Lipid metabolism is differentially modulated by salicylic acid and heptanoyl salicylic acid during the induction of resistance in wheat against powdery mildew

Heptanoyl salicylic acid (HSA) is a salicylic acid (SA) derivative obtained by esterification of 2-OH benzoic acid with heptanoic acid. In wheat, the protection levels obtained against Blumeria graminis f. sp. tritici (Bgt) increased from 50% with SA to 95% with HSA. Using molecular, biochemical and cytological approaches, we investigated here how wheat lipid metabolism is differentially activated by SA and HSA in both infectious and non-infectious conditions, and how Bgt infectious process is altered by both inducers. First, in the absence of Bgt, continuous lipoxygenase (LOX)-encoding gene expression and corresponding activity were specifically induced by HSA. Moreover, compared to SA, HSA treatment resulted in earlier up-regulations of the phospholipase C2-encoding gene expression and it specifically affected the expression of a lipid transfer protein-encoding gene. In infectious context, both HSA and SA sprayings impaired penetration events and therefore haustorium formation, leading to less frequent fungal colonies. While this alteration only slowed down the evolution of Bgt infectious process in SA-sprayed leaves, it completely impaired the establishment of successful infectious events in HSA-sprayed leaves. In addition, HSA induced continuous increases of a LOX-encoding gene expression and of the corresponding LOX activity when compared to SA-sprayed leaves. Lipid metabolism is therefore overall highly responsive to HSA spraying and could represent effective defence mechanism triggered during the induction of resistance in wheat toward Bgt. The concepts of priming and energy costs of the defences induced by SA and HSA are also discussed.     

Changes in lipid composition of Blumeria graminis f.sp. tritici conidia produced on wheat leaves treated with heptanoyl salicylic acid

Treatment of wheat leaves with heptanoyl salicylic acid (HS) and trehalose at concentrations of 0.1 and 15 g l(-1), prior to fungal inoculation, resulted in 40% and 60% protection, respectively, against powdery mildew. The total lipid composition of Blumeria graminis f.sp. tritici (Bgt) conidia, the causal agent of wheat powdery mildew, was compared when produced on wheat leaves, respectively, untreated and treated with the two elicitors, HS and trehalose. An obvious effect was observed on lipid composition (sterol and fatty acid (FA)) of Bgt conidia produced on wheat leaves treated with HS. A total of 16 FA (C12-C24 saturated and unsaturated) as well as unusual methoxylated Fatty Acids (mFA) (3-methoxydocosanoic and 3-methoxytetracosanoic acids) were detected in the conidia. Medium chain FA were predominant in HS treated conidia (64.65%) while long chain fatty acids constituted the major compounds in untreated conidia (62%). The long chain/medium chain FA ratio decreased from 1.8 in the conidia produced on untreated leaves to 0.5 in the conidia obtained from HS treated leaves. When comparing the sterol composition of Bgt conidia produced on leaves treated with HS versus conidia obtained from untreated ones, very important changes within the two major classes can be seen. In particular, 24-methylsterols, e.g., 24-methylenecholesterol and 24-methylcholesta-7,24-dien were reduced by about 82% whereas 24-ethylsterols, e.g., 24-ethylcholesterol and 24-ethylcholesta-5,22-dienol were increased by about 85%. The 24-methylsterols/24-ethylsterols ratio was reduced by ninefold in the conidia produced from HS treated leaves.     

Inheritance of partial resistance to powdery mildew in spring wheat

Summary Four spring wheat (Triticum aestivum L.) cultivars exhibiting partial resistance to powdery mildew induced by Erysiphe graminis f.sp. tritici were crossed to a common susceptible cultivar to study the inheritance of resistance. The genetic parameters contributing to resistance were estimated by generation means analyses. Additive gene action was the most important genetic component of variation among generation means in all four crosses. Additive by additive effects were significant in one cross and both additive by additive and additive by dominance effects were significant in another. Dominance effects were not significant. The F2/F3 correlations in three crosses ranged from 0.27 to 0.43. Three additional crosses among resistant cultivars were employed to study the effectiveness of selection in improving resistance. By selecting the most resistant plants from the F2 and evaluating the progenies in the F4, increases in resistance ranging from 21% to 31% were obtained. In all crosses, there was transgressive segregation in both directions indicating that the genes conferring resistance to these cultivars differ and exhibit additive effects.     

