Environmental factors influencing the infection of wheat by Puccinia graminis

Germination rate and total germination of Puccinia graminis uredospores were directly related to pustule age and duration between spore collections. Partial drying of the spores enhanced germination rate; keeping them for 18 h at 100% r.h. reduced both rate and total germination. Spores germinated in polystyrene dishes between 4 and 29 °C and optimally between 15 and 23 °C Light (3 cal/cm²/h) had little effect on germination on moist surfaces but inhibited germination on the leaf. In Hybrid 229/8 wheat this effect was more pronounced than in var. Little Club. The number of primary infections increased linearly with duration of surface wetness with a narrow temperature optimum at 23.5 °C. Two phases of infection could be distinguished: germination (requiring darkness and capable of taking place over a wide temperature range) and penetration (requiring light and slightly higher temperature than for germination). Stomatal closure caused by subjecting the plants to water stress led to proporational reductions in infection. The results are discussed in relation to dew formation.     

The ultrastructure of the uredospore of Puccinia recondita

Dry, fresh uredospores ofPuccinia recondita that have been killed by infra-red radiation showed no striking ultrastructural differences from dry, fresh, viable uredospores. Numerous spherical and elipsoidal mitochondria with distinct and deep cristae, ribosomes, endoplasmic reticulum and convoluted plasmalemma were well shown by both.When moistened and incubated, killed uredospores lost ultrastructural organization, whereas moistened, viable, fresh uredospores imbibed moisture, became more spherical and germination commenced. A more or less centrally located nucleus was seen with the double membrane invaginated at some points. The advance of the germ tube was initially by enzymic degradation and concluded by mechanical disruption of the degraded pore plug. The mitochondria and the endoplasmic reticulum increased in number and the stored lipid bodies were gradually depleted as germination progressed.Features known as the foamy cytoplasm and folded membranes were seen in the germinating uredospores only. It was suggested that the foamy cytoplasm could be functionally similar to the glyoxisome because of the close association of the former to lipid bodies. The folded membranes may be accumulated endoplasmic reticulum being transported to the site of wall formation.Abbreviations used in electronmicrographs CW Cell wall - er Endoplasmic reticulum - f folded membranes - GP Germ pore - L Lipid bodies - M Mitochondria - P Plasmalemma - Pm foamy cytoplasm - R Ribosomes - T Germ tube     

The ultrastructure of the uredospore of Puccinia recondita

Dry, fresh uredospores ofPuccinia recondita that have been killed by infra-red radiation showed no striking ultrastructural differences from dry, fresh, viable uredospores. Numerous spherical and elipsoidal mitochondria with distinct and deep cristae, ribosomes, endoplasmic reticulum and convoluted plasmalemma were well shown by both. When moistened and incubated, killed uredospores lost ultrastructural organization, whereas moistened, viable, fresh uredospores imbibed moisture, became more spherical and germination commenced. A more or less centrally located nucleus was seen with the double membrane invaginated at some points. The advance of the germ tube was initially by enzymic degradation and concluded by mechanical disruption of the degraded pore plug. The mitochondria and the endoplasmic reticulum increased in number and the stored lipid bodies were gradually depleted as germination progressed. Features known as the ‘foamy cytoplasm’ and ‘folded membranes’ were seen in the germinating uredospores only. It was suggested that the ‘foamy cytoplasm’ could be functionally similar to the glyoxisome because of the close association of the former to lipid bodies. The ‘folded membranes’ may be accumulated endoplasmic reticulum being transported to the site of wall formation.     

Effect of Ophiobolus graminis infection on the growth of wheat and barley

Glasshouse experiments are reported, in which the development of young wheat and barley plants was examined following inoculation with Ophiobolus graminis (Sacc.) Sacc. The dry weight, leaf area, tiller number and water content of the shoot were reduced by infection. Reductions were equally severe in wheat and barley. The seminal root system of both was severely attacked and its growth retarded. Inoculated plants, however, translocated a greater proportion of their total assimilates to the root system and produced more adventitious roots than healthy plants. As a result there was an increase in both the number and proportion of healthy roots on these plants following the initial infection of their root systems. This effect was more pronounced in barley than in wheat. It is suggested that this may in part account for the reported relative tolerance of barley to take-all attack under field conditions.     

The relationship between hypersensitive tissue and resistance in wheat seedlings infected with Puccinia graminis tritici

Data obtained during the first 120 hr. of the infection process in several resistant and susceptible varieties of wheat infected with Puccinia graminis tritici did not show any consistent relationship between the growth of the rust colony and the rate of increase of host tissue showing hypersensitive necrosis. Furthermore, no apparent relationship existed between the area of the rust colony at any given time after inoculation, and the amount of hypersensitive necrotic tissue per unit area of the leaf colonized. Thus, it is suggested that hypersensitive necrosis of the host tissue is a consequence and not the cause of resistance of wheat to infection by P. graminis tritici.     

