Polyamines in discrete regions of barley leaves infected with the powdery mildew fungus, Erysiphe graminis

The concentrations of putrescine, spermidine and spermine and the activities of arginine decarboxylase (ADC; EC 4.1.1.19) and ornithine decarboxylase (ODC: EC 4.1.1.17) were determined in discrete regions of barley leaves ( Hordeum vulgare L. cv. Golden Promise) infected with the powdery mildew fungus ( Erysiphe graminis f.sp. hordei Marchal). Polyamine concentrations and the activities of both enzymes were always greatest within the region surrounding the fungal pustule, with the lowest values always being found in the region furthest away from the pustule. Although the concentrations of the three amines and ADC and ODC activities within the fungal pustule were always less than values from the zone surrounding the pustule, these differences were never significant. Polyamine concentrations and ODC activity were not significantly reduced, and ADC activity remained unchanged in mildewed leaves with all surface fungal growth removed. It would appear therefore that not only does most of the increase in amines and ODC activity reside in the leaf itself, but that very little of this increase is due to fungal growth and sporulation. Furthermore, it seems possible that the increase in polyamines in mildewed barley could be involved in 'green-island' formation, where regions around mildew pustules remain green and physiologically active while the rest of the leaf senesces.     

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.     

Metabolic regulation in diseased leaves. I. The respiratory rise in barley leaves infected with powdery mildew

Photosynthetic and respiratory activities have been measured in leaves of Hordeum vulgare L. var. Manchuria (barley) after infection with Erysiphe graminis var. hordei (powdery mildew). Two isogenic lines, one resistant to infection and the other highly susceptible, were examined.

These isogenic lines showed very different physiological responses following infection. Photosynthesis and the chlorophyll content of resistant leaves was unaffected by infection. Respiration increased slightly and this was accompanied by small increases in activities of enzymes of glycolysis, the pentose-P pathway and the tricarboxylic acid cycle.

The infection of susceptible leaves resulted in a slight increase in photosynthesis 48 hours after inoculation, but subsequently there was a progressive decrease in the photosynthesis of these leaves compared with that of noninfected leaves. The capacity of infected leaves for partial reactions of photosynthesis such as the Hill reaction and the photoreduction of nicotinamide adenine dinucleotide phosphate (NADP¹) decreased during the later stages of infection. The levels of chlorophyll, NADPH-diaphorase and aldolase also declined. There was no detectable difference in the respiration of infected and noninfected leaves until 48 hours after inoculation. After this time, the infected leaves showed a higher respiration, the maximum difference occurring about 144 hours after inoculation. The respiratory increase was not accompanied by significant changes in the levels of enzymes of glycolysis and the tricarboxylic acid cycle with the exception of malate dehydrogenase which was lower in infected leaves. In contrast, the activities of glucose-6-P dehydrogenase and 6-P-gluconate dehydrogenase showed changes similar to that observed for respiration.

The respiration and the activities of glucose-6-P dehydrogenase and 6-P-gluconate dehydrogenase did not increase in infected leaves of etiolated plants, even when excellent growth of the fungus was established by growing the plants in White's basal medium supplemented with sucrose. The respiration of a susceptible mutant barley (the yellow-green virescent mutant of the variety Himalaya) when grown in the light at 11° was not changed by infection although the characteristic respiratory rise occurred in plants grown at 15°. At the lower temperature chloroplasts fail to develop in this mutant, although development is normal at 15°.

It is suggested that the pathogen is not directly responsible for the increase in respiration in green leaves, rather that this is a response in the host cells to a loss of photosynthetic capacity.

     

Competitive ability and ethirimol sensitivity in strains of barley powdery mildew

Strains within field populations of barley powdery mildew (Erysiphe graminis f. sp. hordei) varied greatly in their response to ethirimol. Each strain remained stable whether the fungicide was present or not, and no evidence for adaptation was obtained. Strains of intermediate sensitivity were the most frequent within the pathogen population, and these also dominated model populations maintained in the laboratory. Ethirimol eliminated sensitive strains from laboratory mixtures, and increased the relative fitness of insensitive ones, but not sufficiently to oust the intermediate strains. Mildew from treated field plots was less sensitive than that from untreated plots, but only early in the epidemic. Insensitivity was not related to the level of ethirimol used and, at four times the rate used commercially, insensitive strains were no more frequent than at lower rates. As the complexity of mildew populations increased, changes in ethirimol sensitivity in response to selection became less pronounced, and it is suggested that strains of intermediate sensitivity to ethirimol exert a stabilising effect within natural populations. This could alter if the fitness of insensitive strains were to increase, perhaps through recombination. Consideration should be given to the effect ethirimol might have on the composition of the pathogen population if applied when sexual recombination occurs, and to the role ascospores play in disease development.     

