Quantitative Differences in Powdery Mildew Resistance Among Spring Barley Cultivars

Quantitative powdery mildew resistance in compatible host-pathogen-combinations was measured by the number of pastules/cm² leaf area. Spring barley cultivar ‘Proctor’ was significantly less infected than ‘Golden Promise”. Using these two cultivars (having no effective major resistance gene) as controls, MO- and AR-resistant cultivars were inoculated with virulent mildew isolates. ‘Mona”, ‘Grit’ and ‘Nudinka’ had a higher or, at least, the same level of quantitative resistance as ‘Proctor”. None of the remaining cultivars showed the high susceptibility expressed by ‘Golden Promise”. Ranking of host genotypes was nearly constant while that of mildew isolates varied considerably. Only a small portion of the observed variance was due to interaction between host cultivars and pathogen isolates. ‘Triesdorfer Diva’ gave a resistant infection type after inoculation with different AR-virulent isolates, indicating that this cultivar has major resistance other than that conditioned by gene Ml-a12.     

Induction of resistance mechanisms in barley by yeast-derived elicitors

Yeast cell-wall extracts (YE) had high phytoalexin elicitor activity on soybeans and induced resistance against barley powdery mildew in the normally highly susceptible cv. Golden Promise. Following treatment with YE there was rapid stimulation of phenylalanine ammonia-lyase (PAL) activity and faster formation of papillae in response to attempted fungal penetration. The spray adjuvants Agral and LI-700 each had poor phytoalexin elicitor activity and caused a slight enhancement of papilla formation and a low level of mildew control. The addition of the adjuvant to YE did not affect phytoalexin elicitation on soybeans but significantly enhanced the speed and the extent of PAL activity, papilla formation and mildew control. It is proposed that this is due to greater coverage and improved persistence enabling increased uptake of YE into the barley leaves.     

Selection for Aggressiveness in Erysiphe graminis f. sp. Hordei Towards Partial Resistance in Barley

Isolates of powdery mildew were subjected to recurrent selection on partially resistant barley cultivars for 76 inoculation cycles over three years. Previous intermediate tests on up to 32 cycles for adaptation to overcome the host resistance revealed a trend towards adaptation to the cv. 7204 recurrent host in infection frequency data but not towards cv. Golden Promise or 9319, Subsequent cycles demonstrated adaptation to these latter hosts also.     

Genetic Adaptation of Erysiphc graminis f. sp. Hordei to Barley with Partial Resistance

Ten Isolates of powdery mildew were tested on three spring barley cultivars known to express partial resistance and on Golden Promise. Measurements of colony lengths were made under several different environments and compared with similar data produced from several cultivars containing one of two major resistance genes. Significant isolate, cultivar and environment interactions were recorded and one isolate in particular showed adaptation to a partially resistant cultivar. Measurements of infection frequency were also made under two temperature regimes. Again significant isolate, cuitivar and temperature interactions were recorded and the same isolate showed adaptation to the same partially resistant cultivar. This indicates that adaptation to a source of partial resistance can be found amongst unselected isolates and that pleiotropically adapted genotypes may rapidly overcome such resistance in the field following its widespread deployment. Other genes for resistance were detected in the major gene cultivars, including partial resistance in the ml-v genotypes.     

Germination of Erysiphe gratninis f.sp. hordei conidia on barley leaves

Germination of Erysiphe graminis f.sp. hordei conidia on leaves of several barley cultivars was studied in the laboratory. On both detached leaves and intact plants, within 48 h of inoculation a higher proportion of conidia had germinated on the basal and middle portions of the adaxial leaf surface than on the corresponding portions of the abaxial surface. Such differences between surfaces were not observed near the leaf tip. Similar results were obtained with all the cultivars and growth stages tested, and with five isolates of E. graminis, and are consistent with the observation that there is usually less powdery mildew on the abaxial than the adaxial surface of barley leaves. With most of the barley genotype/mildew isolate combinations tested, within 48 h of inoculation higher proportions of conidia germinated on seedlings and juvenile plants than on older plants. Inherited characteristics which affect spore germination on the leaf surface may be important factors in the development of adult-plant resistance of barley to powdery mildew, particularly in certain genotypes.     

