The Interaction of Humidity and Resistance Elicitors on Expression of Polygenic Resistance of Barley to Mildew

Elicitor-active glucan was applied to leaves of genotypes of barley expressing different levels of resistance to powdery mildew ( Erysiphe graminis f. sp. hordei )which was assessed by measuring mildew colonies per leaf or speed of papilla induction response to mildew challenge. The influence of humidity was also assessed by treating leaves with glucan, inoculating with mildew and immediately sealing the leaves in Perspex containers in an apparatus which enabled temperature, air speed and humidity to be controlled accurately. In water-treated controls low humidity alone reduced colonies per leaf, and at 60% r.h. genotype 7204 showed significantly less effect of humidity compared with other genotypes, although at 40% it responded as other genotypes. At low humidity glucan stimulated resistance for genotypes Proctor and 7204 but not for Golden Promise or 9855; the effect of humidity or elicitor alone was only significant on genotype 7204, Chitin also proved an effective elicitor, but at high and not at low humidity. Time of pre-treatment before mildew challenge, leaf stage, type of elicitor, concentration of elicitor, post-inoculation light regime, mildew isolate and barley genotype all had major influences on papilla response. Other factors were less important. Based on papilla formation, the barley cultivar Proctor was most and Golden Promise the least responsive to glucan as an elicitor. The potential for exploiting the genetic basis of inducible resistance is discussed.     

Effects of elevated atmospheric CO2 and temperature on the management of powdery mildew of zucchini

The impact of combined environmental factors, such as temperature and CO₂, on the control of the powdery mildew of zucchini, caused by Podosphaera xanthii, and of different control measures has been studied on plants grown in phytotrons. Five experimental trials were conducted, and the powdery mildew severity of both treated and untreated zucchini plants was found to be significantly affected by the interaction between temperature (three different regimes: 16–18; 18–22; 22–26°C), CO₂ (two concentrations: 400–450 and 800–850 ppm) and the treatments. However, at the end of the trials, the efficacy of all the products was not affected by the different, tested environmental conditions. Sulphur consistently provided the highest disease control (75%–85% efficacy). Among the resistant inducers that were tested, calcium oxide was the most effective, in terms of powdery mildew control under all the conditions tested in phytotrons, reducing disease severity from 46% to 61%. Foliar applications of phosphite (14%–28% efficacy), Ampelomyces quisqualis (12%–23% efficacy) and potassium silicate (13%–24% efficacy) only slightly reduced the disease severity for all the tested temperature regimes and CO₂ concentrations, compared to the untreated control. The results obtained under our experimental conditions show that a possible increase in CO₂ concentration and temperature, which is expected for the next few years, should not influence the efficacy of the tested resistance inducers or of sulphur against powdery mildew on zucchini. Moreover, the suppressive effect of calcium oxide is in light of its possible use in greenhouses for zucchini powdery mildew control under 400–450 ppm of CO₂ and under enriched condition of 800–850 ppm of CO₂.     

Evidence for Resistance-inducing Compounds in Conidia of Erysiphe graminis f.sp. hordei

Preparations of Erysiphe graminis f.sp. hordei conidia were spray-applied to the first leaf of barley plants which were subsequently challenge inoculated with virulent powdery mildew. The powdery mildew reducing effect of the preparations was assessed by scoring the outcome of the challenge inoculation. Homogenates of ungerminated conidia, germinated conidia, and methanol-water extracts of germinated conidia reduced the number of powdery mildew colonies. Cell wall fragments from ungerminated conidia, germinated conidia, and conidial germination fluid obtained from conidia germinated in aqueous suspension did not reduce the number of powdery mildew colonies. Microsconical analysis of the infection course following challenge inoculation indicated that the powdery mildew reducing effect is due partly to induced resistance.     

