Pathogenicity to Wheat of Isolates of Pseudocercosporella herpotrichoides (Fron) Deighton from Five Graminaceous Hosts

Eleven isolates of Pseudocercosporella herpotrichoides were obtained from field material of the Gramineae Agropyron repens, Alopecurus myosuroides, Apera spica-venti, Hordeum vulgare and Triticum aestivum. Four of these isolates belonged to the variety acuformis and 7 isolates to the variety herpotrichoides of the fungus. In a greenhouse experiment, winter wheat was inoculated with these isolates. All isolates were pathogenic on wheat. They differed in virulence, but these differences could not be related to the nature of their original hosts nor to their classification as variety acuformis or herpotrichoides.     

Dispersal of Pseudocercosporella herpotrichoides Spores from Infected Wheat Straw

Evidence from spore samples collected amongst infected straw spread on fallow ground supported the conclusion that spores of Pseudocercosporella herpotrichoides are dispersed mostly by rainsplash. Most spores travelled a short distance in the larger ballistic splash droplets, although some may have travelled further in smaller airborne droplets. Weekly spore counts from microscope slides under rainshields, a funnel and an impinger, evaluated as samplers for spores of P. herpotrichoides, showed a similar seasonal pattern. The funnel, as the largest sampler, generally collected most spores, but the impinger collected more spores per unit area of sampling surface. Slides sometimes collected spores when none was recovered from other samplers. Young wheat plants, exposed with the samplers and changed weekly, subsequently developed eyespot symptoms for most of the season.     

Production and Regeneration of Protoplasts from Pseudocercosporella herpotrichoides (Fron) Deighton

This paper describes the development of a system for protoplast isolation from this pathogen using the commercially available, hydrolytic enzyme preparations Rhozyme HP150, Driselase and Cellulase CP. An analysis of the active components of these enzymes believed to be involved in protoplast release has been made. Factors affecting protoplast release including mycelial age, enzyme concentration and choice of osmotic stabilizer have been optimised, and conditions for high frequency protoplast regener, ation have been determined. The possible significance of the interaction of the several components of each enzyme is discussed. The developments described in this paper can now be exploited in genetic studies in this imperfect fungus.     

Detection of Pseudocercosporella herpotrichoides (Fron) Deighton in Wheat by Indirect ELISA

An indirect ELISA for quantitative detection of P. herpotrichoides in fections in wheat is presented. All tested isolates of the virulent varieties P. herpotrichoides var. herpotrichoides, P. h. var. acuformis or the W- and R-type react on a high level in the test, while the less virulent P. anguioides is assessed only with 40% and the avirulent P. aestiva with 20% of the homologous reaction. No crossreactions occur with extracts of 11 other species of in, vitro cultivated fungi nor with plant material infected with other pathogens. The infection profile throughout the leaf-sheaths was clearly reflected by ELISA. The examination of 24 stembase samples from the field showed that the values assessed by ELISA correlate well also with the disease indices of naturally infected plant material.     

Recovery of Pseudocercosporella herpotrichoides Spores from Rain-Splash Samples

Methods for recovery of P. herpotrichoides spores from rain-splash were tested, initially with pure spore suspensions and then with field samples. Provided surfactant was added and samples were processed immediately, centrifugation gave good recovery or spores from field samples which contained few soil particles. Foam-flotation, glycerol and sucrose methods for separation of spores from soil particles were not satisfactory for P. herpotrichoides spores. A paraffin oil method proved to be the most reliable because it gave good recovery of P. herpotrichoides spores from field samples containing many soil particles.     

Development of Pseudocercosporella herpotrichoides (Fron) Deighton var. herpotrichoides and var. acuformis on Wheat Plants Measured by ELISA

Using the ELISA method, the development of Pseudocercosporella herpotrtchoides var. herpotrichoides and var. acuformis in a susceptible cultivar of winter wheat was compared under controlled and held conditions. In the greenhouse, var. acufornis grew less vigorously, was slower in penetrating the coleoptile and the successive leaf sheaths and in colonizing the stem tissue than var. herpotrichoides. In the field, these differences were confirmed on the last leaf sheaths and the stem. At ripening stage, however, identical ELISA values were measured for both varieties. Moreover, a significant variation was observed between the individual isolates of each variety. Comparison of the effect of both varieties of P. herpotrichoides on 20 wheat cultivars characterized by different resistance levels showed significant interactions. The cultivars carrying the Pch-1 gene always remained the less diseased genotypes. In general, var. acuformis developed less antigen in the cultivars than var. herpotrichoides. It is concluded, that in tests for resistance to P. herpotrichoides mixtures of many highly pathogenic isolates of both fungus varieties should be used. Less complex mixtures or single isolates may result in wrong estimates of resistance.     

