis f. sp. tritici ), dwarf smut ( Tilletia contraversa ), flag smut ( Urocystis tritici ), take-all ( Gaeumannomyces graminis ) and a wheat mosaic virus (recently observed in a province of Central Anatolia).     

Assessment of the loose smut fungi (Ustilago nuda and U. tritici) in tissues of barley and wheat by fluorescence microscopy and real-time PCR

Loose smut fungi of barley and wheat (Ustilago nuda and U. tritici, respectively) colonize the plant without causing obvious disease symptoms before heading. The availability of diagnostic methods to detect and follow the growth of these pathogens in the plant would therefore be highly advantageous for both resistance breeding and the development of effective seed treatments. Using seed lots of barley and wheat highly infected with loose smut, we studied the early establishment of the loose smut pathogens in the plant by fluorescence microscopy. In hand-cut sections stained with the fluorochrome Blankophor?, fungal hyphae were observed to invade the shoot apical meristem and leaf primordia during the first days after the onset of germination. At the first node stage the ear and leaf primordia were generally extensively colonized. Hyphae of U. nuda were also regularly observed in high density in the nodes. A protocol was developed for the specific amplification of U. nuda and U. tritici DNA extracted from infected plant tissue. PCR screening of U nuda in seedlings from infected and healthy seed lots was compared to ELISA, microscopy and ultimately head infection of mature plants derived from tillers of the tested seedlings. The results indicated that a prediction of loose smut infection by real-time PCR is possible at the second leaf stage, and that the assay is equally suited for use with spring and winter varieties of barley and wheat.     

Genetic analysis and molecular mapping of wheat genes conferring resistance to the wheat stripe rust and barley stripe rust pathogens

ABSTRACT Stripe rust is one of the most important diseases of wheat and barley worldwide. On wheat it is caused by Puccinia striiformis f. sp. tritici and on barley by P. striiformis f. sp. hordei Most wheat genotypes are resistant to P. striiformis f. sp. hordei and most barley genotypes are resistant to P. striiformis f. sp. tritici. To determine the genetics of resistance in wheat to P. striiformis f. sp. hordei, crosses were made between wheat genotypes Lemhi (resistant to P. striiformis f. sp. hordei) and PI 478214 (susceptible to P. striiformis f. sp. hordei). The greenhouse seedling test of 150 F(2) progeny from the Lemhi x PI 478214 cross, inoculated with race PSH-14 of P. striiformis f. sp. hordei, indicated that Lemhi has a dominant resistance gene. The single dominant gene was confirmed by testing seedlings of the F(1), BC(1) to the two parents, and 150 F(3) lines from the F(2) plants with the same race. The tests of the F(1), BC(1), and F(3) progeny with race PSH-48 of P. striiformis f. sp. hordei and PST-21 of P. striiformis f. sp. tritici also showed a dominant gene for resistance to these races. Cosegregation analyses of the F(3) data from the tests with the two races of P. striiformis f. sp. hordei and one race of P. striiformis f. sp. tritici suggested that the same gene conferred the resistance to both races of P. striiformis f. sp. hordei, and this gene was different but closely linked to Yr21, a previously reported gene in Lemhi conferring resistance to race PST-21 of P. striiformis f. sp. tritici. A linkage group consisting of 11 resistance gene analog polymorphism (RGAP) markers was established for the genes. The gene was confirmed to be on chromosome 1B by amplification of a set of nullitetrasomic Chinese Spring lines with an RGAP marker linked in repulsion with the resistance allele. The genetic information obtained from this study is useful in understanding interactions between inappropriate hosts and pathogens. The gene identified in Lemhi for resistance to P. striiformis f. sp. hordei should provide resistance to barley stripe rust when introgressed into barley cultivars.     

Macroscopic leaf symptoms in wheat infected by Tilletia tritici

Journal of Plant Diseases and Protection - Common bunt (Tilletia tritici) infects the wheat plant during germination of the seed. For experiments with common bunt, it is conventional to examine...