The transfer of a powdery mildew resistance gene from Hordeum bulbosum L to barley (H. vulgare L.) chromosome 2 (2I)

Hordeum bulbosum L. is a source of disease resistance genes that would be worthwhile transferring to barley (H. vulgare L.). To achieve this objective, selfed seed from a tetraploid H. vulgare x H. bulbosum hybrid was irradiated. Subsequently, a powdery mildew-resistant selection of barley phenotype (81882/83) was identified among field-grown progeny. Using molecular analyses, we have established that the H. bulbosum DNA containing the powdery mildew resistance gene had been introgressed into 81882/83 and is located on chromosome 2 (2I). Resistant plants have been backcrossed to barley to remove the adverse effects of a linked factor conditioning triploid seed formation, but there remains an association between powdery mildew resistance and non-pathogenic necrotic leaf blotching. The dominant resistance gene is allelic to a gene transferred from H. bulbosum by co-workers in Germany, but non-allelic to all other known powdery mildew resistance genes in barley. We propose Mlhb as a gene symbol for this resistance.     

Effects of fungal infection and wounding on the expression of chitinases and β-1,3 glucanases in near-isogenic lines of barley

Chitinases (EC 3.2.1.14) and β -1.3 glucanases (EC 3.2.1.39) have been known to play a vital role in the defense of plants against fungal pathogens. The pattern of induction of these two enzymes subsequent to infection by powdery mildew was studied in 10 pairs of near-isogenic lines of barley ( Hordeum vulgare L.) which possess powdery mildew resistance genes. These isogenic lines have been grotiped according to their reaction to the fungus. The induction patterns varied between the resistant and the susceptible cultivars within each group and between different groups. More tsozymcs were induced in susceptible varieties of highly resistant groups and the overall levels and the number of isozymes of chitinases and β -1.3 glucanases were lower in groups with low resistance. The effect of powdery mildew infection and mechanical wounding on the cellular localization of chitinases and β -1.3 glucanases in barley leaves has also been studied. The 31 kDa leaf chitinase, L-CH2, and trace amounts of a 25 kDa chitinase. L-CH3. were present in healthy leaves. Wounding increased the levels of L-CH3 within I ft h. Powdery mildew infection increased the levels of L-CH3 both in intercellular fluid and in intracellular extract of leaves. A /3-I.3 glucanase. GH, also increased after infection and wounding. In infected barley leaves, GL-1 was present both in intercellular space and intracellular extract. It is concluded that powdery mildew resistance genes exhibit qualitative and quantitative differences in the expression of chitinases and β -1.3 glucanases. Further, chitinases and β -1.3 glucanases appear to be a response to active infection rather than the factors responsible for disease resistance.     

Accumulation of stress-proteins in intercellular spaces of barley leaves induced by biotic and abiotic factors

Accumulation of the extracellular proteins localized in intercellular spaces of barley primary leaves was examined after inoculation with powdery mildew (Erysiphe graminis f. sp. hordei) as biotic stress factor and after abiotic stresses such as heat shock, low temperature and heavy metal (Mg,Zn, Cu, Al, Cd and Co) treatment using native polyacrylamide gel electrophoresis. Six to eight major pathogen-induced proteins (bands on native gel) have been identified. Their accumulation at host-parasite incompatibility was more expressive than at compatibility interaction. Elevated temperature did not induce pathogenesis-related (PR) proteins while low temperature induced three of them. Cu, Al, Cd and Co induced accumulation pattern of extracellular proteins was very similar to that in powdery mildew inoculated leaves. Mg and Zn had no effect on the induction of protein accumulation in the intercellular spaces of leaves. Induction of PR proteins by different stresses indicated their general function in the resistance of plants to changing environment. This revised version was published online in July 2006 with corrections to the Cover Date.     