Development of Eyespot and Brown Foot Rot in Wheat Plants Inoculated with Mixtures of Pseudocercosporella herpotrichoides Types and Fusarium Species

The effects of Fusarium avenaceum, F. culmorum and Microdochium nivale on eyespot development and of types of the eyespot fungus Pseudocercosporella herpotrichoides on brown foot rot caused by the Fusarium or Microdochium spp. were investigated by sequential inoculation of wheat plants grown in pots in a controlled environment. The W-type of the eyespot fungus induced more severe disease than the R-type, but its symptons were suppressed to a greater extent by Fusarium spp., especially F. avenaceum. Brown foot rot symptoms were sometimes suppressed by P. herpotrichoides but were occasionally more severe when the Fusarium inoculum was applied after the P. herpotrichoides. There, sults are discussed in relation to observations of natural infections.     

Expression of the Escherichia coli beta-glucuronidase gene in Pseudocercosporella herpotrichoides.

The plant-pathogenic fungus Pseudocercosporella herpotrichoides has been successfully transformed by using two different positive selection systems in combination with the Escherichia coli gusA gene. The selectable markers used in this study were the hygromycin B phosphotransferase gene (hph) from E. coli and the gene (bml) for beta-tubulin from a benomyl-resistant mutant of Neurospora crassa. A lower transformation rate was obtained with the bml system than with the hph system. Conversely, cotransformation frequencies, as determined with medium plates containing the chromogenic substrate 5-bromo-4-chloro-3-indolyl-beta-D-glucuronic acid, were higher with bml than with hph as the selectable marker. The hygromycin-resistant transformants were mitotically stable, and both the selectable gene and gusA were maintained through conidiation. The vector DNA was integrated into the genome, and the number and sites of insertion varied among transformants. Enzyme assays of mycelial extracts showed that beta-glucuronidase activity was highest in transformants with a high gusA copy number. Expression of gusA during growth of the fungus on plants was easily detectable and did not affect pathogenicity. These results form the basis for construction of a versatile and sensitive reporter gene system for P. herpotrichoides.     

Expression of the Escherichia coli beta-glucuronidase gene in Pseudocercosporella herpotrichoides.

The plant-pathogenic fungus Pseudocercosporella herpotrichoides has been successfully transformed by using two different positive selection systems in combination with the Escherichia coli gusA gene. The selectable markers used in this study were the hygromycin B phosphotransferase gene (hph) from E. coli and the gene (bml) for beta-tubulin from a benomyl-resistant mutant of Neurospora crassa. A lower transformation rate was obtained with the bml system than with the hph system. Conversely, cotransformation frequencies, as determined with medium plates containing the chromogenic substrate 5-bromo-4-chloro-3-indolyl-beta-D-glucuronic acid, were higher with bml than with hph as the selectable marker. The hygromycin-resistant transformants were mitotically stable, and both the selectable gene and gusA were maintained through conidiation. The vector DNA was integrated into the genome, and the number and sites of insertion varied among transformants. Enzyme assays of mycelial extracts showed that beta-glucuronidase activity was highest in transformants with a high gusA copy number. Expression of gusA during growth of the fungus on plants was easily detectable and did not affect pathogenicity. These results form the basis for construction of a versatile and sensitive reporter gene system for P. herpotrichoides.     

