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.     

酿酒高粱品种、组合及亲本的丝黑穗病抗性鉴定

为了明确不同类型的酿酒高粱材料对西南地区丝黑穗病的抗性和促进抗丝黑穗病品种的选育,采用土壤接菌法对107份材料进行了丝黑穗病抗性鉴定;根据抗性鉴定结果,比较了酿酒高粱杂交组合与其亲本对丝黑穗病的抗性,并对供试菌种进行了致病力分析.结果显示:在感病对照恢1植株平均病株率为67.65％时,有13个不育系、6个保持系、10个恢复系、28个杂交组合的病株率为0,达免疫水平,其中,酿酒高粱不育系和杂交组合所占比例较大,分别为86.67％和75.68％;恢复系中免疫类型所占比例较小,为24.39％;常规品种和组合中无免疫类型;杂交高粱对丝黑穗病的抗性属显性遗传;泸州丝黑穗病菌具有较强的致病力.     

3种防御酶在水稻抗稻曲病中的活性变化

不同水稻品种对稻曲病抗性存在明显差异。本研究选用12个不同抗、感病水稻品种,在抽穗前7~10 d,用注射法接种稻曲病菌分生孢子悬浮液。结果表明,在抗病品种中苯丙氨酸解氨酶（PAL）、过氧化物酶（POD）和多酚氧化酶（PPO）3种酶活性接种后迅速提高,变化幅度较大;而感病品种的酶活性在接种后缓慢增加,变化幅度小。由此说明,这3种防御酶与水稻抗稻曲病有一定的关系。     

Comparison of responses to floret and seedling inoculation in wheat- Ustilago tritici and barley- U. nuda combinations

The floret and seedling methods for inoculating wheat and barley with Ustilago tritici and U. nuda, respectively, were optimized and then compared. Floret inoculation gave higher infection levels than did seedling inoculation, especially for wheat, but problems of plant mortality and standardization of tissue age were greater with the former method. Floret inoculation identified wheat and barley varieties exhibiting embryo resistance (class 1 or 2 resistance) or seedling resistance (class 3), but only class 3 wheat and barley varieties (and one class 2 wheat variety) were resistant to seedling inoculation. Using near-isogenic wheat lines expressing different levels of partial resistance to floret inoculation, positive correlations were obtained between percentage infection levels resulting from the two inoculation protocols. In addition, race-specific host-pathogen interactions such as hypersensitivity and partial smutting were expressed following both floret and seedling inoculation, indicating that these interactions operate in the developing plant. The potential value of the seedling inoculation method in fundamental and applied research into the cereal-loose smut interaction is discussed.     

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.     

Pheromone-Related Inhibitors of Ustilago hordei Mating and Tilletia tritici Teliospore Germination

ABSTRACT Ustilago hordei, the causal agent of barley covered smut, produces mating pheromones that break down to smaller peptide compounds that act as potent inhibitors of mating and germination in several fungi. The pheromones are members of the farnesylated family of proteins. Synthetic peptide analogs of the pheromone derivatives, ranging in size from 4 mers to full length pheromones, were farnesylated, methyl esterified, or both and tested for mating or teliospore germination inhibition with U. hordei or Tilletia tritici, respectively. N-Acetyl-S-farnesylcysteine, which inhibits processing of Ras, and other sulfur-containing compounds such as homocysteine or methionine, were likewise modified and tested. The most potent inhibitors were methionine methyl ester and modified 4-mer peptides from both pheromones. Alanine scanning of the inhibitory 4 mers determined that the native amino acid sequence was specific for a high level of activity. The sulfur amino acids appear to be required for inhibition. Glasshouse studies using selected antagonists of mating and teliospore germination as seed treatments inhibited covered smut of barley and common bunt of wheat, although the level of control was inconsistent. The use of pheromone-related antagonists to mating or teliospore germination is a promising, novel strategy for control of smut and bunt diseases.     

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.     

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.     