A pattern recognition tool for quantitative analysis of in planta hyphal growth of powdery mildew fungi

The development of fungal pathogens can be quantified easily at the level of spore germination or penetration. However, the exact quantification of hyphal growth rates after initial, successful host invasion is much more difficult. Here, we report on the development of a new pattern recognition software (HyphArea) for automated quantitative analysis of hyphal growth rates of powdery mildew fungi on plant surfaces that usually represent highly irregular and noisy image backgrounds. By using HyphArea, we measured growth rates of colonies of the barley powdery mildew, Blumeria graminis f. sp. hordei, on susceptible and induced-resistant host plants. Hyphal growth was not influenced by the resistance state of the plants up to 48 h postinoculation. At later time points, growth rate increased on susceptible plants, whereas it remained restricted on induced-resistant plants. This difference in hyphal growth rate was accompanied by lack of secondary haustoria formation on induced-resistant plants, suggesting that induced resistance in barley against Blumeria graminis is caused mainly by reduced penetration rates of primary as well as secondary appressoria leading, finally, to fewer and less-developed fungal colonies. No evidence was found for reduced nutrient-uptake efficiency of the primary haustoria in induced-resistant leaves, which would be expected to have resulted in reduced hyphal growth rates during the first 48 h of the interaction.     

Effects of soil water level on the development of adult plant resistance to powdery mildew in barley

Barley grown in dry soil developed greater adult plant resistance (APR) to powdery mildew (Erysiphe graminis DC. f. sp. hordei Mérat) than barley grown in wet soil. Conidial germination and appressorium formation were less, and fungal development between formation of appressoria and elongating secondary hyphae on upper leaves was inhibited, when adult plants were grown in dry soil. Mildew colonies expanded more slowly on leaves of adult plants than on leaves of seedlings, especially if adult plants had grown in dry soil. APR was reduced if plants, previously grown in dry soil, were well watered more than 32 h before inoculation. Conidia originating from plants grown in dry soil had a lower solute potential and greater ability to infect plants grown in dry but not wet soil than conidia originating from plants grown in wet soil. APR could not be attributed simply to increased cell wall or cuticle thickness, nor to lowered leaf solute potentials, as has sometimes been suggested for powdery mildew diseases. Increasing plant age and water stress induced increases in cell wall and cuticle thickness, but these changes did not always coincide with changes in disease resistance. Increasing plant age and water stress also lowered leaf solute potentials but fungal solute potentials were lower than leaf solute potentials and, more importantly, were lower than leaf water potentials. Thus, fungal growth was not limited by the availability of water from the host during penetration and hyphal establishment. It is suggested that resistance levels may be determined not by the thickness of epidermal structures, nor by lowering of solute potential per se, but by specific substances harmful to the fungus which accumulate in either cell wall, cuticle or sap, and whose concentration is dependent on the age and water stress of leaves.     

Acquired resistance functions in mlo barley,which is hypersusceptible to Magnaporthe grisea

Barley plants carrying a mutation in the Mlo (barley [Hordeum vulgare L.] cultivar Ingrid) locus conferring a durable resistance against powdery mildew are hypersusceptible to the rice blast fungus Magnaporthe grisea. It has been speculated that a functional Mlo gene is required for the expression of basic pathogen resistance and that the loss of Mlo function mediating powdery mildew resistance is an exception for this particular disease. Here, we report that the onset of acquired resistance (AR) after chemical as well as biological treatments is sufficient to overcome the hypersusceptible phenotype of backcross line BCIngridmlo5 (mlo) barley plants against M. grisea. Moreover, even barley plants bearing a functional Mlo gene and thus showing a moderate infection phenotype against rice blast exhibit a further enhanced resistance after induction of AR. Cytological investigations reveal that acquired resistance in mlo genotypes is manifested by the restoration of the ability to form an effective papilla at sites of attempted penetration, similarly to wild-type Mlo plants. In addition, the rate of effective papillae formation in Mlo plants was further enhanced after the onset of AR. These results demonstrate that treatments leading to the AR state in barley function independently of the Mlo/mlo phenotype and suggest that the Mlo protein is not a component of the AR signaling network. Moreover, it seems that only concomitant action of Mlo together with AR permits high level resistance in barley against blast. Higher steady state levels of PR1 and barley chemically induced mRNA correlate with higher disease severity rather than with the degree of resistance observed in this particular interaction.     

