Species and Mating-Type Distribution of Tapesia yallundae and T. acuformis and Occurrence of Apothecia in the U.S. Pacific Northwest

ABSTRACT Eyespot of wheat is caused by the discomycete fungi Tapesia yallundae and T. acuformis. T. yallundae is considered the most important causal agent of the disease in this region but no apothecia of either species have been found in the U.S. Pacific Northwest (PNW). Two compatible isolates of T. yallundae from the PNW were used to inoculate a field plot in the fall of 1998 and apothecia developed in the spring and fall of 2000 on standing wheat stubble. In the spring of 2000, wheat stubble from eight naturally infected fields was examined for the presence of apothecia of T. yallundae and T. acuformis. Apothecia of T. acuformis were found in two fields but no apothecia of T. yallundae were found. This is the first report of apothecia of the eyespot pathogens occurring in the PNW. Species and mating-type distribution of T. yallundae and T. acuformis in the PNW were determined from 817 isolates collected from diseased wheat over 3 years at spatial scales ranging from within fields to across states. In all, 460 isolates were identified as T. yallundae and 357 isolates were identified as T. acuformis with MAT1-1/MAT1-2 ratios not significantly different from 1:1 based on chi(2) tests at most scales tested. The apparent increase in frequency of T. acuformis from previous surveys may indicate a shift in the predominant species causing eyespot. The occurrence of apothecia under field conditions, along with the widespread distribution of mating types of both species, suggests that sexual reproduction may be occurring in both species.     

Population Genetic Structure of Tapesia acuformis in Washington State

ABSTRACT Eyespot of wheat is caused by Tapesia yallundae and T. acuformis. Historically, T. yallundae has been considered the more important causal agent of the disease in Washington state and consists of a large homogeneous population with a genetic structure consistent with both sexual and asexual reproduction. T. acuformis has increased significantly in Washington in the past 10 years and apothecia were found recently under natural field conditions, indicating that T. acuformis may have a more important role in eyespot of wheat than previously was thought. To determine the genetic structure of T. acuformis in Washington, 141 single conidial isolates were sampled from four subpopulations in the eastern wheat-growing region of the state. Isolates were scored for mating type and six amplified fragment length polymorphism markers. All markers segregated in a 1:1 ratio and were determined to be unlinked based on genetic analysis of 24 progeny from an in vitro cross. No significant differences in allele frequencies (0.127 < P < 0.809) were found among individual loci across the four subpopulations and over all loci based on contingency table analysis of the log-likelihood ratio statistic G(2). Likewise, no overall differences between subpopulations were detected using the population differentiation statistic theta (theta = -0.004, P = 0.537). Random mating could not be rejected within each subpopulation or for the combined data using clone-corrected data sets based on (i) 1:1 ratio of mating-type, (ii) multilocus gametic disequilibrium analyses (index of association), (iii) phylogenetic analyses (parsimony tree length permutation test), and (iv) genotypic diversity analyses. T. acuformis has a genetic structure similar to that of sympatric populations of T. yallundae in Washington, with both sexual and asexual reproduction contributing to the structuring of this species.     

Isolation of R-type progeny of Tapesia yallundae from apothecia on wheat stubble in England

Apothecia of Tapesia yallundae were collected from a set-aside straw stubble site in Lincolnshire in March 1993. Single ascospore isolates were obtained which produced colonies with morphologies and growth rates characteristic of the R-pathotype of Pseudocercosporella herpotrichoides. Isolates were confirmed to be R-type by the use of randomly amplified polymorphic DNA (RAPD) markers. These observations confirm that T. yallundae is the teleomorph of the R-type of P. herpotrichoides and represent the first detection of the sexual stage of the R-type in the UK.     