Host and environmental effects on the penetration of oats by Puccinia graminis avenae and Puccinia coronata avenae

The frequency of penetration from appressoria of Puccinia graminis avenae and P. coronata avenae varied among Avena species and between oat cultivars, although both rusts produced susceptible infection type pustules on the cultivars tested. Penetration on cv. Garry was significantly less than that on the Avena species (A. barbata, A.fatua and A. sterilis) studied and penetration of these Avena species was significantly less than on the cvs Algerian and Fulmark. When the rusts were allowed to develop into pustules on seedlings which had been inoculated with fixed amounts of inoculum, there was a direct relationship between number of pustules produced and penetration frequency. The effects of temperature, light and dew period on penetration from appressoria of ‘single race’ and ‘mixed race’ inocula was also studied on these cultivars and species. Penetration by P. graminis avenae was greatest at 30–35 °C and at light intensities of 5625 lux and above, whereas that by P. coronata avenae was greatest at 20 °C and was unaffected by artificial light intensities up to n 250 lux. Maximal penetration by P. graminis avenae and P. coronata avenae was observed after inoculated plants had been exposed to dew periods of 16 and 12 h respectively. Some penetration was observed after a dew period of 8 h. The time taken for each rust to attain maximum penetration varied from 36 to 52 h after inoculation, depending on the environment, and was usually less for P. coronata avenae than for P. graminis avenae.     

Population divergence in the wheat leaf rust fungus Puccinia triticina is correlated with wheat evolution

Co-evolution of fungal pathogens with their host species during the domestication of modern crop varieties has likely affected the current genetic divergence of pathogen populations. The objective of this study was to determine if the evolutionary history of the obligate rust pathogen on wheat, Puccinia triticina, is correlated with adaptation to hosts with different ploidy levels. Sequence data from 15 loci with different levels of polymorphism were generated. Phylogenetic analyses (parsimony, Bayesian, maximum likelihood) showed the clear initial divergence of P. triticina isolates collected from Aegilops speltoides (the likely B genome donor of modern wheat) in Israel from the other isolates that were collected from tetraploid (AB genomes) durum wheat and hexaploid (ABD genomes) common wheat. Coalescence-based genealogy samplers also indicated that P. triticina on A. speltoides, diverged initially, followed by P. triticina isolates from durum wheat in Ethiopia and then by isolates from common wheat. Isolates of P. triticina found worldwide on cultivated durum wheat were the most recently coalesced and formed a clade nested within the isolates from common wheat. By a relative time scale, the divergence of P. triticinia as delimited by host specificity appears very recent. Significant reciprocal gene flow between isolates from common wheat and isolates from durum wheat that are found worldwide was detected, in addition to gene flow from isolates on common wheat to isolates on durum wheat in Ethiopia.     

Interactions of Puccinia hordei and Erysiphe graminis on seedling barley

The development of Puccinia hordei on the first leaf of barley seedlings previously inoculated with Erysiphe graminis was compared with that on uninoculated leaves of comparable age. On cv. Zephyr, more rust pustules developed when leaves were inoculated with both fungi within 24 h but fewer pustules if the period between the two inoculations was longer than 2 days. The reduction in numbers of rust pustules was especially marked where leaves were previously inoculated with many conidia of E. graminis. The size of rust pustules was reduced whatever the period between the two inoculations. Arresting mildew development by applying ethirimol as a soil drench to pots of seedlings inoculated with E. graminis 6 days previously, or floating segments of leaves inoculated with both fungi on 2% sucrose, in part counteracted these effects on rust pustule size. Similar effects were observed with cv. Mazurka where inoculations with E. graminis produced only small necrotic flecks but did induce premature loss of chlorophyll. On this cultivar (in contrast to Zephyr) the inoculation of one leaf surface affected the development of P. hordei on the other. In comparable experiments using Zephyr, E. graminis produced smaller colonies with fewer conidiophores on leaves previously inoculated with P. hordei. These effects could be alleviated by arresting rust development with a spray containing benodanil or by floating segments of leaves inoculated with both fungi on 2% sucrose. Germination of the conidia of E. graminis, formation of appressoria and initiation of colonies were not affected by the presence of P. hordei.     

Performance and survival of different races of Puccinia graminis f.sp. tritici on detached leaves of wheat

The detached leaf culture of Puccinia graminis f.sp. tritici was tested with eight races and two biotypes of the pathogen. The inherent characteristics of the races were similar to those in intact plants. Continuous maintenance of races in pure culture on detached leaves showed the usefulness of the technique for multiplication of inoculum as well as for maintenance of races in other basic studies.     