Gramine increase associated with rapid and transient systemic resistance in barley seedlings induced by mechanical and biological stresses.

Systemic acquired resistance (SAR) is one of the intriguing issues for studying the mechanism in signal transduction system in a whole plant. We found that SAR and increase of an antifungal compound were induced rapidly and transiently in barley (Hordeum vulgare L. cv. Goseshikoku) by mechanical and biological stresses. One of the major antifungal compounds was identified as an indole alkaloid, gramine (N,N-dimethyl-3-aminomethylindole), by mass spectrum and NMR analyses. Gramine is well known as a constitutive compound of barley, but it increased significantly in the primary and secondary leaves of barley seedlings within 12 h after pruning or inoculating with the powdery mildew fungi of barley (Blumeria graminis f.sp. hordei) and wheat (B. graminis f.sp. tritici). However, in the leaf detached from unwounded seedlings or in the leaf inoculated with the barley powdery mildew fungus, gramine did not increase at all. In the water droplets contacted with barley leaves, the amount of leaked gramine increased dependently upon the time after the seedling was injured mechanically. We also found a tight correlation between gramine increase and enhancement of resistance to the barley powdery mildew fungus in barley leaves treated with an endogenous elicitor. Furthermore, such a systemic resistance was not observed in a barley cultivar Morex that lacks the biosynthetic pathway of gramine. From these results, we conclude that gramine is the excellent marker in rapid and transient systemic acquired resistance in barley.     

Correlation of gibberellin-induced growth, polyamine levels and amine oxidases in epicotyl, root and leaf blade of barley seedlings

The potential role of diamine oxidase (DAO) and polyamine oxidase (PAO) in relation to polyamines was investigated in epicotyls, roots and leaf blades at 3 and 6 days after gibberellic acid (GA) application in barley (Hordeum vulgare L.) seedlings of cvs. Maythorpe (non-mutant parent) and Golden Promise (semi-dwarf mutant). There was a significant increase in epicotyl and leaf-blade elongation rates in GA-treated seedlings of cv. Maythorpe as compared to cv. Golden Promise. DAO and PAO were detectable in all segments of the leaf blade, but the highest activities were present in basal segments. These enzymes, which are thought to have a role in the elimination of cellular polyamines, increased in activity following GA application compared to controls. Application of 10⁻⁶ M GA to the first leaf, significantly increased endogenous bound putrescine (Put) levels in both the epicotyl and leaf blade of cv. Maythorpe. In contrast, there was only a slight increase in cv. Golden Promise. Levels of soluble Put increased in roots and leaf blades of both cultivars following GA treatment but the effect was greatest in leaves of cv. Maythorpe. It is suggested that polyamines may play a role in GA-induced epicotyl and leaf-blade elongation in barley.     

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.     

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.     

Components of adult plant resistance to powdery mildew (Erysiphe graminis f. sp. avenae) in oats

The adult plant reaction to mildew (Erysiphe graminis f. sp. avenae) of four oat genotypes was studied. A negative relationship existed in these genotypes between length of latent period and the percentage leaf area with mildew. The very susceptible CI 4877 had the shortest latent period and the largest area of leaf infected, while the highly resistant Cc 4761 had the longest latent period and the least leaf area covered with mildew. The same association also occurred between leaves within a genotype, particularly those genotypes with adult plant resistance, i.e., Maldwyn and Cc 4761. The more recently developed upper leaves showed the longest incubation period and least percentage area affected compared with lower leaves. A visual score of pustule sporulation indicated that CI 4877, Milford and Maldwyn had similar well-sporulating foci, but in Cc 4761 the pustules showed significantly less sporulation. Estimates of the relative number of spores produced per day over a 15 day period reflected the level of resistance. Thus the most susceptible genotype CI 4877 released over 50 times more spores than Cc 4761, while Milford and Maldwyn respectively produced 9 and 4.5 times more spores than the highly resistant Cc 4761. The longer latent period of Cc 4761 was reflected in the delay this genotype showed in reaching its relatively low peak production of spores. The possible use of the latent period as a factor when selecting for adult plant resistance in early growth stages is discussed.     

