A comparison of cultural, morphological and DNA markers for the classification of Pseudocercosporella herpotrichoides

Isolates of Pseudocercosporella herpotrichoides were classified by a variety of established techniques including colony morphology, colour, pathogenicity on seedlings and spore morphology. In all cases, certain isolates could not be clearly classified and correlation between the systems was incomplete. In particular, the typing system of Nirenberg in which two varieties of P. herpotrichoides (var. herpotrichoides and var. acuformis ) are recognized did not correlate with the widely used UK system based on colony morphology on PDA: fast, even (F E) and slow, feathery (S/F). Restriction fragment length polymorphisms in total DNA and rDNA indicated groupings related to morphology (and pathogenicity) with considerable polymorphisms in rDNA of F/E isolates. Generally, good correlation was found between RFLP markers and other tests.     

The identification of a pathotype-specific DNA probe for the R-type of Pseudocercosporella herpotrichoides

A 6·7-kb DNA fragment has been isolated from an R-type isolate of Pseudocercosporella herpotrichoides that exhibits specific hybridization to R-type isolates and not to W, C or S pathotypes or to P. anguioides. R-type isolates are polymorphic with respect to this fragment and three sizes were present (11·6, 6·7 and 4·5 kb) in Eco RI-digested DNA from isolates of a world-wide collection. Infection of rye seedlings was demonstrated by hybridization of this probe to DNA extracted from infected plants.     

Identification of W-type and R-type isolates of Pseudocercosporella herpotrichoides

W-type isolates of Pseudocercosporella herpotrichoides grown on a maize-based agar and exposed to near- ultra-violet radiation at c . 13°C produced a greenish black colour, whilst R-type isolates produced a pink or pale brown colour in the agar medium. More colonies from directly plated lesions or from spore suspensions could be recognized as P. herpotrichoides and could be more easily differentiated as W-type or R-type or as mixtures of both by colour production on maize agar (MA) than by colony morphology on potato dextrose agar (PDA), despite the presence of other fungi. Isolates with intermediate morphology on PDA were positively identified as W-type or R-type on MA; their pathogenicities to wheat and rye seedlings were usually similar to those of W-type or R-type isolates with typical colony morphology, confirming their identification on MA. Drops of mixed suspensions of W-type and R-type spores on PDA formed fast-growing colonies with smooth margins which sometimes had slow-growing sectors with feathery margins. Drops of the same mixtures on MA formed greenish black colonies which sometimes had pink or pale brown sectors. However, when these mixtures were spread onto MA, W-type and R-type colonies could easily be differentiated by colour.     

Molecular characterization of Pseudocercosporella herpotrichoides isolates by amplification of ribosomal DNA internal transcribed spacers

In order to determine whether a given isolate of Pseudocercosporella herpotrichoides. the cause of eyespot disease of cereals, belongs to the W-type or the R-type, we have developed a test based on the polymerase chain reaction (PCR). Sequences of the internal transcribed spacer (ITS) region of the ribosomal genes were determined for three different isolates of each pathotype. Primers complementary to variable sequences were specifically chosen to amplify differentially DNA from W- and R-types. Specific reaction conditions of the PCR assay were experimentally determined by testing four isolates of each type that exhibit a wide range of sensitivity to fungicides.     

Development of a monoclonal antibody immunoassay for the eyespot pathogen Pseudocercosporella herpotrichoides

A double-antibody-sandwich ELISA test has been developed for the detection of Pseudocercosporella herpotrichoides using a highly specific monoclonal antibody PH-10 as the capture antibody and genus-specific rabbit polyclonal antiserum as the detector antibody. The assay recognizes extracts from plants both artificially and naturally infected with P. herpotrichoides giving at least three-fold higher absorbance values with extracts from Pseudocercosporella-infected tissue than with extracts from healthy tissues or from tissues naturally infected with Microdochium nivale, Rhizoctonia cerealis or material artificially inoculated with P. anguioides. The assay tested positively against all isolates of P. herpotrichoides , including both W-type and R-type isolates. In this assay system, extraction of the antigen from the stem bases of infected plants is a one-step process not requiring any dilution procedures. The assay can be used to detect the pathogen in presymptomatic infected seedlings. The immunogen used to generate the specific monoclonal antibody and the rabbit antiserum was a mycelial extract from which the high-molecular-weight proteins and glycoproteins had been removed by ammonium sulphate precipitation. The high-molecular-weight fraction was shown to contain cross-reactive antigens; it induced antiserum in mice that cross-reacted with the other stem-base fungi even at high dilutions. The monoclonal antibody PH-10 is an IgM antibody. Heat and periodate treatment of the antigen indicate that it is a glycoprotein and that the epitope recognized by the antibody is a protein.     