A refined inoculation method to evaluate false smut resistance in rice

False smut, caused by Ustilaginoidea virens, is a serious disease of rice worldwide. To evaluate false smut resistance in rice, we developed a method combining the cultivation of the main culm of rice plants in the greenhouse and rapid preparation of a conidial suspension to inject into the leaf sheath. The method was used to evaluate false smut resistance in 18 varieties/lines of rice. For comparison, field trials were also carried out in 2007 and 2008. The results indicated that the greenhouse method was more reproducible than field trials: commercial varieties tested were resistant; almost all the forage varieties were highly susceptible; and blast-resistant varieties/lines were mostly resistant to false smut. Thus, this inoculation method will be useful for determining the level of false smut resistance in rice and for breeding resistant varieties.     

78个水稻不育系对稻粒黑粉病的抗性评价

 水稻稻粒黑粉病主要为害水稻不育系,是限制我国南方杂交水稻制繁种产量和质量的主要病害因素。为挖掘抗稻粒黑粉病的水稻不育系品种,提供具有抗稻粒黑粉病育种价值的水稻不育系材料,本研究于2014-2016年,采用田间接种稻粒黑粉病菌的方法,对四川、湖北、福建的78个水稻不育系品种进行了稻粒黑粉病菌抽穗期抗性评价。连续3年接种稻粒黑粉病菌试验表明,共有4个水稻不育系品种对稻粒黑粉病表现中抗及以上抗性,大部分为感病品种,其中4766A接种稻粒黑粉病菌后未发病,对稻粒黑粉病表现稳定高抗,为我国首次报道的高抗稻粒黑粉病水稻品种。     

玉米瘤黑粉病抗病性研究

调查研究了沈阳地区田间玉米瘤黑粉病2004年自然发病情况,结果表明:供试的10个玉米品种的产量损失率为1%~10%,都是玉米瘤黑粉病的抗病品种;其中辽单37和辽单120是抗病品种,产量损失较大;新铁单10、沈农1号、丹玉90、铁单17和沈单12是中抗品种;铁单16、掖单2和沈单14是高抗品种,产量损失较小。玉米育种时,应当先从铁单16、掖单2和沈单14等抗病品种中选取,缩短育种周期,加快玉米品种的选育。     

The spread oi Erysiphe graminis f. sp. hordei in mixtures of barley varieties

A model is proposed of mechanisms which might affect the progress of Erysiphe graminis f. sp. hordei in mixtures of barley varieties. Results obtained from two field trials indicate that the efffect of mixtures may be panitioned into three categoriesof the influence of the reduced density of the susceptible plants, the barrier effect of the resistant plants, and the induced resistance due to the non-virulent pathogen biotypes. In the early stages of plant growth the lower density of susceptible plants accounted for most of the reduction in pathogen development in mixtures. As the epidemic progressed, the barrier and induced resistance effects increased in importance and the total mixture effect was at a maximum mid-way through epidemic development. Towards the end of the trials the overall mixture effect declined though the influence of induced resistance was at its maximum. The reasons for these changes and their implications for the use of host varietal mixtures in disease control are discussed.
Mixtures also protected the crop against a pathogen other than the target organism.     

Molecular mapping of the crown rust resistance gene rpc1 in barley

ABSTRACT Crown rust of barley, caused by Puccinia coronata var. hordei, occurs sporadically and sometimes may cause yield and quality reductions in the Great Plains region of the United States and Canada. The incompletely dominant resistance allele Rpc1 confers resistance to P. coronata in barley. Two generations, F(2) and F(2:3), developed from a cross between the resistant line Hor2596 (CIho 1243) and the susceptible line Bowman (PI 483237), were used in this study. Bulked segregant analysis combined with random amplified polymorphic DNA (RAPD) primers were used to identify molecular markers linked to Rpc1. DNA genotypes produced by 500 RAPD primers, 200 microsatellites (SSRs), and 71 restriction fragment length polymorphism (RFLP) probes were applied to map Rpc1. Of these, 15 RAPD primers identified polymorphisms between resistant and susceptible bulks, and 62 SSR markers and 32 RFLP markers identified polymorphisms between the resistant and susceptible parents. The polymorphic markers were applied to 97 F(2) individuals and F(2:3) families. These markers identified 112 polymorphisms and were used for primary linkage mapping to Rpc1 using Map Manager QT. Two RFLP and five SSR markers spanning the centromere on chromosome 3H and one RAPD marker (OPO08-700) were linked with Rpc1 and, thus, used to construct a 30-centimorgan (cM) linkage map containing the Rpc1 locus. The genetic distance between Rpc1 and the closest marker, RAPD OPO08-700, was 2.5 cM. The linked markers will be useful for incorporating this crown rust resistance gene into barley breeding lines.     