Forecasting barley mildew development in West Scotland

Observations on plots of spring barley cv. Golden Promise during May-July 1973–79 showed that escalation of mildew commenced in mid-June 1976, in late June in 1973 and 1974, in early July in 1978 and 1979, and failed to develop in 1975 and 1977. By mid-July there was 40% infection in 1976, and 13–21% infection in 1973, 1974, 1978 and 1979. Less than 2% mildew occurred in 1975 and 1977. Using as a basis the method of mildew forecasting formulated by Polley & King (1973), it was found that once mildew was established in the crop there was moderately good association between the incidence of high mildew risk according to the Polley weather criteria and the major peaks of spore concentration above the crops. It was not possible to correlate these two features during May and early June each year because of a lack of inoculum, but it is considered that the failure of mildew to develop during the entire growing seasons of 1975 and 1977, in spite of frequent high risk periods, was due in part to low atmospheric humidity during the early part of the season. The relative number of accumulated Polley factors satisfied on days when the relative humidity approx. 1 m above ground was 78% or more (at 0900 GMT) during the period mid-May to mid-June, separated the low mildew years of 1975 and 1977 from the moderate ones of 1973, 1974, 1978 and 1979, and the severe mildew year of 1976. This procedure may provide a practical means of forecasting the level of mildew build-up during the later growth stages of the barley crop.     

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.     

The influence of photoperiod on growth patterns of Erysiphe graminis f. sp. hordei

Detached seedling leaf segments of five spring barley genotypes were inoculated with an isolate of barley mildew to which they possessed different levels of resistance. Segments of each host genotype were then incubated under either continuous light or treatments with 2, 4, 8 or 16 h darkness per 24 h cycle. Macroscopic observation showed that the latent period of infection was reduced slightly in treatments with at least 4 h darkness/24 h. Yellowing of detached segments occurred fastest under continuous light and slowest under a 16 h dark/8 h light cycle. Microscope observation of segments fixed 4·5 days after inoculation showed that as the length of the dark period increased, so the number of haustoria formed per colony also increased. This increase in haustorial production appeared to be associated with an accentuation in the synchrony of production of the secondary and tertiary haustorial generations. Varietal differences in susceptibility were also more marked in segments incubated under short days. It is concluded that under the conditions of temperature and light intensity used, a 16 h dark/8 h light system is most desirable for quantifying mildew resistance because it allows confident identification of distinct haustorial generations, accentuates differences in varietal susceptibility and delays chlorophyll degradation in detached barley leaves infected with mildew.     

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.     

Transformation of different barley (<Emphasis Type="Italic">Hordeum vulgare</Emphasis> L.) cultivars by <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis> infection of in vitro cultured ovules

Most cultivars of higher plants display poor regeneration capacity of explants due to yet unknown genotypic determined mechanisms. This implies that technologies such as transformation often are restricted to model cultivars with good tissue characteristics. In the present paper, we add further evidence to our previous hypothesis that regeneration from young barley embryos derived from in vitro-cultured ovules is genotype independent. We investigated the ovule culture ability of four cultivars Femina, Salome, Corniche and Alexis, known to have poor response in other types of tissue culture, and compared that to the data for the model cultivar, Golden Promise. Subsequently, we analyzed the transformation efficiencies of the four cultivars using the protocol for Agrobacterium infection of ovules, previously developed for Golden Promise. Agrobacterium tumefaciens strain AGL0, carrying the binary vector pVec8-GFP harboring a hygromycin resistance gene and the green fluorescence protein (GFP) gene, was used for transformation. The results strongly indicate that the tissue culture response level in ovule culture is genotype independent. However, we did observe differences between cultivars with respect to frequencies of GFP-expressing embryos and frequencies of regeneration from the GFP-expressing embryos under hygromycin selection. The final frequencies of transformed plants per ovule were lower for the four cultivars than that for Golden Promise but the differences were not statistically significant. We conclude that ovule culture transformation can be used successfully to transform cultivars other than Golden Promise. Similar to that observed for Golden Promise, the ovule culture technique allows for the rapid and direct generation of high quality transgenic plants.     

