Resistance to powdery mildew (Blumeria graminis f.sp. avenae) in oat seedlings and adult plants

In this work, 165 Avena sativa and Avena byzantina accessions were screened for resistance to powdery mildew caused by Blumeria graminis f.sp. avenae and the defence mechanisms of resistant plants were further characterized. Ten resistant and moderately resistant accessions were selected according to macroscopic assessment. A detailed histological study of selected genotypes showed a range of defence mechanisms, acting alone or in combination, that impeded fungal development at different stages. Since the resistance observed in the collection was scarce, a study of adult plant resistance was carried out in 45 genotypes selected from field trials. Nine oat landraces and two commercial varieties showed very high levels of adult plant resistance. A detailed study of the components of the adult plant resistance revealed a high increase of penetration and post‐haustorial resistance in the fifth compared to the first leaves. Identification of the resistance sources and characterization of underlying defence mechanisms will be useful for future breeding programmes and for further cellular and molecular studies to unravel the genetic basis of resistance, in this species in particular and in cereal–powdery mildew interactions in general.     

Development and validation of a quantitative PCR assay method of assessing relative resistance of oat (Avena sativa) to crown rust (Puccinia coronata f. sp. avenae)

Evaluation of oat crown rust resistance is usually based on visual assessment of disease severity or infection types. Visual assessment is subjective, prone to rater bias and requires expert knowledge. PCR-based quantitative assays can overcome challenges associated with visual assessment. New TaqMan primers and probes were designed from Puccinia coronata f. sp. avenae (Pca) sequences. The primer–probe sets were specific to Pca, amplified using as little as 0.5 pg fungal DNA (fDNA) and allowed for scaling to variation in sample total DNA quantity. The quantitative PCR (qPCR) assay was validated using oat recombinant inbred lines (RILs) from the Provena × 94197A1-9-2-2-2-5 cross evaluated under a controlled environment. For comparison with fDNA load, inoculation with the Pca race LCBB provided segregation data on the hypersensitive response, while Pca race LSLG provided data on segregation for reduced pustule number. fDNA content was positively correlated with both pustule number and infection type (IT). Composite interval mapping identified two quantitative trait loci (QTLs) on oat linkage groups Mrg12 and Mrg20 using visual and qPCR assessments (pustule number, IT and fDNA). In this study a qPCR assay method that can be used to assess the relative resistance of oat to crown rust was refined and validated, and single nucleotide polymorphisms (SNPs) closely linked with two QTLs derived from the crown rust resistant line 94197A1-9-2-2-2-5 were identified.     

Temperature-sensitive wheat stem rust resistance gene Sr15 is effective against Puccinia graminis f. sp. tritici race TTKSK

The wheat stem rust fungus, Puccinia graminis f. sp. tritici (Pgt), race TTKSK and related races pose a serious threat to world wheat production. Knowing the effectiveness of wheat stem rust resistance (Sr) genes against Pgt race TTKSK is fundamental in mitigating this threat through resistance breeding. Sr15 was previously identified as being ineffective against Pgt race TTKSK. Here, multirace disease phenotyping data, linkage analyses, allelism testing and haplotype analyses are presented to support the conclusion that Sr15 is effective against Pgt race TTKSK. Resistance to race TTKSK was mapped to Sr15 in a biparental population. Thirty-two accessions with Sr15 displayed seedling resistance phenotypes against race TTKSK. However, these accessions were susceptible as seedlings at high temperatures (22–25 °C), consistent with previous reports that the interaction between avirulent Pgt isolates and Sr15 is temperature-sensitive. Markers STS638, wri4 and KASP_IWB30995 were found to predict the presence of Sr15, suggesting the utility of these assays for marker-assisted selection in breeding programmes. The effectiveness of Sr15 to specific Pgt races and temperatures makes it a less-desirable TTKSK-effective gene. Wheat lines assayed as resistant to race TTKSK at the seedling stage may possess Sr15 and breeders should be aware of the limitations of Sr15 for conferring stem rust resistance.     

