Stability of Adult‐plant Resistance to Septoria tritici blotch in 24 European Winter Wheat Varieties Across Nine Field Environments

Septoria tritici blotch (STB) is one of the most important leaf diseases in wheat worldwide. Objectives of this study were (i) to compare inoculation and natural infection; (ii) to evaluate the level of adult‐plant resistance to STB using four isolates; and (iii) to analyse environmental stability of 24 winter wheat (Triticum aestivum L.) varieties in inoculated vs. non‐inoculated field trials across 3 years including nine environments (location × year combinations). Field trials were sown in split‐plot design inoculated with four aggressive isolates of S. tritici plus one non‐inoculated variant as main factor and 24 wheat varieties as subfactor. Septoria tritici blotch severity was visually scored as percentage flag leaves covered with lesions bearing pycnidia. Overall STB rating ranged from 8% (Solitär) to 63% (Rubens) flag leaf area affected, resulting in significant (P < 0.01) genotypic variance. Variance of genotype × environment interaction amounted to approximately 50% of the genotypic variance. Genotype × isolate interaction variance was significant too (P < 0.01) but of minor importance. Therefore, environmental stability of varieties should be a major breeding goal. The varieties Solitär, History and Florett were most resistant and stable as revealed by a regression approach, and the susceptible varieties were generally unstable. Hence, STB resistance and stability are correlated (P < 0.01), but there were some exceptions (Tuareg, Ambition). Promising candidates for an environmentally stable, effective adult‐plant resistance have been identified.     

The reaction of wheat genotypes to Septoria tritici

Seedling leaves, flag leaves, culms and heads of wheat genotypes were evaluated under glasshouse and field conditions for reaction to Septoria tritici. Parameters used for resistance screening were the incubation and latent periods, disease and sporulation levels, and 1000 grain weight. Significant correlations were obtained between reactions of different plant organs and between field and glasshouse tests; significant interspecific and intraspecific differences occurred for all screening parameters. Triticum aestivum varieties showed a wide range of disease reaction ranging from the high resistance of Elite Lepeuple, Maris Dove, Maris Ensign, Chalk and Tommy to the high susceptibility of Rothwell Sprite, Sovereign, Cardinal, Maris Ranger and Maris Templar.     

The Use of Detached Seedling Leaves of Triticum aestivum to Study Pathogenicity in Puccinia recondita f. sp. tritici

A method is described for establishing isolates of Puccinia recondita f. sp. tritici (causal agent of brown rust of wheat) on detached seedling leaf segments. The method was used to compare the responses of leaf segments and intact seedling leaves for 28 differential genotypes inoculated with eight rust isolates. Leaf segments were incubated at two post-inoculation temperatures (17 and 23C) and intact seedlings at 20–25 C. Reliable determinations of isolate pathogenicity was obtained using detached leaf segments of wheats with genes Lr l. Lr2a, Lr3a, Lr3bg., Lr3ka, Lr9, Lr15, Lrl9. Lr20, Lr24, Lr25. Lr26, Lr28, and Lr30 at both post-inoculation temperatures, and for wheats with genes Lr2b. Lr2c, Lrl7, Lr23, Lr27 + Lr31 and LrH, at 23°C. Differences between leaf segments and intact leaves for the remaining eight differentials were attributed to inconsistent or poor expression of genes in detached leaf segments. By repeating tests with detached leaf segments, it was possible to establish the pathogenicities of the isolates on all of these differentials except those carrying Lr13, Lr14a, Lr16 and Lr18. Potential uses and limitations of the technique in studies of Puccinia recondita f. sp. tritici are discussed.     

Changes in Wheat Leaf Polysomal Messenger RNA Populations during the Early Stages of Rust Infection: EFFECTS OF CHLORAMPHENICOL AND LINCOMYCIN ON CELL-FREE TRANSLATION BY POLYSOMES FROM HEALTHY AND INFECTED LEAVES