Appressorium morphogenesis and cell cycle progression are linked in the grass powdery mildew fungus Blumeria graminis

Conidial germination and differentiation – the so-called prepenetration processes – of the barley powdery mildew fungus (Blumeria graminis f. sp. hordei) are essential prerequisites for facilitating penetration of the host cuticle. Although the cell cycle is known to be pivotal to cellular differentiation in several phytopathogenic fungi there is as yet no information available concerning the relationship between cell cycle and infection structure development in the obligate biotroph B. graminis. The timing of specific developmental events with respect to nuclear division and morphogenesis was followed on artificial and host leaf surfaces by 4′,6-diamidino-2-phenylindole (DAPI) staining in combination with a pharmacological approach applying specific cell cycle inhibitors. It was found that the uninucleate conidia germinated and then underwent a single round of mitosis 5–6 h after inoculation. During primary germ tube formation the nucleus frequently migrated close to the site of primary germ tube emergence. This nuclear repositioning was distinctly promoted by very-long-chain aldehydes that are common host cuticular wax constituents known to induce conidial differentiation. The subsequent morphogenesis of the appressorial germ tube preceded mitosis that was spatially uncoupled from subsequent cytokinesis. Blocking of S-phase with hydroxyurea did not inhibit formation of the appressorial germ tube but prevented cytokinesis and appressorium maturation. Benomyl treatment that arrests the cell cycle in mitosis inhibited nuclear separation, cytokinesis, and formation of mature appressoria. Thus, we conclude that a completed mitosis is not a prerequisite for the formation and swelling of the appressorial germ tube, which normally provides the destination for one of the daughter nuclei, while appressorium maturation depends on mitosis.     

Two stable QTL involved in adult plant resistance to powdery mildew in the winter wheat line RE714 are expressed at different times along the growing season

Powdery mildew (Blumeria graminis f. sp. tritici) is one of the major diseases of wheat (Triticum aestivum). Adult plant resistance (APR) to powdery mildew is considered more durable than resistance conferred by major race-specific resistance genes. The objective of the present study was a better understanding of the genetic basis of APR in RE714 by means of QTL analysis of several resistance scores along the growing season. A population of 160 recombinant inbred lines obtained from the cross between RE714 and Hardi (susceptible) was assessed for APR under natural infection conditions during 3 years and a genetic map with whole genome coverage was developed with microsatellite and AFLP markers in this population. Two major QTL on chromosomes 5D and 6A were detected each year, and 6 minor QTL were detected only in 1 or 2 years. The QTL on chromosome 5D was detected during all the growing season each year and its R ² value varied between 8.5 and 56.3%, whereas the QTL on chromosome 6A was detected at 1–4 scoring dates in the 3 years, and its R ² value varied between 6.1 and 20.5%. The two QTL explained between 24.4 and 52.1% of the phenotypic variance for AUDPC, depending on the year. The models including QTL and cofactors in the composite interval mapping explained between 29 and 72% of the variance. The molecular markers linked to the two major QTL could be used in marker-assisted selection for adult plant resistance to powdery mildew. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Transgressive segregation for very low and high levels of basal resistance to powdery mildew in barley