Disease development and genotypic diversity of Puccinia graminis f. sp. avenae in Swedish oat fields

The disease development and population structure of Puccinia graminis f. sp. avenae, which causes stem rust on oats, were studied to investigate if sexual reproduction plays an important role in the epidemiology of the disease. The genetic population structure of P. graminis f. sp. avenae in Sweden was investigated by sampling 10 oat fields in July and August 2008 and seven fields during the same period in 2009. Nine single‐pustule isolates were first used to test simple sequence repeat (SSR) markers developed for P. graminis f. sp. tritici. Eleven of the 68 tested SSR markers were useful for genotyping P. graminis f. sp. avenae. For the main study, DNA from single uredinia was extracted and the SSR markers were used to genotype 472 samples. Both allelic and genotypic diversity were high in all fields, indicating that P. graminis f. sp. avenae undergoes regular sexual reproduction in Sweden. No significant relationship between genetic and geographic distances was found. Disease development was studied on two farms during 2008 and 2009. The apparent infection rates ranged between 0·17 and 0·55, indicating the potential for rapid disease development within fields. The incidence of oat stem rust has increased recently in Sweden. One possible explanation is a resurgence of its alternate host, barberry (Berberis spp.), after the repeal of the barberry eradication law in 1994. Barberry is present in several grain‐producing areas in Sweden, which supports the conclusion that P. graminis f. sp. avenae undergoes regular sexual reproduction there.     

Identification of a new pathotype of Puccinia hordei with virulence for the resistance gene Rph7

Barley leaf rust resistance gene Rph7, derived from barley accession Cebada Capa, is the most effective R-gene for resistance to Puccinia hordei. Virulence for this gene was known in the USA, Israel and Morocco but not yet in Europe. We found an unexpected leaf rust infection in the field at Córdoba, Spain in 2004 on Rph7 carrying lines. This virulence for Rph7 was confirmed in growth chamber experiments, being the first report of Rph7 virulence in European populations of P. hordei. A collection of 680 barley accessions was screened for resistance against this new isolate. Twelve accessions showed segregation with individual plants showing resistance based on hypersensitivity (low infection type). These individual resistant plants were selected and grown in the greenhouse to obtain seeds.     

Disease severity and pathotype dynamics of Puccinia striiformis f.sp. tritici in Denmark

Disease severity of wheat yellow rust, Puccinia striiformis f.sp. tritici , was analysed in Denmark from 1985 to 1999 in relation to the effects of weather on winter survival, distribution of host cultivars and pathotype dynamics. Below-average temperatures in January and February (midwinter) reduced yellow rust on the susceptible cv. Anja, and in three of four growth seasons following cold winters no yellow rust was observed on any cultivar under natural conditions. The agronomic consequences of dispersal of yellow rust urediniospores from external sources to Denmark, in a period during which large areas were planted with relatively few wheat cultivars, were demonstrated in several cases, most evidently when the Yr9 and Yr17 resistance genes became ineffective. Yr9 was overcome by the pathogen in a period with severe yellow rust epidemics on commercial cultivars, while virulence for Yr17 was first observed in a year with almost no yellow rust. In contrast, the resistance in cv. Kraka ( Yr1, CV ) was increasingly effective in controlling yellow rust, because pathotypes with the matching combination of virulence declined in the pathogen population. Pathotype frequency dynamics were thus influenced by selection forces within the country, and by selection forces in areas where spores were spread to Denmark from outside. The importance of a sufficient level of partial resistance in the wheat germplasm to prevent too much damage by yellow rust epidemics, in the event that the resistance genes are overcome by the pathogen population, is emphasized.     