Polysomes isolated from a susceptible variety of wheat leaves (cultivar W2691) and those inoculated with the wheat stem rust fungus (f. sp. tritici, race 126-ANZ-6, 7) were incubated in a cell-free protein-synthesizing system. Under these conditions, different size classes of polypeptides, ranging in molecular weight from 10,000 to 80,000, are radiolabeled. Using double-isotope labeling technique, we show that some discrete size classes of polypeptides are synthesized in significantly greater quantitites by polysomes from inoculated leaves compared to the corresponding size classes synthesized by polysomes from healthy leaves. These results confirm our previous observation that there are significant changes in the wheat leaf polysomal messenger RNA populations at 3 days after inoculation with the rust fungus.The effects of the organelle-specific inhibitors of protein synthesis, chloramphenicol and lincomycin, on in vitro polysomal messenger RNA translation were investigated. The polypeptides synthesized by polysomes from healthy and inoculated leaves in the presence of chloramphenicol were compared. The results show that, even in the presence of this antibiotic, the polysomes from inoculated leaves synthesize greater quantities of some size classes of polypeptides. These data indicate that changes in polysomal messenger RNA populations involve, at least in part, cytoplasmic messenger RNA.     

Screening of Jatropha curcas genotypes to anthracnose caused by Colletotrichum gloeosporioides

Anthracnose caused by Colletotrichum gloeosporioides is an important physic nut (Jatropha curcas) disease causing damages on leaves, stems and fruits and consequently a decrease in seed quantity and quality. Physic nut is a native plant of Central America that has gained international attention due to its potential as biodiesel crop. However despite its highest relevance, studies concerning the response of different Jatropha genotypes to infection with this disease are very limited. The main objective of this study is the evaluation of susceptibility of 38 Jatropha curcas genotypes from a worldwide germplasm collection to Colletotrichum gloeosporioides. A simple method using wounded detached leaves inoculated with a 5-mm mycelia plug was used. Observations (lesion diameter and % of infection) were made after 3 and 7 days under controlled conditions (25 degrees C and 85% RH). Results showed significantly differences (P < or = 0.001) in both percentage of infection and lesion diameter among the tested accessions. Two genotypes from Brazil and Cape Verde, presented no lesions after 7 days of treatment, while the others presented between 67 and 100% of infection. Lesion diameter showed an increase with time of incubation and proved to be a useful tool for evaluation of plant susceptibility.     

Genetic architecture of resistance to Septoria tritici blotch in European wheat

Background

Septoria tritici blotch is an important leaf disease of European winter wheat. In our survey, we analyzed Septoria tritici blotch resistance in field trials with a large population of 1,055 elite hybrids and their 87 parental lines. Entries were fingerprinted with the 9 k SNP array. The accuracy of prediction of Septoria tritici blotch resistance achieved with different genome-wide mapping approaches was evaluated based on robust cross validation scenarios.

Results

Septoria tritici blotch disease severities were normally distributed, with genotypic variation being significantly (P < 0.01) larger than zero. The cross validation study revealed an absence of large effect QTL for additive and dominance effects. Application of genomic selection approaches particularly designed to tackle complex agronomic traits allowed to double the accuracy of prediction of Septoria tritici blotch resistance compared to calculation methods suited to detect QTL with large effects.

Conclusions

Our study revealed that Septoria tritici blotch resistance in European winter wheat is controlled by multiple loci with small effect size. This suggests that the currently achieved level of resistance in this collection is likely to be durable, as involvement of a high number of genes in a resistance trait reduces the risk of the resistance to be overcome by specific pathogen isolates or races.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-14-858) contains supplementary material, which is available to authorized users.     

苹果炭疽叶枯病菌致病力分析及苹果种质抗病性鉴定

苹果炭疽叶枯病是由胶孢炭疽菌(Colletotrichum gloeosporioides)引起的一种真菌病害,现已上升为世界苹果生产中的主要病害之一。了解不同来源的苹果炭疽叶枯病菌致病力差异及明确苹果种质资源对苹果炭疽叶枯病的抗性,对品种选育、品种合理布局以及控制病害的流行具有重要的参考价值。本研究对不同来源的79株病原菌进行了室内致病力测定,获知该菌致病力差异明显,其中强致病力菌株所占比例大。同时,本研究也对327份苹果种质资源进行了室内抗病性鉴定,其中高抗资源160份,中抗资源6份,中感资源22份,高感资源139份。表明我国现保存的苹果种质资源中存在丰富抗病种质。进一步按苹果分类系统分析发现,抗病资源在当前栽培的主要品种群中均有分布,特别是红玉品种群、富士品种群抗病资源最为丰富。     