The influence of the transpiration rate on uptake and transport of potassium ions in barley plants

Summary The vegetation points of branches of Caralluma frerei (Asclepiadaceae) were treated with 300, 100 and 30 ppm of crude aflatoxin (36% B₁, 38% G₁, 3% B₂, 2% G₂) and with toxin-free control. Application of 300 and 100 ppm aflatoxin resulted in stop of growth and death of the upper leaves and flower buds. Malformations or wilting was not observed in any case. Branches treated with 30 ppm aflatoxin and with control solution developed normally. It is concluced that under the experimental conditions used aflatoxin has an unspecific toxic effect.     

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     

Single-cell transcript profiling of barley attacked by the powdery mildew fungus

In many plant-pathogen interactions, there are several possible outcomes for simultaneous attacks on the same leaf. For instance, an attack by the powdery mildew fungus on one barley leaf epidermal cell may succeed in infection and formation of a functional haustorium, whereas a neighboring cell attacked at the same time may resist fungal penetration. To date, the mixed cellular responses seen even in susceptible host leaves have made it difficult to relate induced changes in gene expression to resistance or susceptibility in bulk leaf samples. By microextraction of cell-specific mRNA and subsequent cDNA array analysis, we have successfully obtained separate gene expression profiles for specific mildew-resistant and -infected barley cells. Thus, for the first time, it is possible to identify genes that are specifically regulated in infected cells and, presumably, involved in fungal establishment. Further, although much is understood about the genetic basis of effective papilla resistance associated with mutant mlo barley, we provide here the first evidence for gene regulation associated with effective papilla-based nonspecific resistance expressed in nominally "susceptible" wild-type barley.     

Olfactory cues from plants infected by powdery mildew guide foraging by a mycophagous ladybird beetle

Powdery mildews (Erysiphales) are economically important plant pathogens that attack many agricultural crops. Conventional management strategies involving fungicide application face challenges, including the evolution of resistance and concerns over impacts on non-target organisms, that call for investigation of more sustainable alternatives. Mycophagous ladybird beetles (Coleoptera: Coccinellidae) feed on powdery mildew and have considerable potential as biological control agents; however, the foraging ecology and behavior of these beetles is not well understood. Here we document the olfactory cues presented by squash plants (Cucurbita moschata) infected by powdery mildew (Podosphaera sp.) and the behavioral responses of twenty-spotted ladybird beetles (Psyllobora vigintimaculata) to these cues. Volatile analyses through gas chromatography revealed a number of volatile compounds characteristic of infected plants, including 3-octanol and its analogues 1-octen-3-ol and 3-octanone. These compounds are typical "moldy" odorants previously reported in volatiles collected from other fungi. In addition, infected plants exhibited elevated emissions of several compounds also observed in collections from healthy leaves, including linalool and benzyl alcohol, which are reported to have anti-fungal properties. In Y-tube choice assays, P. vigintimaculata beetles displayed a significant preference for the odors of infected plants compared to those of healthy plants. Moreover, beetles exhibited strong attraction to one individual compound, 1-octen-3-ol, which was the most abundant of the characteristic fungal compounds identified. These results enhance our understanding of the olfactory cues that guide foraging by mycophagous insects and may facilitate the development of integrated disease-management strategies informed by an understanding of underlying ecological mechanisms.     

Field resistance of spring barley cultivars to powdery mildew in Lithuania

During vegetative period 2004–2005 powdery mildew (Erysiphe graminis DC. f. sp. hordei Em. Marchal) field resistance of spring barley cultivars was investigated at the Lithuanian Institute of Agriculture. The spring barley genotypes tested were Lithuania-registered cultivars, cultivars from genetic resources collection, and the new cultivars used for initial breeding. In total, 23 resistance genes were present in the 84 cultivars studied. Among mono-genes only mlo and 1-B-53 showed very high resistance. Slight powdery mildew necroses (up to 3 scores) formed on cultivars possessing these genes. The maximal powdery mildew (PM) severity reached a score of 8.5 and the area under disease progress curve (AUDPC) a value of 1216.8. The cultivars ‘Primus’, ‘Astoria’, ‘Power’, ‘Harrington’ and ‘Scarlett’ were the most resistant among the non mlo cultivars. Severity of PM on ‘Primus’ reached a score of 3.5 (3.0 of PM necrosis) in average, the other cultivars were diseased from 4.5 (3.0) to 5.0 (2.0). The AUDPC values for these cultivars except ‘Scarlett’ were the lowest (85.0–145.3) among the other cultivars. The highest contrast in development of the other leaf diseases was between highly resistant and susceptible to PM cultivar groups. The fast development of PM depressed development of the other diseases 4.7 times.     