SSR-based genetic linkage analysis of resistance to crown rust (Puccinia coronata f. sp. lolii) in perennial ryegrass (Lolium perenne)

Crown rust (caused by Puccinia coronata f. sp. lolii) is a serious foliar disease of the pasture and turfgrass perennial ryegrass (Lolium perenne). Previous genetic studies have detected both qualitative and quantitative resistance mechanisms, and interpretation of the genetic system is complicated by variation within the sexually reproducing pathogen. Resistant and susceptible parental genotypes of ryegrass were identified using a composite urediniospore population collected from three geographically distinct locations. A two-way pseudo-testcross mapping population was obtained as the F₁ progeny of the pair-cross between ryegrass parental genotypes Vedette₆ and Victorian₉. Both parents showed intermediate resistance against a pathogen population collected in a single geographical zone (Hamilton, Victoria), but in the F₁ population, significant variation for a range of resistance-associated characters was detected. Statistical analysis of phenotypic data suggested a major gene effect, hence bulked segregant analysis with map-assigned simple sequence repeat (SSR) markers was used to scan the genome. A marker showing strong association with resistance was assigned to linkage group (LG) 2 of perennial ryegrass. Analysis of 11 LG2 SSR markers defined an interval between loci xlpssrh03f03 and xlpssrk02e02 as containing the gene or genes (LpPc1) conferring crown rust resistance. Resistance gene determinants were inherited from both parents, with up to 80% of the total phenotypic variation explained by markers segregating from Vedette₆ and up to 26% of the variation explained by markers segregating from Victorian₉. The two contributions together resulted in an additive increase in effect, with fully resistant individuals requiring determinants from both parents. A conserved syntenic relationship was observed with linkage group B of Avena strigosa, which is the location of a cluster of resistance genes to the oat form of crown rust. The implications of this study for marker-assisted selection of disease resistance in perennial ryegrass are discussed.     

Temperature adaptation in Australasian populations of Puccinia striiformis f. sp. tritici

Stripe rust, caused by Puccinia striiformis f. sp. tritici, is one of the major fungal pathogens of wheat. A new pathotype was introduced to Australia in 2002 and several derivative pathotypes were detected in subsequent seasons. It has been suggested that the severity of stripe rust outbreaks in Australia since 2002 could be as a result of traits other than virulence in the pathogen population. This study was conducted to investigate the hypothesis that the stripe rust pathogen population dominant in Australia since 2002 was better adapted to warm temperature conditions compared to previous pathogen populations. Sixteen pathotypes were selected to examine the influence of two contrasting temperature regimes during the 24 h incubation (10°C and 15°C) and the subsequent post‐inoculation (17°C and 23°C) periods on latent period and infection efficiency on four susceptible wheat cultivars. In addition, the effect of two contrasting incubation temperatures on urediniospore germination was examined. The results indicated that pathotypes of P. striiformis f. sp. tritici detected after 2002 did not show evidence of adaptation to high temperatures, which suggests that other factors contributed to the observed increased aggressiveness.     

Genetic analysis and molecular mapping of resistance to Puccinia striiformis f. sp. pseudo‐hordei in common wheat

This is the first genetic study reporting on the interaction and molecular mapping of resistance to the barley grass stripe rust pathogen (Puccinia striiformis f. sp. pseudo‐hordei, Psph) in common wheat. Seedlings of 638 wheat accessions were tested and it was determined that wheat is a near‐nonhost to Psph based on rare susceptibility observed in <2% of commercial cultivars and <5% of wheat landraces. As previously observed for P. striiformis f. sp. tritici (Pst), the Australian cultivar Teal was highly susceptible to Psph. In contrast, a selection of cv. Avocet carrying complementary resistance genes Yr73 and Yr74 (Avocet R; AvR) was resistant. The Teal × AvR (T/A) doubled haploid (DH) population was used to map resistance in AvR to Psph. Infection types on the T/A DH lines inoculated with Psph and Pst indicated that all DH lines carrying both Yr73 and Yr74 were also resistant to Psph; however, fewer DH lines were susceptible to Psph than expected, suggesting the resistance was more complex. QTL analysis using 9053 DArT‐Seq markers determined that resistance to Psph was polygenically inherited and mapped to chromosomes 3A, 3D, 4A and 5B. The 3DL and 5BL markers co‐located with Yr73 and Yr74, suggesting an overlap between host and non‐host resistance mechanisms.     