Observations of apothecia of Tapesia yallundae and the cultural phenotypes of their progeny

Apothecia of Tapesia yallundae, the perfect state of Pseudocercosporella herpotrichoides, were found in March 1989 on wheat and barley stubble at nine of 37 naturally infested sites and also at one inoculated site in Germany. Apothecia were found on about 1% of stems collected from the naturally infested sites and 5% of stems from the inoculated site, and were first found in the second week of March, coinciding with the onset of an early spring. Apothecia were not found on either green plants or second-year stubble. In total, 143 single-spore isolates were obtained from the apothecia and described according to a range of morphological characteristics. The majority were ascribed to P. herpotrichoides var. herpotrichoides. This is the first isolation of this variety from T. yallundae apothecia in Germany. Even single-conidial isolates from one apothecium showed much variation, some being like var. herpotrichoides, some like var. acuformis and some intermediate.     

Changes in populations of the eyespot fungi Tapesia yallundae and T. acuformis under different fungicide regimes in successive crops of winter wheat, 1984–2000

Effects of regular treatments with the fungicides carbendazim and prochloraz applied to whole plots divided into subplots with different initial population mixtures of carbendazim-sensitive or carbendazim-resistant Tapesia yallundae or T. acuformis were studied in successive crops of winter wheat from 1984/85 to 1999/2000. In unsprayed and carbendazim-sprayed whole plots, a stable coexistence of about 50% each of T. yallundae and T. acuformis developed within five seasons, but in whole plots sprayed with prochloraz or prochloraz plus carbendazim, the proportion of T. acuformis increased to > 80%. A discrete time difference equation model was derived from knowledge of the biology of eyespot and competition theory to describe the population changes. The model was fitted to the data from treatments where coexistence occurred [subplots in unsprayed (1985–92) and carbendazim-sprayed (1985–89) whole plots], using nonlinear least squares regression. The optimized value of the resource overlap coefficient was small, suggesting niche differences between the two species. Populations were nearly 100% carbendazim-resistant in carbendazim-sprayed whole plots by July 1985 (one season) and in whole plots sprayed with prochloraz plus carbendazim by July 1986 (two seasons). In prochloraz-sprayed whole plots, the proportion of carbendazim-resistant isolates decreased more rapidly than in unsprayed whole plots in the 1980s, but by July 1992 a shift in populations in unsprayed and prochloraz-sprayed whole plots towards predominantly carbendazim-resistant strains had occurred.     

Development of apothecia of the eyespot pathogen Tapesia on cereal crop stubble residue in England

A reservoir of infection of Tapesia yallundae may exist after harvest in bases of cereal stems due to the presence of apothecia capable of discharging infective ascospores. Apothecia of T. yallundae developed in a seasonal pattern on winter barley inoculated with the pathogen, with maximum numbers of apothecia produced on stubble 5–7 months after harvest. A similar pattern of development was observed on infected winter wheat. However, the peak in numbers of mature apothecia was observed 2 months later than in winter barley. Apothecia capable of discharging ascospores were present for up to 6 months on stubble. Apothecia of T. acuformis were not detected on spring or winter barley, or spring or winter wheat stubble, despite inoculation of growing crops with isolates of compatible mating type.     

Incidence of apothecia of Tapesia yallundae at setaside sites in England and sensitivity of the ascospore offspring to the fungicides benomyl and prochloraz

A total of 45 field sites in England were surveyed once for the presence of apothecia of Tapesia yallundae from 1992 to 1994. Apothecia were found at 21 locations and were mainly present on less than 3% of stems. However, apothecia were found on 1532% of stems at four sites. Analysis of the growth characteristics of ascospore isolates from seven sites showed that most produced colonies characteristic of the W-type of T. yallundae , with only one site yielding the R-type, Most ascospore isolates were resistant to the fungicide benomyl and effectively all remained sensitive to prochloraz. The results of the study are discussed in relation to the fungicidal control and epidemiology of T. yallundae , and the risk of spread of disease from set-aside sites.     

First record of Tapesia yallundae as the teleomorph of Pseudocercosporella herpotrichoides var. acuformis, and its occurrence in the field in the Federal Republic of Germany

Tapesia yallundae is reported for the first time in the Federal Republic of Germany. Apothecia were found on wheat and rye stubble at two sites in the north of the country and one in the centre, in April 1988. Isolates of Pseudocercosporella herpotrichoides var. acuformis were obtained from germinating single ascospores, identified on the basis of colony morphology, mode of conidial production and pathogenicity to wheat and rye.     