Influence of primary callus induction conditions on the establishment of barley cell suspensions yielding regenerable protoplasts

Summary With the aim of the development of a culture method for efficient plant regeneration from barley (Hordeum vulgare L.) protoplasts, we examined several culture conditions for primary calli from immature embryos of cvs. Dissa and Igri, which were used for initiation of cell suspensions. Among the primary callus culture conditions tested, growth condition of donor plants had a great impact on these efficiencies; Igri protoplasts derived from embryos of plants grown in a greenhouse gave rise to albino plants and few green shoots while several cell lines originating from embryos of plants grown in a growth chamber (16h light, 12°C) yielded protoplasts developing into green plants. In contrast, cell suspensions were produced at higher frequencies from calli derived from embryos of greenhouse-grown Dissa plants. In Igri, increased levels of 2,4-dichlorophenoxyaceticacid (2,4-D) significantly reduced the efficiency of cell suspension establishment and plant regeneration from protoplasts was achieved only with suspension cells derived from calli induced at the lowest level (2.5 mg/l), while the effect of the 2,4-D concentration was not clear in Dissa. The developmental stage of immature embryos also affected the efficiency of cell suspension establishment, and the optimal embryo size was determined to be approximately 1mm in diameter. These results demonstrate the importance of callus induction conditions for successful barley protoplast culture.     

Endogenous Elicitor Present in Barley Seeds

Endogenous elicitor(s) present in barley seeds induce the accumulation of antifungal substances effective against powdery mildew fungi. Antifungal substances induced by the treatment with barley seeds extract coincides with the one induced by the inoculation with compatible or incompatible races of powdery mildew fungi on thin-layer chromatography analysis. The resistance induced by the treatment with barley seeds extract correlates to the accumulation of the antifungal substances in barley leaves. Furthermore, the activity of phenylalanine ammonia-lyase was also induced by the treatment with barley seeds extract.     

Molecular marker analyses of powdery mildew resistance in barley

Powdery mildew, caused byEryisphe graminis f. sp.hordei, is one of the most important diseases of barley (Hordeum vulgare). A number of loci conditioning resistance to this disease have been reported previously. The objective of this study was to use molecular markers to identify chromosomal regions containing genes for powdery mildew resistance and to estimate the resistance effect of each locus. A set of 28 F₁ hybrids and eight parental lines from a barley diallel study was inoculated with each of five isolates ofE. graminis. The parents were surveyed for restriction fragment length polymorphisms (RFLPs) at 84 marker loci that cover about 1100 cM of the barley genome. The RFLP genotypes of the F₁s were deduced from those of the parents. A total of 27 loci, distributed on six of the seven barley chromosomes, detected significant resistance effects to at least one of the five isolates. Almost all the chromosomal regions previously reported to carry genes for powdery mildew resistance were detected, plus the possible existence of 1 additional locus on chromosome 7. The analysis indicated that additive genetic effects are the most important component in conditioning powdery mildew resistance. However, there is also a considerable amount of dominance effects at most loci, and even overdominance is likely to be present at a number of loci. These results suggest that quantitative differences are likely to exist among alleles even at loci which are considered to carry major genes for resistance, and minor effects may be prevalent in cultivars that are not known to carry major genes for resistance.     

兼抗抗白粉病和黄矮病小麦育种材料的创造与鉴定

白粉病和黄矮病是小麦生产上的重要病害,近几年来这两种病害经常在我国一些小麦产区同时发生。为解决该问题,本研究通过杂交、回交方法将抗黄矮病的Bdv2基因（源自于YW642）和抗白粉病的Pm21基因（源自于CB037）聚合在一起,育成了兼抗黄矮病和白粉病的小麦新材料。通过田间抗病性鉴定与分子标记辅助选择相结合,得到聚合了Bdv2基因和Pm21基因的BC1代小麦22株,F2代小麦51株。农艺性状调查显示,这些含Pm21和Bdv2基因的双抗白粉病和黄矮病小麦新材料的农艺性状优于感病植株和原先的亲本,可以在小麦白粉病和黄矮病兼性抗病育种中作为优异种质资源加以利用。     

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.     