Resveratrol stimulates phenolic metabolism and PSII efficiency in wheat infected with powdery mildew

The beneficial effects of trans-resveratrol on pathogen-response and its physiological mechanisms were studied in two wheat cultivars differing in the level of resistance against powdery mildew (Blumeria graminis (DC.) Speer). Resveratrol induced phenolics metabolism in a manner that lead to increased phenylalanine ammonia-lyase (PAL) and β-D-glucosidase activity in leaf regions showing no symptoms of pathogen infection. In less-resistant cultivar, similar alterations were additionally observed in regions invaded by the pathogen. Resveratrol also stimulated photosynthetic efficiency during pathogenesis and this effect was more prominent in the resistant cultivar. Regulatory function of resveratrol on wheat growth and development was also observed (stimulation of generative induction). High performance liquid chromatography (HPLC) analysis detected a low amount of compound with retention time corresponding to trans-resveratrol in healthy leaves and increased the content of this compound in infected foliage. The multidirectional properties of resveratrol activity in wheat plants are discussed.     

Host surface properties affect prepenetration processes in the barley powdery mildew fungus

The initial contact between Blumeria graminis f.sp. hordei and its host barley (Hordeum vulgare) takes place on epicuticular waxes at the surfaces of aerial plant organs. Here, the extent to which chemical composition, crystal structure and hydrophobicity of cuticular waxes affect fungal prepenetration processes was explored. The leaf surface properties of barley eceriferum (cer) wax mutants were characterized in detail. Barley leaves and artificial surfaces were used to investigate the early events of fungal infection. Even after epicuticular waxes had been stripped away, cer mutant leaf surfaces did not affect fungal prepenetration properties. Removal of total leaf cuticular waxes, however, resulted in a 20% reduction in conidial germination and differentiation. Two major components of barley leaf wax, hexacosanol and hexacosanal, differed considerably in their ability to effectively trigger conidial differentiation on glass surfaces. While hexacosanol, attaining a maximum hydrophobicity with contact angles of no more than 80 degrees, proved to be noninductive, hexacosanal significantly stimulated differentiation in c. 50% of B. graminis conidia, but only at contact angles > 80 degrees. These results, together with an observed inductive effect of highly hydrophobic, wax-free artificial surfaces, provide new insights into the interplay of physical and chemical surface cues involved in triggering prepenetration processes in B. graminis.     

Relationship between transpiration and respiration in plants during the dark period

Energetic aspects of the relation between transpiration and respiration during the dark period were evaluated. One-year old seedlings of three trees, one bush and one annual plant were grown in controlled conditions. Experiments were performed under uniform environment during the day and two regimes of air relative humidity (RH) during the night, low (50 - 65 %) and high (95 %). For all investigated plant species the dark transpiration rate (E), the free energy of respiratory substrate, the entropy production and the free energy balance (FEB) of the dark respiration were higher at low than at high RH. E was linearly related to the FEBr ² ranged between 0.63 and 0.90)     

A high-throughput screening system for barley/powdery mildew interactions based on automated analysis of light micrographs

Background  

To find candidate genes that potentially influence the susceptibility or resistance of crop plants to powdery mildew fungi, an assay system based on transient-induced gene silencing (TIGS) as well as transient over-expression in single epidermal cells of barley has been developed. However, this system relies on quantitative microscopic analysis of the barley/powdery mildew interaction and will only become a high-throughput tool of phenomics upon automation of the most time-consuming steps.     

Chemical-induced resistance against powdery mildew in barley: the effects of chitosan and benzothiadiazole

Chitosan (CHT), a deacetylated chitin derivative, and benzo-(1,2,3)-thiadiazole-7-carbothioic acid S-methyl ester (BTH), a non toxic synthetic functional analogue of salicylic acid, were applied as foliar spray to barley plants (Hordeum vulgare L.), to compare their effectiveness in inducing resistance against Blumeria graminis f. sp. hordei and to investigate the underlying defence response. After an induction phase of 3 days (IP, time elapsed between treatment and fungal inoculation) both compounds reduced significantly the infection on the primary leaf, namely of 55.5% for CHT and of 68.9% for BTH, showing the induction of a good level of local resistance (LAR). A 5-day IP further reduced the infected areas in BTH treated plants (−77.2%) but not in CHT treated ones (−47.1%). Furthermore, both CHT and BTH also induced SAR, being the infection in the second non treated leaves reduced of 57% and 76.2%, respectively, as evaluated at 10-day IP. Both BTH and CHT induced oxidative burst and phenolic compound deposition in treated leaves, creating an hostile environment that slowed down the fungal spreading by impairing haustorium development. However, the greater efficacy of BTH was possibly due to: i) a greater reinforcement of papilla; ii) a higher level and the more homogeneous diffusion of H₂O₂ in the treated leaf tissues and iii) an induced hypersensitive-like response in many penetrated cells.