Dispersal of Pseudocercosporella herpotrichoides and Pyrenopeziza brassicae spores in splash droplets

Splash dispersal of conidia which differ in shape and size, those of Pseudocercosporella herpo ichoides (needle-shaped, 52×2 μm) and Pyrenopeziza hrassicae (cylindrical, 12×3 μm), was studisd by allowing 5 mm drops to fall 13 m on to suspensions (depth 0.5 mm, concentration 120, 000 spores/ml) of each. The resulting splash droplets were collected on photographic film. The pattern of spore dispersal with distance and the distribution of spore-carrying droplets and spores within droplet-size categories were similar for both fungi. Regressions of square root spore number) on droplet diameter gave lines with slopes of 0.0138 for P. herpotrichoides and 0.0167 for P. brassicae.     

Mechanism of selectivity to fenpropimorph in Fusarium genus and Pseudocercosporella herpotrichoides

Algorithms developed for use in the study of artificial intelligence also show promise in such quite different fields as the physical and life sciences. These methods may have advantages in speed, scope, or both, over more conventional scientific methods, and expand the range of algorithmic techniques open to the scientist. The mechanism of one of these methods, the genetic algorithm, which is already finding application in a number of areas in science, is outlined.     

Screening for potential antagonists of Pseudocercosporella herpotrichoides, the causal agent of eyespot disease of cereals

Bacteria isolated from wheat seedlings, plants or straw from several field sites were screened for antagonism towards the cereal eyespot pathogen Pseudocercosporella herpotrichoides on several media of differing nutrient status. Thirteen out of 348 isolates inhibited pathogen growth on low-nutrient media and several also prevented spore germination or reduced germ tube extension. These were selected for further tests on wheat seedlings inoculated with the eyespot fungus. Twelve known bacterial antagonists of other fungal plant pathogens were tested in vitro using the same methods, and the majority showed some activity towards P. herpotrichoides. Selected isolates were equally inhibitory to both W and R pathotypes of the fungus. Effects of potential antagonists on disease development were assessed by scoring lesions or by counting the number of infection plaques formed by the fungus on leaf sheaths. Two isolates of Pseudomonas fluorescens , along with a commercial strain of Streptomyces griseoviridis , showed activity both in vitro and in subsequent infection trials with plants and may therefore be of potential value as antagonists of P. herpotrichoides.     

Isozyme polymorphism in pathotypes of Pseudocercosporella herpotrichoides and related species from cereals

Isolates of Pseudocercosporella herpotrichoides , causal agent of the eyespot disease of cereals, are usually classified as W-type or R-type on the basis of differential pathogenicity towards wheat and rye hosts, a distinction often correlated with cultural morphology. Polyacrylamide gel electrophoresis (PAGE) of soluble proteins was used to compare 101 isolates of Pseudocercosporella spp. including both W-types and R-types, and the related species P. anguioides and P. aestiva . Of 16 enzyme activities detected, seven were differential for the pathotypes. Isozyme polymorphisms for five enzymes clearly differentiated W-type from R-type isolates, a result confirmed by pathogenicity tests. Within each pathotype, isozyme patterns were similar for isolates of diverse geographical origins, including Europe, North America, South Africa and New Zealand. Isolates of P. anguioides and P. aestiva could also be distinguished on the basis of isozyme patterns. The mechanisms maintaining these polymorphisms, and the value of PAGE for population studies of this pathogen, are discussed.     