Infestation of transgenic powdery mildew-resistant wheat by naturally occurring insect herbivores under different environmental conditions

A concern associated with the growing of genetically modified (GM) crops is that they could adversely affect non-target organisms. We assessed the impact of several transgenic powdery mildew-resistant spring wheat lines on insect herbivores. The GM lines carried either the Pm3b gene from hexaploid wheat, which confers race-specific resistance to powdery mildew, or the less specific anti-fungal barley seed chitinase and β-1,3-glucanase. In addition to the non-transformed control lines, several conventional spring wheat varieties and barley and triticale were included for comparison. During two consecutive growing seasons, powdery mildew infection and the abundance of and damage by naturally occurring herbivores were estimated under semi-field conditions in a convertible glasshouse and in the field. Mildew was reduced on the Pm3b-transgenic lines but not on the chitinase/glucanase-expressing lines. Abundance of aphids was negatively correlated with powdery mildew in the convertible glasshouse, with Pm3b wheat plants hosting significantly more aphids than their mildew-susceptible controls. In contrast, aphid densities did not differ between GM plants and their non-transformed controls in the field, probably because of low mildew and aphid pressure at this location. Likewise, the GM wheat lines did not affect the abundance of or damage by the herbivores Oulema melanopus (L.) and Chlorops pumilionis Bjerk. Although a previous study has revealed that some of the GM wheat lines show pleiotropic effects under field conditions, their effect on herbivorous insects appears to be low.     

Suppression by Red Light Irradiation of Corynespora Leaf Spot of Cucumber Caused by Corynespora cassiicola

The effect of red light irradiation on development of Corynespora leaf spot of cucumber plants (Cucumis sativus L. cv. Hokushin) caused by Corynespora cassiicola (Berk. & Court.) was investigated in greenhouses. In a greenhouse without red light (?Red), lesions enlarged, coalesced, and finally covered the entire leaves of cucumber. In a greenhouse with red light (+Red), however, lesion appearance was delayed relative to that under ?Red and its development was also significantly suppressed. Such difference in disease development was also observed in cucumbers grown under +Red and ?Red in the same greenhouse. Disease suppression under red light was also observed in glasshouse‐grown C. cassiicola‐inoculated cucumbers. Red light did not inhibit the infection behaviour of the pathogen. Our results indicated that the delay and suppression of Corynespora leaf spot of cucumber under +Red were due to induction of resistance in cucumber, and not to differences in environmental conditions or fungal population between the two greenhouses. Red light‐induced resistance might contribute to the development of new methods for controlling Corynespora leaf spot of greenhouse‐grown cucumber.     

Characterisation of powdery mildew resistance in a segregating diploid rose population

Powdery mildew (Podosphaera pannoso) is one of the most serious fungal diseases on both greenhouse and field grown roses. Improvement of disease resistance is a major selection aim for garden rose breeders. For rose cultivars, being mostly tetraptoid, it is complicated to develop molecular markers for resistance. Hence, a segregating diploid population was established from a cross between 'Yesterday', a commercial available rose variety susceptible to powdery mildew, and R. wichurana, a rose species with resistance to certain isolates of powdery mildew. A progeny of 94 seedlings was planted in the field. The segregation of powdery mildew resistance was studied in this population by means of a bioassay with two different monoconidial isolates of powdery mildew. Based on the response to these inoculations different groups were selected: a first group of genotypes was susceptible to both isolates, other groups were susceptible to one of both isolates and a last group was resistant to both tested isolates. The disease resistance inherits for both isolates in a quantitative way. A genetic map based on AFLP and SSR markers was established and will be used for QTL analysis of powdery mildew resistance.     