In vitro evaluation of sugarcane resistance to gumming disease and of Xanthomonas campestris pv. vasculorum aggressiveness

Sugarcane plantlets were sectioned halfway between the base and the youngest ligule and then inoculated by soaking the wound in a suspension of Xanthomonas campestris pv. vasculorum. The infection caused rapid necrosis of the inoculated leaves, chlorosis of uninoculated leaves, or death of the inoculated plantlet. New tillers sometimes showed chlorosis or white streaks. The effects of the inoculum concentration, the cultivar, and the bacterial strain on symptom severity were determined. The ranking of cultivars depended on the inoculum concentration, and strains were found to differ with regard to aggressiveness. However, cultivars and strains were more effectively classified in greenhouse trials. The poor expression of leaf resistance appeared to limit the use of the in vitro test.     

Two Mycosphaerella graminicola French isolates differ in symptoms, in planta sporulation and cell wall degrading enzymes in vitro production

The pathogenicity level of two French Mycosphaerella grominicola field isolates (T0414 and T0251) was evaluated on Soissons wheat cultivar using two methods: detached wheat leafs assay in a climatic chamber and wheat seedlings assay in a greenhouse. Both methods revealed that chlorosis and necrosis caused by the T0414 isolate were larger than those caused by the T0251 isolate. Indeed, in the detached leaf assay, the first symptoms on leaves inoculated by T0414 were observed 12 days post inoculation and 50% of the leaves were infected on day 18, with a maximum of 84% of leaves on day 22. On the other hand, the first symptoms were observed on day 18 on leaves inoculated with T0251 with a maximum of only 10% of leaves that were infected on day 22. In addition, both methods showed that necrosis surfaces caused by T0414 were covered by large number of pycnidia, while no pycnidia were observed on the reduced necrosis caused by the T0251 isolate. To understand the pathogenicity variation between these two isolates, their ability to produce cell wall degrading enzymes, xylanases and polygalacturonases was investigated in vitro every 2 days for 20 days. The results showed similar time course for production of polygalacturonases for the two isolates, with non significant higher production for T0414. However, a peak of maximum production of xylanases by T0414 (343 +/- 52 mU mL(-1)) was observed on day 12 post inoculation, while the maximum production (265 +/- 72 mU mL(-1)) by T0251 was observed only on day 20 post inoculation. This result shows a relationship between the beginning of the appearance of symptoms on detached leaves inoculated by T0414 on day 12 and the maximum of xylanases production on the same day in enzymes assays. In conclusion, this study suggests pathogenicity variability between M. graminicola isolates and the role of xylanases in the pathogenicity of this fungus.     

Phenotypic and genetic analysis of the Triticum monococcum-Mycosphaerella graminicola interaction

Here, the aim was to understand the cellular and genetic basis of the Triticum monococcum-Mycosphaerella graminicola interaction. Testing for 5 yr under UK field conditions revealed that all 24 T. monococcum accessions exposed to a high level of natural inocula were fully resistant to M. graminicola. When the accessions were individually inoculated in the glasshouse using an attached leaf seeding assay and nine previously characterized M. graminicola isolates, fungal sporulation was observed in only three of the 216 interactions examined. Microscopic analyses revealed that M. graminicola infection was arrested at four different stages post-stomatal entry. When the inoculated leaves were detached 30 d post inoculation and incubated at 100% humidity, abundant asexual sporulation occurred within 5 d in a further 61 interactions. An F(2) mapping population generated from a cross between T. monococcum accession MDR002 (susceptible) and MDR043 (resistant) was inoculated with the M. graminicola isolate IPO323. Both resistance and in planta fungal growth were found to be controlled by a single genetic locus designated as TmStb1 which was linked to the microsatellite locus Xbarc174 on chromosome 7A(m). Exploitation of T. monococcum may provide new sources of resistance to septoria tritici blotch disease.     