Occurrence of Tapesia yallundae apothecia on field and laboratory-inoculated material and evidence for recombination between isolates

Tapesia yallundae was observed in early July on wheat straws, which had been inoculated with isolates of Pseudocercosporella herpotrichoides (W-type) 9 months previously and placed in plots of winter wheat, to act as inoculum in a resistance screen. A perfect state was not observed on straws inoculated with R-type isolates in the same experiment. One hundred and thirty-four single-ascospore isolates were obtained and observations on morphology in culture, pathogenicity to wheat and rye seedlings, resistance to benomyl and the pattern of restriction fragment length polymorphisms demonstrated that all isolates were W-type, although some grew at half the expected rate on agar, and at least two isolates were involved in the production of apothecia. Apothecia were produced in vitro on straws inoculated with single isolates of P. herpotrichoides only when they were incubated in contact with other straws similarly inoculated with certain other isolates. No apothecia were produced on straws with only isolate present, suggesting the need for at least two strains, possibly representing different mating types, for apothecium formation. Apothecia of T. yallundae were produced in vitro on straw pieces which had been inoculated individually with W-type isolates, 14-16 weeks after straws of different isolates were mixed. Evidence for genetic recombination between isolates was obtained when single-ascopore colonies were analysed for resistance to benomyl in culture and for restriction-length polymorphisms (RFLPs).     

Development of a Specific Polymerase Chain Reaction-Based Assay for the Identification of Fusarium oxysporum f. sp. ciceris and Its Pathogenic Races 0, 1A, 5, and 6

ABSTRACT Specific primers and polymerase chain reaction (PCR) assays that identify Fusarium oxysporum f. sp. ciceris and each of the F. oxysporum f. sp. ciceris pathogenic races 0, 1A, 5, and 6 were developed. F. oxysporum f. sp. ciceris- and race-specific random amplified polymorphic DNA (RAPD) markers identified in a previous study were cloned and sequenced, and sequence characterized amplified region (SCAR) primers for specific PCR were developed. Each cloned RAPD marker was characterized by Southern hybridization analysis of Eco RI-digested genomic DNA of a subset of F. oxysporum f. sp. ciceris and nonpathogenic F. oxysporum isolates. All except two cloned RAPD markers consisted of DNA sequences that were found highly repetitive in the genome of all F. oxysporum f. sp. ciceris races. F. oxysporum f. sp. ciceris isolates representing eight reported races from a wide geographic range, nonpathogenic F. oxysporum isolates, isolates of F. oxysporum f. spp. lycopersici, melonis, niveum, phaseoli, and pisi, and isolates of 47 different Fusarium spp. were tested using the SCAR markers developed. The specific primer pairs amplified a single 1,503-bp product from all F. oxysporum f. sp. ciceris isolates; and single 900- and 1,000-bp products were selectively amplified from race 0 and race 6 isolates, respectively. The specificity of these amplifications was confirmed by hybridization analysis of the PCR products. A race 5-specific identification assay was developed using a touchdown-PCR procedure. A joint use of race 0- and race 6-specific SCAR primers in a single-PCR reaction together with a PCR assay using the race 6-specific primer pair correctly identified race 1A isolates for which no RAPD marker had been found previously. All the PCR assays described herein detected up to 0.1 ng of fungal genomic DNA. The specific SCAR primers and PCR assays developed in this study clearly identify and differentiate isolates of F. oxysporum f. sp. ciceris and of each of its pathogenic races 0, 1A, 5, and 6.     