The recent history of Puccinia striiformis f.sp. tritici in Denmark as revealed by disease incidence and AFLP markers

Disease observations and amplified fragment length polymorphism (AFLP) markers were used to study recent developments in the Puccinia striiformis f.sp. tritici population in Denmark. The fungus appeared spontaneously at 10 locations in Denmark in 1997 after it was not observed under natural conditions in 1996. The pattern of disease development and prevailing winds suggested that the fungus reappeared by airborne spores from the south or west. In 1998, disease incidence was more evenly distributed throughout the country. Forty-eight single lesion isolates were collected from most crops where the disease was observed in these years; all except one from 1997 belonged to two pathotypes that were not previously detected in the country, and both possessed the newly discovered Yr17 virulence. The isolates were characterized with AFLP markers together with 28 isolates representing eight of 13 pathotypes observed prior to 1996. Initial screening of 240 Pst I/ Mse I AFLP primer combinations on four isolates showed that a primer combination, on average, revealed 0·4 polymorphisms between any isolate pair. A selection of 21 primer combinations resulted in 28 AFLP markers, which revealed 16 AFLP phenotypes among all 76 isolates. The two Yr17- virulent pathotypes consisted of three AFLP phenotypes, which were observed in both 1997 and 1998; the two most frequent AFLP phenotypes occurred at most sampling locations and often within the same crop. AFLP diversity was larger among samples collected prior to 1996, and also in this period most AFLP phenotypes were observed at different sampling locations. These results are consistent with the features of an entirely asexually reproducing pathogen dispersed by aerial spores across large areas.     

普通小麦品种陕旱8675中抗条锈病基因的分子定位

 利用SSR标记技术对小麦品种陕旱8675的抗条锈病基因进行了分子标记定位。通过对290对微卫星引物的筛选,发现Xwmc170和Xcfa20432对引物在抗亲、感亲、抗池和感池之间均有多态。群体分析结果表明,Xwmc170和Xcfa2043与陕旱8675中抗病基因相连锁,遗传距离分别为9.8 cM和15.0 cM,基因和标记之间的顺序为着丝点-Xcfa2043-Xwmc170-YrSh,间隔距离分别为19.05、5.2和9.8 cM。根据作图的结果,将陕旱8675中所含有的抗病基因定位于2A染色体长臂上,根据该基因的作图位置与抗谱分析,认为该基因可能是1个新的抗条锈基因,暂定名为YrSh。     

四川省小麦条锈菌耐高温性鉴定及其cDNA文库构建

近年条锈菌越夏调查发现,小麦条锈菌耐高温性增强,越夏海拔下限有所降低。本研究通过在高温(21±0.2)℃条件下接种,评价了119个来自四川省的条锈菌菌株耐高温性,并构建了耐高温菌株SC-PX1-4-3萌发夏孢子的cDNA文库。结果发现来自四川的条锈菌群体中耐高温菌株占35.3%,主要分布在成都市、凉山市、简阳市和广元市。夏孢子萌发率统计分析表明,耐高温菌株在高温条件下比温度敏感菌株具有更高的萌发率。采用SMART技术构建了耐高温菌株萌发夏孢子cDNA文库,库容为2.1×106cfu,插入片段平均多大于1.0kb。本研究结果初步明确了四川省小麦条锈菌群体中耐高温菌株组成和分布区域,不同菌株在夏孢子萌发阶段对高温的耐受性存在显著差异,为进一步研究条锈菌耐高温机制奠定基础。     

A detailed evaluation method to identify sources of quantitative resistance to Fusarium oxysporum f. sp. pisi race 2 within a Pisum spp. germplasm collection