Effects of changes in environmental schedules on the penetration of leaves and epidermal strips of wheat seedlings by Puccinia graminis tritici

The frequency with which intact leaves and epidermal leaf strips of wheat seedlings were penetrated by Puccinia graminis tritici, strain 21 Anz 2, was observed following exposure of inoculated material to various regimes of illumination/darkness and temperature. Epidermal strips, at 24°C, were penetrated most frequently when continuous light (540 ft-c) was commenced at either 4 or 8,20 or 24, or 36 or 40 h after inoculation or when a 4 h period of light was applied at these times. With both treatments, the curve obtained when the length of the dark period preceding illumination was plotted against the frequency of penetration showed a series of alternate peaks and troughs and the periodicity of the curves was significant at the 1 % probability level. In the former curve the best form of regression was linear modified by a cosine function, while in the latter the linear term proved to be non-significant. When dark periods of different lengths were applied, penetration was more variable on intact leaves than on epidermal strips. Penetration of intact leaves was influenced by the light intensity to which the seedlings were exposed after the dark treatment. The time of day when intact leaves were inoculated influenced penetration when they were grown under controlled conditions. Greatest penetration of the variety Little Club occurred when leaves were inoculated at 1.30 a.m. The time of day when epidermal strips were inoculated did not influence penetration. The inhibitory effect of continuous light on penetration of epidermal strips was overcome by changing the temperature from 18·5 to 24°C. Greatest penetration was obtained when the change was made 24 or 28 h after inoculation.     

Effect of bacterial polysaccharides on the growth ofGaeumannomyces graminis var.tritici and wheat roots

Agrobacterium sp. and related species which in the soil and in the rhizosphere of wheat accompany the fungus Gaemannomyces graminis var. tritici and cause take-all of the wheat roots produced polysaccharides in pure cultures (glucans, mannoglucans and galactomannoglucans). These polysaccharides were utilized better by the mycelium of G. graminis than glucose and polysaccharides of plant origin that occurred on the surface of wheat roots (the so-called mucigel). At lower concentrations these bacterial polysaccharides stimulated growth of wheat roots, higher concentrations (more than 0.1%) were inhibitory. Bacteria inoculated on the surface of wheat first inhibited and then stimulated the development of the plants and their growth. Changes in the growth rate of wheat, the rhizosphere of which was colonized by bacteria simultaneously with the fungus G. graminis and also some changes in the course of the disease of wheat roots caused by the fungus can be explained by the inhibitory or stimulatory effect of polysaccharides of accompanying bacteria.     

Effect of temperature, light and host on prepenetration development of Puccinia graminis avenae and Puccinia coronata avenae

The influence of temperature and light on prepenetration development of single and mixed isolates of Puccinia graminis avenae and Puccinia coronata avenae was studied on 0–2% water agar and on leaves of three oat cultivars and on three non-cultivated species of Avena. Germination of uredospores of P. graminis avenae and P. coronata avenae occurred best at 10–30^oC and at 20^oC respectively. The optimum temperature for germ-tube growth and for appressorial formation was 20^oC for both rusts. An inverse relationship was observed between light intensity and prepenetration development with maximal germination of uredospores, germ-tube growth and appressorial formation occurring in darkness. Under optimum conditions maximum percentage germination and appressorium formation of both rusts was attained within 4 and 12 h after inoculation respectively. The proportion of germinated uredospores of crown rust which gave rise to appressoria was about twice that observed for stem rust. No significant differences were observed in prepenetration development between the single and mixed race inocula of the two rusts. Although germination of uredospores was significantly greater on water agar than on oat leaves, there were no significant differences in prepenetration development of the rusts on the various oat cultivars and species examined. Consequently, the data failed to indicate the presence of resistance mechanisms operating during the prepenetration phase of the infection process on the cultivars and species examined.     

Analysis of the wheat and Puccinia triticina (leaf rust) proteomes during a susceptible host-pathogen interaction

Wheat leaf rust is caused by the fungus Puccinia triticina. The genetics of resistance follows the gene-for-gene hypothesis, and thus the presence or absence of a single host resistance gene renders a plant resistant or susceptible to a leaf rust race bearing the corresponding avirulence gene. To investigate some of the changes in the proteomes of both host and pathogen during disease development, a susceptible line of wheat infected with a virulent race of leaf rust were compared to mock-inoculated wheat using 2-DE (with IEF pH 4-8) and MS. Up-regulated protein spots were excised and analyzed by MALDI-QqTOF MS/MS, followed by cross-species protein identification. Where possible MS/MS spectra were matched to homologous proteins in the NCBI database or to fungal ESTs encoding putative proteins. Searching was done using the MASCOT search engine. Remaining unmatched spectra were then sequenced de novo and queried against the NCBInr database using the BLAST and MS BLAST tools. A total of 32 consistently up-regulated proteins were examined from the gels representing the 9-day post-infection proteome in susceptible plants. Of these 7 are host proteins, 22 are fungal proteins of known or hypothetical function and 3 are unknown proteins of putative fungal origin.