Visual disease and PCR assessment of stem base diseases in winter wheat

Stem base disease (eyespot, sharp eyespot and brown foot rot) was assessed visually and by the polymerase chain reaction (PCR) technique on single plants sampled at four-week intervals in two crops of winter wheat grown in the UK in 1992–3. PCR assays were conducted for Fusarium avenaceum , F. culmorum , both varieties of Microdochium nivale , both eyespot-causing species of Tapesia and Rhizoctonia cerealis . PCR diagnoses were compared with visual diagnoses at each time point. Eyespot was caused principally by T. acuformis (R-type) and developed rapidly late in the season. Visual diagnoses of eyespot were largely confirmed by PCR but T. acuformis was detected in many plants lacking eyespot symptoms. R. cerealis was detected at relatively low incidences on both sites, and sharp eyespot visual diagnoses did not correlate with the incidence of any of the pathogens assayed by PCR. Brown foot rot, caused principally by Microdochium nivale var . majus , accumulated earlier in the season than eyespot. Overall, visual diagnoses of stem base disease coincided poorly with PCR data until after growth stage (GS) 30.     

Development of a PCR assay to detect Fusarium poae in wheat

Random amplified polymorphic DNA assays were carried out on a range of isolates of F. poae to identify markers common to all isolates. Two fragments were isolated, cloned and used to probe Southern blots of DNA from F. poae and isolates from a range of wheat seed and stem base pathogens. One fragment, which hybridized preferentially to DNA of F. poae was partially sequenced and two pairs of primers (Fp8 F/R and Fp82 F/R) were generated for use in the polymerase chain reaction (PCR). Amplification of target DNA occurred following PCR of all isolates of F. poae but not from any of a range of other fungal species associated with diseases of cereal ears and seed. The primer pair Fp82 F/R was used to detect F. poae in extracts from wheat seed samples contaminated with Fusarium species. This system offers the potential to determine the presence of F. poae in wheat and avoid problems commonly associated with conventional diagnosis of the disease and isolation of the pathogen.     

Changes in fungicide sensitivity and relative species abundance in Oculimacula yallundae and O. acuformis populations (eyespot disease of cereals) in Western Europe

Changing fungicide sensitivities in populations of Oculimacula yallundae and O. acuformis , the species responsible for cereal eyespot in Western Europe, were determined over a 17 year period between 1984 and 2000. The data were collected by Aventis Crop Science as part of their long-term survey to monitor changes in sensitivity to prochloraz and the methyl benzimidazole carbamate (MBC) fungicides in eyespot populations. The results show evidence for reduced sensitivity to both fungicides over the period of the survey. The decline in MBC sensitivity is in agreement with reports of practical resistance (a detectable loss of disease control in the field) to this fungicide which were widely reported from the mid 1980s onward. Prochloraz sensitivity was more complex, with the emergence of a higher resistance category of isolates in the late 1980s and early 1990s which then decreased in frequency towards the end of the survey. This may be partly explained by the introduction and increased use of cyprodinil in the mid 1990s. Although all trends were similar across Europe, differences were observed between the two eyespot species. A higher frequency of O. yallundae isolates showed decreased sensitivity to MBC, whereas decreased sensitivity to prochloraz was at a higher frequency in O. acuformis populations. The relative abundance of the two eyespot species was influenced by their differential levels of fungicide sensitivity, with the ratio increasing toward the species with the highest level of resistance to the prevailing fungicide.     

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.     

PCR-based assays to detect and quantify <Emphasis Type="Italic">Phomopsis sclerotioides</Emphasis> in plants and soil

We developed polymerase chain reaction (PCR) assays to detect and quantify Phomopsis sclerotioides, the causal agent of black root rot of cucurbits. We used internal transcribed spacers 1 and 2 of the ribosomal DNA (rDNA) from representative isolates to search for target sequences. Primer pairs were selected after testing against 40 fungal isolates including 13 Ph. sclerotioides isolates, 9 Phomopsis isolates other than Ph. sclerotioides, and 18 soilborne fungi that were either pathogenic or nonpathogenic to cucurbits. Conventional PCR assays with the primer pair of CPs-1 (forward) and CPs-2 (reverse) produced target DNA amplicons from all Ph. sclerotioides isolates but none of the other isolates tested. From soil and root samples collected from fields naturally infested with black root rot of cucumber and melon, the CPs-1/CPs-2 primer pair successfully amplified target DNA fragments in conventional PCR assays. Moreover, we applied the CPs-1/CPs-2 primer pair in a real-time PCR assay with SYBR Green I, and PCR-amplified products were successfully quantified by reference to a standard curve generated by adding known amounts of target DNA. Target Ph. sclerotioides DNA fragments were similarly detected in artificially inoculated roots of cucumber, melon, pumpkin, and watermelon, but quantities of Ph. sclerotioides DNA in their hypocotyls of the hosts varied as follows: melon ≥ cucumber ≥ watermelon > pumpkin. These results suggest that Ph. sclerotioides infection is not species-specific but the rate of infection may differ among host species.     