Basal resistance of barley to powdery mildew is a quantitatively inherited trait that limits the growth and sporulation of barley powdery mildew pathogen by a non-hypersensitive mechanism of defense. Two experimental barley lines were developed with a very high (ErBgh) and low (EsBgh) level of basal resistance to powdery mildew by cycles of convergent crossing and phenotypic selection between the most resistant and between the most susceptible lines, respectively, from four mapping populations of barley. Phenotypic selection in convergent crossing was highly effective in producing contrasting phenotypes for basal resistance and susceptibility. In ErBgh, almost 90% of infection units failed to form a primary haustorium in the epidermal cells in association with papilla formation, but in EsBgh only 33% of infection units failed to form a primary haustorium. The contrast between ErBgh and EsBgh for successful formation of secondary and subsequent haustoria was much less obvious (69% versus 79% successful secondary haustorium formation). In an earlier investigation, we determined seven QTLs for basal resistance in the four mapping populations. Checking the peak markers of these QTLs indicated that only four out of seven QTLs were confirmed to be present in the selected resistant lines and only four QTLs for susceptibility were confirmed to be present in the selected susceptible lines. Surprisingly, none of the expected QTLs could be detected in the resistant line ErBgh. We discuss some reasons why marker aided selection might be less efficient in raising levels of basal resistance than phenotypic selection. The very resistant and susceptible lines developed here are valuable material to be used in further experiments to characterize the molecular basis of basal resistance to powdery mildew.     

山西省草地早熟禾白粉菌分离株BGP(TG)的鉴定及致病力分析

【背景】草地早熟禾(Poa pratensis)是一种被广泛使用的兼具观赏性和设施性的冷季型草坪草,白粉病的发生严重降低了草地早熟禾的质量和使用年限。【目的】对山西农业大学温室草地早熟禾白粉病病原物进行分离、纯化、鉴定和致病力分析,为草地早熟禾白粉病的防治提供理论依据。【方法】采用形态特征观察、核糖体内源转录间隔区(ribosomal DNA internally transcribed spacer, rDNA-ITS)序列分析进行分离株BGP(TG)的鉴定;通过分生孢子密集抖落接种法测定BGP(TG)对23份草地早熟禾材料的致病力。【结果】草地早熟禾白粉菌分离株BGP(TG)分生孢子串生、无色、椭圆形,长和宽分别为(23.31−34.67) µm和(11.33−15.77) µm,成熟吸器为指状;rDNA-ITS序列分子发育分析结果表明分离株BGP(TG)与禾布氏白粉菌早熟禾专化型(Blumeria graminis f. sp. poae)聚于同一分支;BGP(TG)对23份草地早熟禾的致病力测定结果显示,白粉病发病率为49.47%−77.72%,病情指数为14.41−53.12;仅‘太行’草地早熟禾对BGP(TG)表现为中抗,‘应县’ ‘黑杰克’和‘浑源’草地早熟禾表现为中感,‘探险家’和‘奖品’草地早熟禾表现为极感,其余17个品种表现为高感,占比达73.91%。【结论】山西省草地早熟禾白粉菌分离株BGP(TG)的致病力较强,进一步挖掘‘太行’草地早熟禾的抗白粉病基因,对草地早熟禾抗白粉病育种工作具有重要意义。     

Wheat cells accumulate a syringyl-rich lignin during the hypersensitive resistance response

The stem rust fungus Puccinia graminis f.sp. tritici is an obligately biotrophic pathogen attacking wheat (Triticum aestivum). In compatible host/pathogen-interactions, the fungus participates in the host's metabolism by establishing functional haustoria in the susceptible plant cells. In highly resistant wheat cultivars, fungal attack is stopped by a hypersensitive response of penetrated host cells. This mechanism of programmed cell death of single plant cells is accompanied by the intracellular accumulation of material with UV-fluorescence typical of phenolic compounds. A similar reaction can be induced in healthy wheat leaves by the application of a rust-derived elicitor. We analysed the biochemical composition of this defense-induced phenolic material. Contents of total soluble and cell wall esterified and etherified phenolic acids were determined in rust-inoculated and elicitor-treated leaves of the fully susceptible wheat cultivar Prelude and its highly resistant, near-isogenic line Prelude-Sr5. While no resistance-related changes occured in any of these fractions, the lignin content as determined by the thioglycolic acid and the acetyl bromide methods increased after elicitor treatment. Nitrobenzene oxidation revealed that the entire increase can be explained by an increase in syringyl units only. These biochemical data were confirmed by fluorescence emission spectra analyses which indicated a defense-induced enrichment of syringyl lignin for cell wall samples both from elicitor-treated wheat leaves and single host cells undergoing a hypersensitive response upon fungal penetration.     