Effects of wheat crop debris on the sporulation and survival of Pseudocercosporella herpotrichoides

Three separate experiments showed that wheat tissue infected with the cereal eyespot fungus, Pseudocercosporella herpotrichoides , produced fewer spores in the presence of chopped wheat straw than when it was absent. This provides a possible explanation for earlier observations that there was often less eyespot in plots where straw had been incorporated than where it had been burnt. Sporulation on eyespot infected wheat tissue was not closely correlated with the viability of P. herpotrichoides , which could usually be isolated from infected tissue for some time after it had apparently lost the ability to produce spores. Many of the colonies that were isolated from such tissue also failed to sporulate under the conditions used but non sporulating colonies were less common amongst those isolated from tissue that had been mixed with chopped straw than from tissue that had not.     

Occurrence of Tapesia yallundae, teleomorph of Pseudocercosporella herpotrichoides, on unharvested wheat culms in England

Apothecia of Tapesia yallundae are reported for the first time in the UK. They were observed on decaying stems and leaf sheaths of unharvested wheat near Bristol in January 1989. Isolates from single ascospores produced cultures typical of the W-type form of the anamorph, Pseudocercosporella herpotrichoides . Pathogenicity on wheat was confirmed by inoculation and re-isolation from glasshouse-grown wheat seedlings.     

Classification of a world-wide collection of isolates of Pseudocercosporella herpotrichoides by RFLP analysis of mitochondrial and ribosomal DNA and host range

Two profiles of mitochondrial DNA (mtDNA) were detected among 59 isolates of Pseudocercosporella herpotrichoides from diverse geographical sources. One profile (type II) was present only among isolates pathogenic on rye (R-type), and a second profile (type I) was present in W, C and S-pathotypes of the fungus. Mitochondrial DNA from P. herpotrichoides hybridized strongly to DNA of P. anguioides , but very weakly to DNA from P. aestiva , suggesting that the latter is not closely related to P. herpotrichoides. Fifteen profiles of ribosomal DNA (rDNA) were observed among isolates with type I mtDNA, and five of these were only found among seven isolates from Ireland. The other profiles present among type-I isolates appeared to be largely independent of geographic origin. Three rDNA profiles were observed among R-type (type II mtDNA) isolates.     

Negative cross-resistance of benzimidazole-resistant strains of Botrytis cinerea,Fusarium nivale and Pseudocercosporella herpotrichoides to various pesticides

Among benzimidazole-resistant strains ofFusarium nivale andPseudocercosporella herpotrichoides negative cross-resistance to N-phenylcarbamates like barban or chlorpropham was more common than to diethofencarb. Such differences were also observed with N-methylcarbamate or organophosphorus insecticides and with triazine herbicides. Several compounds belonging to these various groups of pesticides were highly toxic against the most common benzimidazole-resistant isolates ofBotrytis cinerea. They were not active, however, against isolates recently found in French vineyards that were treated with a mixture of diethofencarb and carbendazim. Some diphenylether derivatives seemed to be effective against all the benzimidazole-resistant phenotypes of this fungus.     

Quantitative studies on dispersal of Pseudocercosporella herpotrichoides spores from infected wheat straw by simulated rain

Splash dispersal ofPseudocercosporella herpotrichoides spores from infected wheat straw was investigated using simulated rainfall (rate 13.8l h^–1 m^–2, volume mean diameter 2.9 mm) and wind (2 m sec^–1) in a raintower/wind tunnel complex. Spores were deposited on the floor of the wind tunnel up to 1 m upwind and 2.5 m downwind from the centre of the straw and impacted on vertical surfaces at heights up to 36 cm above it. Fewer spores were collected with increasing distance from the straw and with increasing height. Most spore-carrying splash droplets were in the size range 400–600 m and very few were less than 200 m.Our results show that these spores are generally dispersed over short distance, which is consistent with field observations.Samenvatting Spetterverspreiding vanPseudocercosporella herpotrichoides van besmet tarwestro werd onderzocht met behulp van nagebootste regen (13,8 l uur^–1 m^–2, gemiddelde druppel diameter 2,9 mm) en wind (snelheid 2 m sec^–1) in een proefopstelling van een regentoren en een windtunnel. Sporen werden op de vloer van de windtunnel gedeponeerd tot 1 m afstand tegen de wind in, gerekend vanaf het centrum van het tarwestro, en met de wind mee tot 2,5 m afstand daarvan. Op verticale vlakken werden sporen op een hoogte van 0 tot 36 cm boven de vloer van de windtunnel opgevangen. Naarmate de afstand en de hoogte toenamen werden er minder sporen gevonden. De meeste spetters met sporen hadden een diameter van 400–600 m en slechts enkele waren kleiner dan 200 m.Onze resultaten tonen aan dat de sporen in het algemeen slechts over korte afstanden verspreid worden, hetgeen overeenkomt met veldwaarnemingen.     