The use of species-specific DNA probes for the identification of Mycosphaerella fijiensis and M. musicola, the causal agents of Sigatoka disease of banana

The polymerase chain reaction (PCR)-based technique of random amplification of polymorphic DNA (RAPD) was used to differentiate DNA from species of the genus Mycosphaerella. DNA from two pathogens which cause Sigatoka leafspot diseases of banana, M. fijiensis and M. musicola , and two other Mycosphaerella species which are commonly found on banana, M. musae and M. minima , gave distinct RAPD banding patterns with all PCR primers tested. PCR, using primer RC07, amplified a 1250bp RAPD fragment from all isolates of M. fijiensis obtained from 11 geographical origins. This fragment was absent from the other species of Mycosphaerella. In Southern blots of genomic DNA, this band hybridized exclusively to DNA from M. fijiensis , and the pattern of hybridization suggested that it was binding to repeated DNA. A RAPD band amplified with primer PM06 obtained from M. musicola was also found to be species-specific. Southern analysis suggested that the fragment hybridized to a single-copy sequence in the M. musicola genome. Total genomic DNA from M. musicola was found to be a species-specific hybridization probe. Dot-blots confirmed the specificity of these probes, and could be used to identify isolates of Mycosphaerella which cause Sigatoka disease of banana in south-east Asia.     

Genetic diversity in the mango malformation pathogen and development of a PCR assay

Malformation is a destructive disease of mango, Mangifera indica . Its causal agent possesses the morphological features of Fusarium subglutinans , a species whose taxonomy and nomenclature has recently been in a state of flux. Genetic diversity was examined among 74 F. subglutinans -like isolates from malformed mango in Brazil, Egypt, Florida (USA), India, Israel and South Africa. With nitrate-nonutilizing ( nit ) auxotrophic mutants, seven vegetative compatibility groups (VCGs) were identified. Three of the VCGs were found in a single country, and VCG diversity was greatest in Egypt and the USA where, respectively, four and three different VCGs were found. RAPD profiles generated with arbitrary decamer primers were variable among isolates in different VCGs, but were generally uniform for isolates within a VCG. In PCR assays, a 20-mer primer pair that was developed previously to identify F. subglutinans from maize (mating population [MP]-E of the Gibberella fujikuroi complex) also amplified a specific 448 bp fragment for isolates of F. sacchari from sugarcane (MP-B) and what was probably F. circinatum (pine, MP-H). With the exception of three isolates from Brazil, it did not amplify the fragment from F. subglutinans -like isolates from mango. A second pair of 20-mer primers was developed from a unique fragment in the RAPD assays. It amplified a specific 608 bp fragment for 51 of 54 isolates from mango (all but the three Brazilian isolates). It also amplified a smaller, 550 bp fragment from isolates of F. nygamai (MP-G), but did not amplify DNA of isolates of any other taxon of Fusarium that was tested.     

Development of a real-time fluorescence loop-mediated isothermal amplification assay for rapid and quantitative detection of Fusarium mangiferae associated with mango malformation

Fusarium mangiferae is a major causal agent of mango malformation disease (MMD) worldwide. Rapid and accurate detection of the causal pathogen is the cornerstone of integrated disease management. In this paper, a real-time fluorescence loop-mediated isothermal amplification assay (RealAmp) was developed for quantitative detection of F. mangiferae in China. The LAMP primer set was designed based on a RAPD marker sequence and positive products were amplified only from F. mangiferae isolates, but not from any other species tested, showing a high specificity of the primer sets. The detection limit was approximately 2.26 × 10⁻⁴ ng/μl plasmid DNA when mixed with extracted mango DNA. Quantification of the pathogen DNA of MMD in naturally collected samples was no significant difference compared to classic real-time PCR Additionally, RealAmp assay was visual with an improved closed-tube visual detection system making the assay more convenient in diagnostics.     