Stable expression of a defense-related gene in wheat epidermis under transcriptional control of a novel promoter confers pathogen resistance

Tissue-specific or regulated expression of transgenes is desirable in order to prevent pleiotropic side effects of putatively harmful transgene products as well as loss of energy resources due to unnecessary accumulation of transgene products. Epidermis-specific expression would be useful for many defense-related genes directed against attack by fungal pathogens that enter the plant body by direct penetration through the epidermis. In an approach to enhance resistance of wheat to the powdery mildew fungus Blumeria graminis f.sp. tritici, a novel epidermis-specific promoter was developed and used for expression of two defense-related genes. A 2.3 kb fragment of the wheat GstA1 promoter in combination with an intron-containing part of the wheat WIR1a gene was found to drive strong and constitutive transient expression in wheat epidermis. This promoter-intron combination was used for overexpression of oxalate oxidase 9f-2.8 and TaPERO peroxidase, two defense-related wheat genes expressed in inner leaf tissues. Expression studies of several transgenic lines by in situ oxalate-oxidase staining, RNA and protein blot analyses, as well as real-time PCR, demonstrated strong and constitutive transgene expression in the shoot epidermis. Transient as well as stable over-expression of the TaPERO peroxidase gene in wheat epidermis under the control of the GstA1i promoter resulted in enhanced resistance against Blumeria graminis f.sp. tritici, whereas oxalate-oxidase overexpression had no effect in either system. The data suggest that the wheat GstA1 promoter in combination with the WIR1a intron is useful for transgenic approaches to fungal disease resistance in cereals.     

A storage-protein marker associated with the suppressor of Pm8 for powdery mildew resistance in wheat

A suppressor of resistance to powdery mildew conferred by Pm8 showed complete association with the presence of a storage-protein marker resolved by electrophoresis on SDS-PAGE gels. This marker was identified as the product of the gliadin allele Gli-A1a. The mildewresponse phenotypes of wheats possessing the 1BL.1RS translocation were completely predictable from electrophoretograms. The suppressor, designated SuPm8, was located on chromosome 1AS. It was specific in its suppression of Pm8, and did not affect the rye-derived resistance phenotypes of wheat lines with Pm17, also located in 1RS, or of lines with Pm7.     

Four powdery mildew species with catenate conidia infect Galium: molecular and morphological evidence

The Erysiphaceae are a group of obligately biotrophic fungi that cause powdery mildew disease of angiosperms. Due to their inability to be cultured on artificial media, the taxonomy of the Erysiphaceae has generally been based on the morphological characteristics of fresh and herbarium specimens. Thus, several morphological species with wide host ranges have long been maintained in this family, even though they clearly consist of several biological species. Erysiphe galii has been known as a powdery mildew of Galium spp. Recently, the former E. galii var. galii has been reassessed as Neoerysiphe galii and E. galii var. riedliana as Golovinomyces riedlianus, along with a taxonomic revision of the generic concept of the Erysiphaceae. The present study was conducted to evaluate the validity of the taxonomic revision of the two varieties of E. galii. During the course of this study, we found that the Galium powdery mildews consist of at least four different species, viz. Neoerysiphe galii, Golovinomyces orontii, G. riedlianus, and an unknown species collected in Argentina. The latter species is described as a new species, Golovinomyces calceolariae. The three species belonging to Golovinomyces are morphologically very similar to each other, i.e. the discrimination between them is rather difficult. The morphological differences of the three Golovinomyces species of Galium are discussed.     