Fusarium oxysporum f. sp. pisi (Fop) is an important pathogen of field pea (Pisum sativum) worldwide. The constant evolution of the pathogen drives the necessity to broaden the genetic basis of resistance to Fop. To achieve this, it is important to have a large germplasm collection available and an accurate and efficient method for disease assessment. Here, a detailed evaluation method coupling disease incidence, disease rating over time and its related area under the disease progression curve (AUDPC) was established and used to screen a Pisum spp. germplasm collection against one isolate of Fop race 2. A large variation in the disease response of specific pea accessions ranging from highly resistant to susceptible was observed within the collection, indicating the quantitative expression of the resistance. The repetition of the inoculation experiments on a subset of 19 accessions, including two susceptible accessions, indicated that the scoring method was robust and reproducible and confirmed the highly resistant phenotypes of 11 accessions. To initiate the characterization of resistance mechanisms within these accessions, the external and internal stem symptoms were compared between these selected pea accessions, together with the extent of fungal colonization within plants. All these tests indicated that, in all resistant accessions, the resistance mechanisms efficiently stopped pathogen progression at the crown. Incorporation of these sources of resistance to breeding programmes will contribute to improved Fop resistance in pea cultivars.     

Development,characterization and application of genomic SSR markers for the oat stem rust pathogen Puccinia graminis f. sp. avenae

Oat stem rust, caused by Puccinia graminis f. sp. avenae (Pga), is one of the most severe diseases of oats worldwide. Population studies are scarce for this pathogen, mainly due to the lack of polymorphic molecular markers suitable for genetic analysis. In this study, an Australian Pga isolate was sequenced, the abundance of simple sequence repeats (SSRs) was determined and PCR‐based polymorphic markers suitable for genetic diversity analysis were developed. The amplification of 194 primer pairs was initially assessed using a set of 12 isolates of different cereal rust species and their formae speciales. A high frequency of cross‐species amplification was observed for most markers; however, 36 SSRs were diagnostic for P. graminis only. A subset of 19 genome‐derived SSRs were deemed useful for genetic diversity analysis of Pga and were assessed on 66 Pga isolates from Australia, Brazil and Sweden. Brazilian and Australian isolates were characterized by one and two predominant clonal lineages, respectively. In contrast, the Swedish isolates, previously shown to undergo sexual recombination, were highly diverse (nine distinct genotypes out of 10 isolates) and divided into two subpopulations. The genome‐derived SSR markers developed in this study were well suited to the population studies undertaken, and have diagnostic capabilities that should aid in the identification of unknown rust pathogen species.     

Extended survival of Puccinia graminis f. sp. tritici urediniospores: implications for biosecurity and on‐farm management

Puccinia graminis f. sp. tritici (Pgt), the causal organism of stem rust, is of global importance across wheat‐growing countries. However, some epidemics commence without the obvious presence of ‘alternate’ or ‘green bridge’ hosts, suggesting urediniospores can survive in the absence of suitable host plants for many weeks. Testing a range of inert material types, including metals, plastics, fabrics and woods, highlighted a significant effect of material type and temperature on urediniospore viability (P < 0.001), with urediniospores remaining attached and viable on these materials (aluminium, paper, rubber, all fabric and all woods) for up to 365 days at 23/8 °C day/night. At 36/14 °C day/night, urediniospore viability was retained for a maximum of 300 days on denim and jute. Furthermore, at 45/15 °C day/night, urediniospores remained viable for a maximum of 180 days on cotton and jute. The frequency of recovery of attached urediniospores was also dependent upon the material type, with significant differences between materials in their abilities to retain urediniospores after washing (P < 0.001). Urediniospores recovered even after 300 or 365 days from the lower two temperature regimes successfully initiated infections of wheat seedlings. Results confirm the potential importance of inert materials as long‐term carriers of viable Pgt urediniospores, highlighting risks of spread of new pathotypes and strains across wheat‐growing regions, the significant biosecurity implications for contaminated carrier materials, and its likely survival across seasons without a host.     

Durability of resistance in lily to basal rot: evaluation of virulence and aggressiveness among isolates ofFusarium oxysporum f. sp.lilii

Prospects of durability of resistance in lily to basal rot have been evaluated by testing the virulence and aggressiveness of 31 isolates ofFusarium oxysporum f. sp.lilii towards a number of different resistance sources inLilium spp. Isolates differed strongly in aggressiveness as did species and cultivars ofLilium spp. in resistance. Significant interactions were observed between isolates of the pathogen and genotypes ofLilium spp., but the magnitude was very small compared to the main effects. The interactions were mainly due to a small group of isolates with low aggressiveness. It is argued that the interactions might be based on minor genes. No major break down of the resistance was found. For practical purposes it will be sufficient to use highly aggressive isolates in screening tests.     