Reduced Anthracnose Stalk Rot in Resistant Maize is Associated with Restricted Development of Colletotrichum graminicola in Pith Tissues

Resistance to anthracnose stalk rot (ASR) in maize was investigated for its effects on the development of Colletotrichum graminicola. ASR and fungal presence in pith tissues of resistant and susceptible genotypes, inoculated at time intervals after wounding in the first internodes, were assessed by rating tissue discoloration and by quantifying ergosterol production using high performance liquid chromatography (HPLC) and fungal recovery from tissues, respectively. Slices (30 μm thick) of pith cores (2 mm diam) of first internodes at late‐whorl and kernel blister stages were also inoculated with a suspension of fungal conidia immediately, 2 or 6 h after slicing. Fungal development was observed in tissues by light microscopy. ASR was markedly reduced in resistant genotypes when compared to susceptible genotypes and when inoculation was delayed after stalk wounding. Ergosterol content in tissues was associated with extent of discoloration due to ASR and fungal recovery. Conidial germination, germ tube elongation, appressorium formation and penetration of cortical cells were all markedly delayed in resistant genotypes, in both resistant and susceptible maize at vegetative stages, and by wound healing. C. graminicola macerated more rapidly and to a greater extent pith tissues of susceptible than resistant genotypes. Resistance mediated by maize genotype and ontogeny, and wound healing is expressed at early stages and subsequent events of host–pathogen interaction. Fungal structural development in detached pith tissues and the rapidity and extent of pith maceration in susceptible when compared to resistant genotypes was indicative of genotypic reaction to ASR in maize in the field. Laboratory inoculation and observation of detached pith tissues could be a useful and accurate tool for rapid screening of maize germplasm to identify ASR resistant genotypes that will function well in the field even where pathogen ingress occurs via wounds.     

Effects of Alteria alternata f.sp. lycopersici toxins at different levels of tomato plant cell development

Since the host-specific toxins of Alternaria alternata f. sp. lycopersici play an important role in pathogenesis, they potentially could be applied as selective agents in in vitro selection at the cellular level for disease resistance. Prerequisite for this is that sensitivity to the Alternaria alternata f.sp. lycopersici pathotoxins is manifest at the cellular level. To gain insight into cellular effects of AAL-toxins and into the mechanisms of plant insensitivity to AAL-toxins, effects of AAL-toxins on leaves, leaf discs, roots, calli, suspension cells, minicalli and protoplasts of susceptible and resistant tomato genotypes were studied. In leaves of susceptible genotypes, toxins cause severe necrosis, while in leaves of resistant genotypes necrosis was never observed. Inhibition effects of toxins were observed at all other levels in susceptible and resistant genotypes: toxins inhibited shoot induction on leaf discs, root growth and growth of calli, suspension cells and protoplasts. This indicates a cellular site for AAL-toxins. Differences in sensitivity to AAL-toxins between susceptible and resistant genotypes were observed in leaves and roots, but were not observed during shoot induction on leaf discs, in calli, suspension cells and protoplasts. However, differences in sensitivity to AAL-toxins in roots were at least 20 times less than in leaves. Therefore insensitivity seems related to a higher level of tomato plant differentiation and is most pronounced in leaves.     

Path-coefficient analyses and genetic parameters of the components of field resistance of potatoes to late blight

Variation, genetic parameters, interrelationships and phenotypic and genetic path analyses for components of field resistance of potatoes to Phytophthora infestuns were studied using detached leaves from 16 potato cultivars. Inter-genotypic variability was significant for the components and the Area Under Disease Progress Curve (AUDPC). The resistant cultivars generally had a longer latent period and lower lesion size and spore production than the susceptible cultivars. The correlations between AUDPC and infection efficiency, and between AUDPC and spore density were not significant, but latent period, lesion size and sporulation did correlate significantly with AUDPC. Genetic and phenotypic path-coefficient analyses indicated lesion size to be the most important component of field resistance. The genetic correlation coefficients between the AUDPC and infection efficiency, latent period and spore density arose mainly because of their indirect effects on AUDPC via lesion size. Lesion size and AUDPC had a high genetic coefficient of variation, heritability and genetic advance (genetic gain).     