Prediction of Eyespot Severity on Winter Wheat or Winter Barley Inoculated with W-type or R-type Isolates of Pseudocercosporella herpotrichoides

In crops of winter wheat (1986—88) or winter barley (1987—88) inoculated with W-type or R-type isolates of Pseudocercosporella herpotrichoides and sown on different dates (1986) or at different seed rates (1987,1988) eyespot epidemics developed in different ways. Methods of measuring eyespot incidence/severity during crop growth were compared for their ability to predict eyespot severity at grain filling. Regressions were calculated for eyespot severity score at GS 71 on earlier measurements, either at GS 30/31 (11 methods) or from GS 22 to GS 65 (3 methods). Based on measurements at GS 30/31, all the methods predicted eyespot severity at GS 71 well in plots of winter barley inoculated with W-type isolates (r, 0.83—0.97) but the accuracy of predictions in plots inoculated with R-type isolates was very variable (r, 0.09—0.71). Predictions for 1987 and 1988 were less accurate in wheat than in W-type plots of barley, but did not differ between W-type and R-type plots (r, 0.70—0.89). When the wheat data for 1986 were also included predictions were less accurate, especially in R-type plots (r, 0—0.59). Generally, it was easier to predict eyespot severity at GS 71 in W-type than in R-type plots, especially in barley and in wheat before GS 37/39. Predictions of eyespot severity at GS 71 based on measurements before GS 25 were inaccurate for both wheat and barley. After GS 25 the accuracy of the prediction was generally good in W-type plots and did not improve greatly except in wheat after GS 59. However, there was a steady improvement in the accuracy of the prediction in R-type plots of barley from GS 24 to GS 53. Assessments of eyespot incidence on stems predicted eyespot severity at GS 71 more accurately than assessments on leaf sheaths on wheat after GS 37/39, but were not as good on barley until GS 53.     

Untersuchungen zur Bildung extrazellulärer Hemicellulasen durch Pseudocercosporella herpotrichoides in vitro

Investigations on the production of extracellular hemicellulases by Pseudocercosporella herpotrichoides in vitro For all 15 isolates of Pseudocercosporella herpotrichoides investigated, xylanase as well as arabanase activity could be demonstrated. After cultivation of 3 weeks, the activity of the enzymes reached a peak. The activity of xylanase was considerably increased by addition of xylan in comparison to Maltzin as the sole source of carbohydrate. Also the arabanase activity could be increased significantly by addition of araban or xylan as compared to the Maltzin variant. The optimum temperature with regard to activity and stability of xylanase ranged at 50°C. The pH-optimum for xylanase activity was found to be at pH 5.0, and the enzyme was stable in ° range between pH4.0 and 8.0 (9.0). In case of arabanase, the temperature optimum varied between 40 and 50°C; up to this temperature, the enzyme was also stable. At pH 5.0, the arabanase activity reached its optimum; stability was observed in - pH range between 4.0 and 9.0. In extracts prepared from autoclaved wheat coleoptiles which were inoculated with Pseudocercosporella herpotrichoides, the presence of the enzymes xylanase, arabanase, cellulase and polymethylgalacturonase could be demonstrated. The enzyme activities of the inoculated samples were considerably higher than those of non-inoculated controls. The differences, in most cases, were statistically significant. Der Deutschen Forchungsgemeinschaft danken wir für finanzielle Unterstützung.     

Investigations on the Activity of Cell Wall-degrading Enzymes in Young Wheat Plants After Infection with Pseudocercosporella herpotrichoides (From) Deighton

In earlier investigations, it has been demonstrated that Pseudocercosporella herpotrichoides (Fron) Deighton is capable of producing pectolytic and cellulolytic enzymes as well as hemicellulases in vitro. The investigation of enzyme activity in extracts from wheat plants infected with P. herpotrichoides (isolates 21e and R6) and from non-infected plants revealed the activity of the following enzymes: pectin methylesterase (PME), polymethylgalacturonase (PMG), pectin lyase (PL), carboxymethylcellulase (CMCase), xylanase and arabanase. Compared to non-infected plants, the enzyme activity in infected plants was considerably higher; in some experiments, only traces of enzyme activity could be found in control plants. The difference in the enzyme activity in infected as compared to non-infected plants was, in most cases, statistically significant, especially beginning at the end of the second week after inoculation.
The enzyme activity depended on the temperature during plant cultivation; with the exception of pectin methylesterase (PME), the activity of all investigated enzymes increased with temperature and the highest activity was found in plants grown at 20°C. The highest PME activity was measured in plants grown at 10°C; the activity of this enzyme was generally lower at 15 and 20°C.