Prepenetration stages in infection of clematis by Phoma clematidina

A detailed study of conidial germination, germ-tube growth and the formation of infection structures in Phoma clematidina , the causal agent of clematis wilt, is described for two clematis varieties differing in disease resistance. On both the resistant and susceptible varieties, the fungus entered leaves and stems by direct penetration of the cuticle, often, but not always, following the formation of infection structures. More germ tubes per conidium were formed on the susceptible host, but these germ tubes were on average shorter than on the resistant host. Although germ tubes regularly entered the plant via trichomes, stomata were not found to be sites of entry. Following penetration of the cuticle of resistant plants, germ-tube growth was sometimes restricted to the subcuticular region, and halo formation occurred at the sites where penetration was attempted. Subcuticular growth and halo formation were not observed on susceptible plants. These observations may partly explain the resistance of small-flowered clematis varieties to P. clematidina .     

Sources and genetics of crown rust resistance in barley

ABSTRACT Crown rust, caused by Puccinia coronata var. hordei, is a new disease threat to barley in the Great Plains region of the United States. Deployment of resistant cultivars is the only economically viable option for the control of this disease. Thus, the objective of this study was to investigate the sources and genetics of crown rust resistance in barley. A geographically diverse sample of barley germ plasm collected around the world (526 accessions total) was evaluated at the seedling stage to P. coronata var. hordei, and only 10 accessions (1.9% of the total) were found resistant. These 10 accessions were also resistant at the adult plant stage in a greenhouse test. Three F(2) populations (Bowman x Hor2596, MR x Hor2596, and MD x Hor2596) were developed to study the inheritance of crown rust resistance in the resistant line Hor2596 (CIho 1243). A close fit to a 3:1 ratio of resistant/susceptible plants was observed in all three populations and is consistent with the segregation of a single resistance gene. F(1) plants from the Bowman x Hor2596 population exhibited slightly higher infection types than the resistant parent, indicating incomplete dominance. The locus symbol Rpc1 and allele symbol Rpc1.a were recommended for the crown rust resistance gene in Hor2596. An attempt was made to associate the Rpc1 locus with one of the seven barley chromosomes by analyzing linkage data with previously mapped morphological markers in crosses with multiple recessive (MR) and multiple dominant (MD) morphological marker stocks. However, no close linkages were detected between Rpc1 and the 20 morphological markers present in the marker stocks. The resistant accessions identified in this study should be useful to breeders for developing barley germ plasm with crown rust resistance.     

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.     

云南籼稻主栽品种对白背飞虱的抗性筛选

为明确云南籼稻主栽品种对白背飞虱Sogatella furcifera(Horváth)的抗虫性及其影响产量的水稻关键生育期,为抗虫品种选育和推广提供参考依据,本文利用苗期和分蘖盛期单株接虫鉴定、成株期(移栽期—乳熟期)田间自然种群发生鉴定等方法,测定了云南籼稻区31个水稻主栽品种对白背飞虱的抗虫性及其产量。结果表明,苗期有7个品种表现高抗,23个品种表现中抗—中感,1个品种表现高感;分蘖期有20个品种表现高抗,有11个表现中抗—中感;成株期田间自然种群鉴定,31个品种成株期白背飞虱田间自然种群的累计虫量都超过了对照品种TN1,均表现为感虫。单株接虫鉴定多数品种表现抗虫,而大田期自然种群鉴定则全部表现为感虫,一定程度说明了云南籼稻区主栽品种对白背飞虱具有耐害性而无抗生性。移栽期、孕穗期、抽穗期百丛虫量与有效穗数呈显著的负相关性;移栽期、孕穗期和抽穗期的百丛虫量是造成有效穗数少和产量低的关键因子。移栽期、孕穗期和抽穗期是防治白背飞虱的关键时期。     

Histology of infection by Erysiphe graminis f.sp. hordei in spring barley lines with various levels of partial resistance