Effects of salicylic acid and cold treatments on protein levels and on the activities of antioxidant enzymes in the apoplast of winter wheat leaves

The effects of salicylic acid (SA) and cold on apoplastic protein levels and activities of apoplastic catalase (CAT), peroxidase (POX) and polyphenol oxidase (PPO) were investigated in winter wheat (Triticum aestivum cv. Dogu-88) leaves. The plants were grown with and without 10 microM SA treatment at both control (20/18 degrees C for 30 and 45-day) and cold (10/5 degrees C for 30-day and 5/3 degrees C for 45-day) acclimatisations. Molecular masses of the apoplastic polypeptides were shown ranging in size from 20 to 66 kDa on SDS-PAGE. Accumulation and pattern of the polypeptides were changed by both SA and cold. It is observed that CAT, POX and PPO activities at 45-day control leaves were higher than at 30-day. When the activities with SA and cold treatments are compared to their controls, CAT activities were decreased while POX and PPO activities were increased by both the treatments. When the activities with cold+SA treatment are compared to their cold treatments, CAT and POX activities were decreased while PPO activity was increased by SA. It is concluded that exogenous SA can be involved in cold tolerance by regulating apoplastic proteins and antioxidant enzyme activities.     

Ultrastructural comparison of incompatible and compatible interactions in the barley powdery mildew disease

Summary In the powdery mildew disease of barley,Erysiphe graminis f. sp.hordei forms an intimate relationship with compatible hosts, in which haustoria form in epidermal cells with no obvious detrimental effects on the host until late in the infection sequence. In incompatible interactions, by contrast, the deposition of papillae and localized host cell death have been correlated with the cessation of growth byE. g. hordei. With the advent of improved, low temperature methods of sample preparation, we felt that it was useful to reevaluate the structural details of interactions between barley andE. g. hordei by transmission electron microscopy. The haustoria that develop in susceptible barley lines appear highly metabolically active based on the occurrrence of abundant endoplasmic reticulum, Golgi-like cisternae, and vesicles. In comparison, haustoria found in the resistant barley line exhibited varying signs of degradation. A striking clearing of the matrix and loss of cristae were typical early changes in the haustorial mitochondria in incompatible interactions. The absence of distinct endoplasmic reticulum and Golgi-like cisternae, the formation of vacuoles, and the occurrence of a distended sheath were characteristic of intermediate stages of haustorial degeneration. At more advanced stages of degeneration, haustoria were dominated by large vacuoles containing membrane fragments. This process of degeneration was not observed in haustoria ofE. g. hordei developing in the susceptible barley line.Abbreviations b endoplasmic reticulum extension, blebbing - er endoplasmic reticulum - f fibrillar material - g Golgi-like structure - h haustorium - hb haustorial body - hcw haustorial cell wall - hcy haustorial cytoplasm - hf haustorial finger - hocw host cell wall - hocy host cytoplasm - 1 lipid-like droplet - m mitochondrion - mt microtubule - mve multivesicular body - n nucleus - p papilla - ph penetration site of an infection peg - pl plasma membrane - s sheath - sm extrahaustorial membrane - v vacuole - ve vesicle     

Dynamic changes in cell wall polysaccharides during wheat seedling development

Changes in arabinoxylan content and composition during development of wheat seedlings were investigated. The cell walls isolated from the seedlings showed an increasing content of arabinoxylan during development, which could be correlated to increased activity of xylan synthase and arabinoxylan arabinosyltransferase. Arabinoxylan changed from initially having a high degree of arabinose substitution to a much lower degree of substitution. beta-Glucan was present in the walls at the early stages of development, but was actively degraded after day 4. Increased deposition of arabinoxylan did not take place until beta-glucan had been fully degraded. Ferulic and p-coumaric acid esters were present at all points but increased significantly from day 3 to 6, where lignification began. Ferulic acid dimers did not appear in the cell wall until day three and the different ferulic acid dimers varied in the course of accumulation. The ratio of ferulic acid dimers to free ferulic acid was maximal at the time when the wall had been depleted for beta-glucan, which had not yet been fully replaced by arabinoxylan. This pattern suggests a role for ferulic acid dimers in stabilizing the wall during the transition from a flexible to a more rigid structure. To investigate if the same changes could be observed within a single seedling, 7 day old seedlings were divided into four sections and the walls were analyzed. Some of the changes observed during the seedling development could also be observed within a single seedling, when analyzing the segments from the elongation zone at the base to the top of the leaf. However, the expanding region of older seedlings was much richer in hydroxycinnamates than the expanding region of younger seedlings. Diferulic acids are stabilizing the wall in the transition phase from an expanding to a mature wall. This transition can take place in different manners depending on the cell and tissue type.     