Rapid detection and diagnosis of Septoria tritici epidemics in wheat using a polymerase chain reaction/PicoGreen assay

In order to detect and quantify Septoria tritici infection levels in wheat leaves, a polymerase chain reaction (PCR) assay was developed using the β-tubulin gene as target. Specific PCR primers were designed by aligning and comparing β-tubulin sequences from other fungi. The final primer set was selected after being tested against several fungi, and against S. tritici -infected and uninfected wheat leaves from different localities. A single DNA fragment (496 bp) was amplified from S. tritici, whereas no products were generated from DNA of the host plant or other micro-organisms associated with wheat leaves. Using agarose gel analysis, approximately 2 pg S. tritici genomic DNA could be detected in each assay. However, for rapid quantification of PCR-amplified products, a fluorometric microtitre plate-formatted PicoGreen assay was used; this could detect as little as 10 pg S. tritici DNA in the presence of 200 ng wheat leaf DNA. The PCR/PicoGreen assay was applied successfully to study the colonization, infection and subsequent disease development of S. tritici on wheat, both under controlled conditions in the glasshouse and in the field.     

Calcium Enhanced Zoospore Production of Pythium myriotylum in vitro

Pythium myriotylum is the causal organism of Cocoyam Root Rot Disease (CRRD). Significant numbers of zoospores were induced within 1.5 h in cultures in Petri dishes containing P. myriotylum soaked in 0.01 M Ca⁺⁺ and sterile deionized distilled water. Soaking solutions # 2 and # 3 inhibited the production of zoospores of P. myriotylum. This may be due to the delay in maturation of sporangia and the release of zoospores when the soaking solutions contain sucrose. Significant necrosis of detached cocoyam plantlet roots in 100 ml beakers confirmed the infection of zoospores of two `local white' cocoyam genotypes. Detached `yellow' cocoyam roots in 100 ml beakers of genotype RO3015 resisted infection of P. myriotylum with no necrosis of the inoculated roots, which may indicate resistance. This provides a quick and reliable pathogenicity test of P. myriotylum on susceptible cocoyam detached roots. Necrosis of inoculated detached cocoyam roots could be reliably used to screen cocoyam germplasm for resistance to P. myriotylum.     

THE REACTION OF WHEAT VARIETIES TO LOOSE SMUT AS DETERMINED BY EMBRYO, SEEDLING, AND ADULT PLANT TESTS

The possibility of using an embryo test as a means of determining the reaction of wheat varieties to loose smut ( Ustilago tritici (Pers.) Rostr.) has been investigated. Fifty-seven varieties were inoculated with two physiologic races by the partial vacuum method and the embryos, seedlings, and adult plants were examined for infection. Most of the varieties were fully susceptible to one or both races, and only ten showed real resistance to any one race. Braun R and Molinel proved almost immune to infection. The resistance in other varieties which showed no infection in the field was expressed as embryo susceptibility, i.e. the embryo became infected in much the same way as in field-susceptible varieties.
In the latter varieties infection passes from the embryo into the growing point of the young seedling. In the embryo-susceptible field-resistant varieties, infection does not pass from the scutellum and the growing point is therefore uninfected.
The reaction of most of the varieties tested was straightforward resistance or susceptibility, but in a few varieties a small proportion of the grains reacted differently from the majority. These reactions are discussed.     

Phenotyping of Partial Physiological Resistance to Rice Sheath Blight

Sheath blight, caused by Rhizoctonia solani, is one of the most important rice diseases worldwide especially under irrigated agro‐ecosystems. To date, no rice accession with complete resistance to sheath blight has been reported. However, a number of genotypes with varying levels of resistance have been reported. Twelve genotypes (including mega varieties) viz. Tetep, Jasmine 85, Te‐Qing, Duduruchi, Betichikon, Khatochalani, D‐6766, D‐256, Swarna, Sarju‐52, MTU‐1010 and Samba Mashuri were evaluated for quantitative measurement of partial physiological resistance to sheath blight under controlled conditions using detached tiller method. Three independent experiments, each involving three replications, were conducted. Seven days after inoculation, the following disease variables were measured: number of lesions, lesion length, vertical sheath colonization (VSC) on the tiller, disease severity, relative vertical sheath colonization (RVSC) and survival of the leaf blade. Variation between rice genotypes was observed for all the disease variables. Disease severity and VSC were the two most correlated variables, whereas the number of lesions and mean lesion length were the least correlated variables. The ranking of varieties often differed depending on the disease variable considered. Amongst the genotypes tested, D‐256, Tetep and Jasmin‐85 had the lowest number of lesions and disease severity. Similarly, Tetep and D‐256 showed the lowest levels of RVSC, whilst Jasmine‐85 was found to be intermediate. D‐6766, Samba Mashuri and Betichikon showed the highest levels of disease variables. The fraction of dead leaves ranged from 0.00 to 0.38. No dead leaves were observed in Te‐Qing, Swarna and MTU‐1010. The highest fraction of dead leaves was observed for Betichikon (0.38) followed by Duduruchi and D‐6766 (0.33). Our results suggest that this method in combination with other phenotyping methods could be used to quantify partial resistance to rice sheath blight.     