The histology of resistance to infection by conidia of Erysiphe graminis f.sp. hordei has been studied in barley lines with various levels of partial resistance. Resistance reduced the proportion of conidia which formed haustoria and was expressed more strongly in fifth than in first formed leaves. Where attempted penetration from the first appressorial lobe failed, a second and sometimes a third lobe could be formed. Most penetrations were from first lobes, a few from second lobes and a negligible number from third lobes. Adult plant resistance decreased penetration success from both first and second lobes. An unidentified component of resistance appeared to impede the development of less vigorous conidia before they stimulated a host cell response. However, in all hosts the majority of failed penetration attempts stimulated a localized host response implicating this response in resistance. Some attempts caused hypersensitive death of epidermal cells in both resistant and susceptible hosts; the proportion of dead cells was higher in fifth than first leaves. Stomatal subsidiary cells were more susceptible to penetration than interstomatal cells and in resistant leaves the majority of successful penetrations were into subsidiary cells. The pathogen was also able to avoid resistance to penetration by establishing endophytic infection following entry of germ tubes through stomates.     

Normal germling development by Erysiphe graminis on cereal leaves freed of epicuticular wax

Experiments were conducted to test the hypothesis that recognition of the physical structure of epicuticular leaf waxes by Erysiphe graminis may be important to the development of normal germlings and the formation of functional appressoria. Comparisons of germination rates and characteristics of germling development by E. graminis f.sp. avenae , and in one experiment by f.sp. hordei , were made between intact cereal leaves and leaves from which the epicuticular waxes had been stripped away.
Overall, fungal development was very similar on intact and wax-free leaves: although germination rates were slightly, but significantly, lower, and lengths of appressorial germ tubes slightly greater, on stripped than intact leaves, a very similar proportion of germlings formed apparently normal appressoria in both cases. This was true for f.sp. avenae on first- and fifth-formed leaves of susceptible and adult plant resistant oats, and on barley and wheat first leaves, and for f.sp. hordei on first leaves of barley, oat and wheat. The appressoria formed on stripped leaves not only appeared normal, but also formed haustoria with at least the same frequency as on intact leaves; in several experiments, a higher proportion formed haustoria in stripped than intact leaves. Wax removal did not affect the adult plant resistance of oat cv. Maldwyn, which limits haustorium formation by appressoria, indicating that epicuticular wax was not involved in this resistance. It is concluded that the physical structure of epicuticular wax is not involved in the recognition processes leading to normal germling development.     

Inheritance and molecular mapping of barley genes conferring resistance to wheat stripe rust

ABSTRACT Most barley cultivars are resistant to stripe rust of wheat that is caused by Puccinia striiformis f. sp. tritici. The barley cv. Steptoe is susceptible to all identified races of P. striiformis f. sp. hordei (PSH), the barley stripe rust pathogen, but is resistant to most P. striiformis f. sp. tritici races. To determine inheritance of the Steptoe resistance to P. striiformis f. sp. tritici, a cross was made between Steptoe and Russell, a barley cultivar susceptible to some P. striiformis f. sp. tritici races and all tested P. striiformis f. sp. hordei races. Seedlings of parents and F(1), BC(1), F(2), and F(3) progeny from the barley cross were tested with P. striiformis f. sp. tritici races PST-41 and PST-45 under controlled greenhouse conditions. Genetic analyses of infection type data showed that Steptoe had one dominant gene and one recessive gene (provisionally designated as RpstS1 and rpstS2, respectively) for resistance to races PST-41 and PST-45. Genomic DNA was extracted from the parents and 150 F(2) plants that were tested for rust reaction and grown for seed of F(3) lines. The infection type data and polymorphic markers identified using the resistance gene analog polymorphism (RGAP) technique were analyzed with the Mapmaker computer program to map the resistance genes. The dominant resistance gene in Steptoe for resistance to P. striiformis f. sp. tritici races was mapped on barley chromosome 4H using a linked microsatellite marker, HVM68. A linkage group for the dominant gene was constructed with 12 RGAP markers and the microsatellite marker. The results show that resistance in barley to the wheat stripe rust pathogen is qualitatively inherited. These genes might provide useful resistance against wheat stripe rust when introgressed into wheat from barley.