Comparative study of morphological,cultural and molecular markers for the characterization ofPseudocercosporella herpotrichoides isolates

Nirenberg's classification system and the polymerase chain reaction (PCR) combined with restriction enzyme digestion of an amplified ribosomal DNA fragment, were compared for the characterization of sixty isolates ofPseudocercosporella herpotrichoides, from various geographical areas and with differing fungicide sensitivity. With Nirenberg's system, it was possible to identify most isolates asP. herpotrichoides var.herpotrichoides orP. herpotrichoides var.acuformis. However, identification was slow and sometimes inconclusive as overlap occurred between the two varieties for all criteria examined. Molecular markers identified two distinct types among the isolates tested and generally good correlation was found between the PCR-based assay and Nirenberg's system, but the molecular assay was more accurate and faster.     

Splash dispersal of Pseudocercosporella herpotrichoides spores in wheat monocrop and wheat–clover bicrop canopies from simulated rain

Simulated rain was allowed to fall onto spore suspensions of Pseudocercosporella herpotrichoides . The resulting splash droplets were collected on horizontal traps at the bottom of a canopy and at 12 cm above treatments comprising no-crop, wheat monocrop and a wheat–clover bicrop. The number of conidia collected on horizontal traps declined exponentially with distance from the inoculum source. The mean number of spores collected in the absence of any crop was twice that in a monocrop; in the monocrop it was twice that in the bicrop. Both splash droplet and spore deposition gradients were steeper in the monocrop treatment than in no-crop, and shallower in bicrop than in monocrop. Evidence is presented that suggests the clover canopy acts as a secondary source for the redistribution of previously dispersed droplets and spores.     

Survey of benomyl resistance in Pseudocercosporella herpotrichoides on winter wheat and barley in England and Wales in 1983

Surveys of 528 randomly selected commercial fields in England and Wales in 1983 showed that isolates of Pseudocercosporella herpotrichoides resistant to benomyl were common and widespread on winter wheat and winter barley. During June and July resistant strains were recovered from 16 to 66% of fields and the proportion of resistant isolates was 37–52%. The frequency of occurrence of resistant isolates was positively correlated with the number of applications of carbendazimgenerating (MBC) fungicides during the previous 8 years. Benomyl resistance was found in 21–48% of fields where it was claimed that MBC had never been used, and 7–28% of isolates from those fields, were resistant. Resistance was more frequent in R-type than in W-type isolates of the pathogen. The proportion of R-type isolates decreased as the number of previous winter wheat crops rose and increased with the number of winter barley crops grown and with the number of MBC sprays applied to crops in previous years. The implications of these findings for the control of eyespot in England and Wales are discussed.     

Field performance of Rendezvous, a wheat cultivar with resistance to eyespot (Pseudocercosporella herpotrichoides) derived from Aegilops ventricosa

Rendezvous is a new cultivar of winter wheat with potent eyespot resistance derived from Aegilops ventricosa. In 11 field experiments conducted over 6 years with both natural and artificial inoculum, Rendezvous was significantly more resistant than other commercial wheat cultivars, many of which have resistance derived from Cappelle-Desprez. It was also more resistant than its parent VPM 1, the donor of the potent eyespot resistance, suggesting that it possesses resistance genes from both Ae. ventricosa and Cappelle-Desprez.
In severe eyespot epidemics, Rendezvous lodged less and had a greater mean yield than the moderately resistant cultivar Norman. Treatment with fungicide reduced disease and increased yield in both cultivars, so that they were equal in yield. Rendezvous should not need routine fungicidal treatment for eyespot control, but under exceptional disease pressure may benefit from treatment.