IV. Variation in fungicide tolerance: Comparisons between isolates of Erysiphe graminis f. sp. hordei tolerant and sensitive to the fungicide tridemorph

Forty-five single pustule isolates of barley powdery mildew taken at random from tridemorph treated and untreated plants were maintained in culture on leaf segments of barley cv. Golden Promise placed on agar containing benzimidazole. In repeated assays for tridemorph tolerance over a 10 month period, tolerance was found in 25 of 36 isolates from treated plants, and remained stable in all but four of the isolates wherein tolerance was lost after varying lengths of time. A negative correlation was found between pathogenicity and tolerance. The germination of tolerant isolates was less depressed than the sensitive on treated seedlings, but no differences between isolates were found on untreated seedlings. Colony growth of all isolates was slower on treated than untreated seedlings. On plants of several cultivars kept in growth rooms, mildew progressed more rapidly on treated barley when tolerant isolates were used as inoculum. Visible disease symptoms spread most rapidly on isolated, untreated plants, and most slowly on isolated treated plants, irrespective of the isolate. Disease on treated plants progressed more rapidly when untreated plants were adjacent, suggesting that interactions take place between pathogen populations on treated and untreated plants.     

Screening for Barley Yellow Dwarf Luteovirus Resistance in Barley on the Basis of Virus Movement

The movement of barley yellow dwarf luteovirus (BYDV) was evaluated in susceptible and resistant barley and bread wheat genotypes. After leaf inoculation, the virus infected the root system and the growing point of susceptible earlier than resistant, barley genotypes. No difference in virus movement occurred in resistant and susceptible wheat genotypes. It was possible to reliably differentiate susceptible from resistant genotypes when root extracts of 41 barley genotypes were tested by DAS-ELISA 3 or 4 days after inoculation at the oneleaf stage. When barley plants inoculated at the two- or three-leaf stage were assayed by tissue-blot ELISA on nitrocellulose membrane, virus was detected in the phloem vessels of the growing points of the susceptible, but not of the resistant genotype, 4–6 days after inoculation. Our results thus suggest that screening for BYDV resistance in barley could be done quickly and cheaply especially when assays are made by the tissue-blot test.     

The mildew resistance of Hordeum bulbosum and its transference into H. vulgare genotypes

Mildew from susceptible genotypes (SI and S2) of Hordeum bulbosum was found to be avirulent on all H. vulgare genotypes tested, including such cultivars as Proctor with no known genes for resistance to mildew. The H. bulbosum genotype SI (2n =14) proved resistant to all isolates of mildew from H. vulgare. The mildew isolates used for these tests possessed most of the common virulence factors which attack the current ‘vulgare’ cultivars in Western Europe. Some H. bulbosum genotypes were resistant to the ‘bulbosum’ mildew. Attempts at combining resistance from both species are presented and discussed.     

Dependence of the Effectiveness of Induced Resistance on Environmental Conditions

Resistance induced by microbial metabolites against powdery mildew on barley always proved to be highly effective particularly under practical conditions in field. The results obtained in the present study demonstrate that the successful induction of resistance requires a conditioning of plants by the environmental conditions acting prior to the application of the inducer. If plants were grown in the greenhouse or outdoors, the infection densities were efficiently reduced after the treatment with the inducer. In contrast to this the disease intensities were not reduced on plants grown at constant temperature, light and humidity conditions. Frequently changing temperatures and the daily courses of temperature, normally occurring in greenhouse or outdoors, were substantiated as the most important factors for a successful induction of resistance. The conditioning of the plants under these temperature conditions depended on light energy, especially in just developing leaves. The dependence of the induced resistance on the environmental conditions was proved with other host-pathogen-relationships as well as with barley and powdery mildew.