Inheritance of resistance in carnation against Fusarium oxysporum f.sp. dianthi races 1 and 2, in relation to resistance components

Four carnation cultivars, Novada (resistant to races 1 and 2 ofFusarium oxysporum f.sp.dianthi), Elsy (susceptible to race 1), Lena (susceptible to race 2) and Sam's Pride (susceptible to both races), were selfed and crossed. When three months old, the seedlings were inoculated via the roots or via the stems, after which wilting was recorded weekly according to a 5-point ordinal scale.Analyses were carried out on the proportions of diseased plants. For race 1 variation between the progenies could be described by means of general combining abilities only; GCA values were not affected by the inoculation method used. Also for race 2 GCAs were most important but the GCA values appeared different for the two inoculation methods. It is concluded that resistance to both races is inherited in an additive way.Indications for independently inherited root-specific resistance components (extravascular resistance) were only found with race 2. With both races, the ability to confine the pathogen at the infection site appeared the most important resistance component. Resistant progenies were also characterized by longer latent periods and lower wilting rates.Both race 1 and race 2 induced the accumulation of the phytoalexins dianthalexin and methoxydianthramide S, but race 2 induced higher amounts than race 1. The accumulation of phytoalexins was positively correlated to the resistance level of the progenies against the respective races. The progenies of the double-resistant cultivar Novada appeared to produce particularly high levels of phytoalexins.     

大麦白粉菌种群毒性监测及抗性材料鉴定

2005和2006年从我国冬大麦区采集和分离大麦白粉菌单胞菌株729个,利用Pallas近等基因系进行致病型鉴定和群体毒性频率分析.同时,利用分离得到的不同致病型菌株,通过抗谱分析的方法鉴定了328份大麦品种(系)的白粉病抗性和抗病基因.结果显示:大麦白粉菌群体对抗病基因Mlal Mla(A12)、Mla3、Mla6 Mla14、Mla7 Mla(No3)、Mla7 Ml(Lg2)、Mla9 Mlk、Mla9、Mlal3 MlaRu3、Mlpl、Mlg(Cp)和mlo5的毒性频率为0;对Mla12 MlaEm2、Mla7 Mlk、Mlat Mla8、Mla10MlaDu2和Mlk1的毒性频率很低,分别为0.1%、0.4%、0.9%、2.8%和4.2%.两年共鉴定出不同的致病型21个,致病型000、001和003在两个年度皆为优势致病型.所鉴定的328份材料绝大多数感病,仅37份抗病材料,能明确推导出抗白粉病基因的品种(品系)很少,这些品种(品系)含有的抗白粉病基因为Mr(Bw)Mla8、Mlg、Mira Mla8、Mla9 Mla1、Mla Mla(A12)和mlo5.     

水培致病测定体系在Foc致病机理分析与香蕉品种抗性评价中的应用

为验证已建立的水培苗直接浇菌法的准确性与适用范围,利用此方法分析了5个致病相关基因敲除突变体对巴西蕉的致病力,评价了巴西蕉、皇帝蕉、粉蕉、宝岛蕉4个香蕉品种对4株尖镰孢菌古巴专化型病原菌株的抗性,并观察了接菌后巴西蕉根部与球茎组织的病原菌入侵过程。结果表明,foste12基因敲除突变株△Foste12菌株对巴西蕉的致病力与野生型菌株相比下降41%,且与传统的盆栽苗伤根淋菌法测试的下降趋势基本一致;皇帝蕉、宝岛蕉对4株病原菌均表现为中抗或高抗,粉蕉均表现为感病或高感;且观察到病原菌孢子附着根部、菌丝入侵根细胞间隙、维管束组织内的菌丝纵向生长及球茎组织内的菌丝侵入定殖等入侵过程。研究证实水培苗直接浇菌法可用于病原菌致病机理分析及与不同香蕉种质资源对尖镰孢菌古巴专化型的室内抗性评价。