Nitric oxide, polyamines and Cd-induced phytotoxicity in wheat roots

To further explore the biochemical basis of Cd toxicity in developing wheat seedlings, we studied the possible role of nitric oxide (NO) and polyamines as signaling molecules involved in metal-induced root growth inhibition. When used at 0.1 mM, sodium nitroprusside, a NO-releasing compound, inhibited root growth to a similar extent as Cd and enhanced the polyamine contents as Cd also did. Putrescine and spermidine treatments caused significant decreases in root growth with spermine giving the greatest level of inhibition (77% reduction). The simultaneous addition of Cd and inhibitors of putrescine biosynthesis (DFMA and DFMO) prevented increases in putrescine levels but did not restore normal root growth. NO content, as evidenced by the fluorescent probe DAF-FM diacetate, was found to be significantly increased in the roots of both Cd and polyamine treated plants, especially in those exposed to spermine. The effect was specific for NO since the NO scavenger cPTIO almost suppressed the fluorescent signal. Concerning the oxidative status of the root system, only Cd and spermine enhanced lipid peroxidation in roots. At the same time, all treatments led to a significant increase in levels of the non-enzymatic antioxidant defense glutathione. Our results strongly suggest that Cd and spermine treatments induce NO formation in wheat roots which, in turn, is involved in root growth inhibition.     

Changes in C12:0, C18:1, C18:2 and C20:2 fatty acid content in wheat treated with resistance inducers and infected by powdery mildew

This work presents a global investigation of total fatty acid (FA) content in wheat in relation to treatment with four inducers of resistance and to powdery mildew infection. Linolenic acid (C18:3), linoleic acid (C18:2) and palmitic acid (16:0) were the most abundant FAs in wheat leaves. We investigated the effect of the following inducers of resistance: Iodus40, heptanoyl salicylic acid (HSA), Milsana and trehalose on FA accumulation. Previous studies established that lipid metabolism is altered by these compounds, and we therefore aimed to characterise their impact at the FA level. During a time course experiment, content (quantitative analysis) and percentage (qualitative analysis) of FAs were compared in treated plants and in controls, as well as in plants inoculated with Blumeria graminis f. sp. tritici (i) and non-inoculated (ni) plants. No change in C18:3 content was observed. C18:1 in Iodus 40-treated (ni) plants showed a quantitative 1.2-fold increase. Lauric acid (C12:0) content quantitatively increased after Iodus 40 (2.8-fold), Milsana (4.8-fold) and trehalose (4.0-fold) treatment in (i) plants. However, eicosadienoic acid (C20:2) quantitatively decreased in (ni) plants after Iodus 40 (1.5-fold) and Milsana (2.3-fold) treatment. The amount of C18:2 increased (1.6-fold) after HSA treatment in (i) plants. All these variations in FA content were correlated with variations in the corresponding relative percentages. Our work provides the first evidence for alterations in C12:0, C18:1, C18:2 and C20:2 FA content caused by four resistance inducers. We also compared the amount and percentage of each FA in untreated (i) and (ni) plants. In (i) plants, eicosadienoic acid (C20:2) increased and C18:2 decreased slightly. The potential involvement of these FAs during induced resistance and infection is discussed.     

Phytotoxic and genotoxic effects of silver nanoparticles exposure on germinating wheat seedlings

We investigated the effects of 1 and 10 mg L⁻¹ AgNPs on germinating Triticum aestivum L. seedlings. The exposure to 10 mg L⁻¹ AgNPs adversely affected the seedling growth and induced morphological modifications in root tip cells. TEM analysis suggests that the observed effects were due primarily to the release of Ag ions from AgNPs.