Local and systemic changes in glucosinolates in Chinese and European cultivars of oilseed rape (Brassica nap us L.) after inoculation with Sclerotinia sclerotiorum (stem rot)

Four Chinese Brassica napus lines, generated through a breeding programme to identify Sclerotinia sclerotiorum tolerant and susceptible lines, and three European varieties were analysed for changes in glucosinolates (qualitative and quantitative), and general host reactions, after localised inoculation with a UK S. sclerotiorum isolate. Plants at the fifth leaf stage were either singly inoculated (third leaf) or were inoculated once (third leaf) and then challenged a second time (seventh leaf) 7 days after the first inoculation. The results showed very distinct reactions in the different lines and cultivars to the fungus, both locally and systemically. Of the European lines B. napus cv. Bienvenu showed good resistance (small lesions and less host damage) both 3 and 7 days post-inoculation. Capricorn was the most susceptible followed by Cobra; the third leaves of these cultivars were showing strong chlorotic and necrotic reactions by day 3 and lesions were well developed. By day 7 the third leaves of Capricorn were completely rotten whilst Cobra still had a little healthy tissue. Inoculation of the four Chinese lines showed that two had moderate resistance (014 and 020 — slightly less resistant than Bienvenu) and two were very susceptible (016 and 024 — similar reactions to Capricorn and Cobra), based on lesion size and host tissue damage. Glucosinolate induction in line 014 was good both locally and systemically, with clear local and systemic induction of indolylglucosinolates and 2-phenylethylglucosinolate both 3 and 7 days post-inoculation. Line 020 did not show no particular increases in glucosinolates after inoculation either locally or systemically. In line 016 there was a small local increase and a large systemic reduction in total glucosinolates. Inoculation of line 024 caused no major local changes in glucosinolates and again a big reduction in glucosinolates systemically. The dual inoculation system, with lines 014 and 016, produced comparable results, with line 014 showing good local and systemic induction of glucosinolates (after the first inoculation) and a further local and systemic induction after the second inoculation. This induction in pre-inoculated line 014 plants was associated with a reduction in lesion size of the second inoculum. Line 016 responded poorly both locally and systemically, and there were no real decrease in the lesion size of the second inoculum. It appears that in line 014 glucosinolate induction may be an important part of resistance, whereas in line 020 there are clearly other non-glucosinolate factors involved. The poor local and systemic induction of glucosinolates in lines 016 and 024, and subsequent susceptibility, implies that glucosinolate induction may be an important marker of resistance to S. sclerotiorum in oilseed rape.     

A Possible Role for Ferrous Complexes in Fungal Disease Suppression: Glume Blotch and Net Blotch of Cereals

Germination of spores of Septoria nodorum and Pyrenophora teres was inhibited and germ-tube growth in germinated spores was reduced b y ferrous ions complexed with a number of cheiating agents. No such inhibition was observed with ferric complexes and none of the chelating agents in the desferri form was toxic to the fungi. The germination mechanism in spores suspended in ferrous-2,3-dihydroxybenzoic acid (Fe¹¹-DHBA) for 24 h could not subsequently be released to any great extent by incubation with ethylenediamjne-di (o-hydroxyphenyl-acetic acid) (EDDHA). Lesion development by the fungi in the presence of ferrous complexes on detached leaves of host plants was almost totally suppressed, but compounds which preferentially chelate ferric ions, used in the desferri form significantly stimulated lesion development by S. nodorum on wheat leaves. Cermination, appressorium formation and lesion development on leaves of host plants were also significantly reduced by Fe¹¹-DHBA when plants were sprayed to run-off up to 5 days prior to inoculation. Disease development and subsequent 1000 grain weight loss were reduced by approximately 50 percent in wheat and barley plants when the flag leaves were treated with Fe¹¹-DHBA (5 × 10^?4 M) prior to inoculation with S. nodorum and P. teres respectively, compared with inoculated, untreated plants.