Effect of climate change on infection of grapevine by downy and powdery mildew under controlled environment

Plant responses to elevated CO2 and temperature have been much studied in recent years, but effects of climate change on pathological responses are largerly unknown. The pathosystems grapevine (Vitis vinifera) - downy mildew (Plasmopara viticola) and powdery mildew (Erysiphe necatrix) were chosen as models to assess the potential impact of increased CO2 and temperature on disease incidence and severity under controlled environment. Grapevine potted plants were grown in phytotrons under 4 different simulated climatic conditions: (1) standard temperature (ranging from 18 degrees to 22 degrees C) and standard CO2 concentration (450 ppm); (2) standard temperature and elevated CO2 concentration (800 ppm); (3) elevated temperature (ranging from 22 degrees to 26 degrees C, 4 degrees C higher than standard) and standard CO2 concentration; (4) elevated temperature and CO2 concentration. Each plant was inoculated with a spore suspension containing 5x10(5) cfu/ml. Disease index and physiological parameters (chlorophyll content, fluorescence, assimilation rate) were assessed. Results showed an increase of the chlorophyll content with higher temperatures and CO2 concentration, to which consequently corresponded an higher fluorescence index. Disease incidence of downy mildew increased when both CO2 and temperatures were higher, while an increase in CO2 did not influenced powdery mildew incidence, probably due to the increased photosynthetic activity of plants under such conditions. Considering that the rising concentrations of CO2 and other greenhouse gases will lead to an increase in global temperature and longer seasons, we can assume that this will allow more time for pathogens evolution and could increase pathogen survival, indirectly affecting downy and powdery mildews of grapevine.     

An Arabidopsis mutant with enhanced resistance to powdery mildew.

We have identified an Arabidopsis mutant that displays enhanced disease resistance to the fungus Erysiphe cichoracearum, causal agent of powdery mildew. The edr1 mutant does not constitutively express the pathogenesis-related genes PR-1, BGL2, or PR-5 and thus differs from previously described disease-resistant mutants of Arabidopsis. E. cichoracearum conidia (asexual spores) germinated normally and formed extensive hyphae on edr1 plants, indicating that the initial stages of infection were not inhibited. Production of conidiophores on edr1 plants, however, was <16% of that observed on wild-type Arabidopsis. Reduction in sporulation correlated with a more rapid induction of defense responses. Autofluorescent compounds and callose accumulated in edr1 leaves 3 days after inoculation with E. cichoracearum, and dead mesophyll cells accumulated in edr1 leaves starting 5 days after inoculation. Macroscopic patches of dead cells appeared 6 days after inoculation. This resistance phenotype is similar to that conferred by "late-acting" powdery mildew resistance genes of wheat and barley. The edr1 mutation is recessive and maps to chromosome 1 between molecular markers ATEAT1 and NCC1. We speculate that the edr1 mutation derepresses multiple defense responses, making them more easily induced by virulent pathogens.     

Simultaneous modification of three homoeologs of TaEDR1 by genome editing enhances powdery mildew resistance in wheat

Wheat (Triticum aestivum L.) incurs significant yield losses from powdery mildew, a major fungal disease caused by Blumeria graminis f. sp. tritici (Bgt). enhanced disease resistance1 (EDR1) plays a negative role in the defense response against powdery mildew in Arabidopsis thaliana; however, the edr1 mutant does not show constitutively activated defense responses. This makes EDR1 an ideal target for approaches using new genome‐editing tools to improve resistance to powdery mildew. We cloned TaEDR1 from hexaploid wheat and found high similarity among the three homoeologs of EDR1. Knock‐down of TaEDR1 by virus‐induced gene silencing or RNA interference enhanced resistance to powdery mildew, indicating that TaEDR1 negatively regulates powdery mildew resistance in wheat. We used CRISPR/Cas9 technology to generate Taedr1 wheat plants by simultaneous modification of the three homoeologs of wheat EDR1. No off‐target mutations were detected in the Taedr1 mutant plants. The Taedr1 plants were resistant to powdery mildew and did not show mildew‐induced cell death. Our study represents the successful generation of a potentially valuable trait using genome‐editing technology in wheat and provides germplasm for disease resistance breeding.