Molecular Detection of Phytophthora ramorum, the Causal Agent of Sudden Oak Death in California, and Two Additional Species Commonly Recovered from Diseased Plant Material

ABSTRACT Sudden oak death is a disease currently devastating forest ecosystems in several coastal areas of California. The pathogen causing this is Phy-tophthora ramorum, although species such as P. nemorosa and P. pseudo-syringae often are recovered from symptomatic plants as well. A molecular marker system was developed based on mitochondrial sequences of the cox I and II genes for detection of Phytophthora spp. in general, and P. ramorum, P. nemorosa, and P. pseudosyringae in particular. The first-round multiplex amplification contained two primer pairs, one for amplification of plant sequences to serve as an internal control to ensure that extracted DNA was of sufficient quality to allow for polymerase chain reaction (PCR) amplification and the other specific for amplification of sequences from Phytophthora spp. The plant primers amplified the desired amplicon size in the 29 plant species tested and did not interfere with amplification by the Phytophthora genus-specific primer pair. Using DNA from purified cultures, the Phytophthora genus-specific primer pair amplified a fragment diagnostic for the genus from all 45 Phytophthora spp. evaluated, although the efficiency of amplification was lower for P. lateralis and P. sojae than for the other species. The genus-specific primer pair did not amplify sequences from the 30 Pythium spp. tested or from 29 plant species, although occasional faint bands were observed for several additional plant species. With the exception of one plant species, the resulting amplicons were smaller than the Phytophthora genus-specific amplicon. The products of the first-round amplification were diluted and amplified with primer pairs nested within the genus-specific amplicon that were specific for either P. ramorum, P. nemorosa, or P. pseudo-syringae. These species-specific primers amplified the target sequence from all isolates of the pathogens under evaluation; for P. ramorum, this included 24 isolates from California, Germany, and the Netherlands. Using purified pathogen DNA, the limit of detection for P. ramorum using this marker system was approximately 2.0 fg of total DNA. However, when this DNA was spiked with DNA from healthy plant tissue extracted with a commercial miniprep procedure, the sensitivity of detection was reduced by 100- to 1,000-fold, depending on the plant species. This marker system was validated with DNA extracted from naturally infected plant samples collected from the field by comparing the sequence of the Phytophthora genus-specific amplicon, morphological identification of cultures recovered from the same lesions and, for P. ramorum, amplification with a previously published rDNA internal transcribed spacer species-specific primer pair. Results were compared and validated with three different brands of thermal cyclers in two different laboratories to provide information about how the described PCR assay performs under different laboratory conditions. The specificity of the Phytophthora genus-specific primers suggests that they will have utility for pathogen detection in other Phytophthora pathosystems.     

A PCR-Based Assay by Sequence-Characterized DNA Markers for the Identification and Detection of Aphanomyces euteiches

ABSTRACT Polymerase chain reaction (PCR) products were identified and amplified from isolates of Aphanomyces euteiches and A. cochlioides. The products were cloned and sequenced, and the data were used to design pairs of extended PCR primers to amplify sequence-characterized DNA markers. The primer pair OPC7-FS-30 and OPC7-RS-25 amplified a single 1,332-bp product from all isolates of A. euteiches that were not amplified from any other isolates tested. A single 718-bp product was selectively amplified only from isolates of A. cochlioides with the primer pair OPB10-FS-25 and OPB10-RS-25. A. euteiches was detected in roots of several varieties of field-grown peas collected from a root rot trial site. PCR also detected A. euteiches in the organic fraction of field soil samples. Both pairs of extended primers were used in a multiplex reaction to unambiguously discriminate between A. euteiches and A. cochlioides. Both pairs of primers were used in two-step PCR reactions in which annealing and extension was done in a single step at 72 degrees C. This reduced the time required for amplification of the diagnostic PCR product and its resolution by electrophoresis to less than 3 h.