Ascospore Maturation Research Articles

A New Source of Inoculum for Stemphylium vesicarium: Consequences for the Management of Brown Spot of Pear

Brown spot of pear (BSP), caused by Stemphylium vesicarium, is one of the most dangerous pear fungal diseases, being responsible for huge losses in production. Currently, in order to increase its containment, chemical control is implemented in conjunction with agronomic techniques able to reduce BSP inoculum sources (e.g., orchard grass sanitation, litter removal or application of biocontrol agents). Regardless, despite the introduction of agronomic practices, the complete control of the disease is still rarely possible, which suggests that other sources of S. vesicarium inoculum that are currently neglected may be involved. The aim of this study is to investigate the possible wintering forms of Pleospora allii/S. vesicarium on pear wood and whether any spore-forming productions (conidial or ascosporic) might infect the green tissues of the plant in the following spring. Symptomatic fragments of woody tissue from a commercial pear orchard (in Ferrara, Emilia-Romagna, Italy) with a high BSP pressure (~40% incidence) were analysed. The results prove that pseudothecia and the maturation of ascospores of P. allii also develop on one-year-old branch cankers of pear trees, thus representing an additional source of inoculum. In conclusion, the pruning of affected branches and removal of relative residues should be preventatively performed in order to improve BSP management and control.

RNA-dependent RNA polymerases regulate ascospore discharge through the exonic-sRNA-mediated RNAi pathway.

We found that in addition to Fgrdrp1 and Fgrdrp2, Fgrdrp5 also plays important roles in ascospore maturation and ascospore discharge of Fusarium graminearum. These three RNA-dependent RNA polymerases participate in the biogenesis and accumulation of exonic small interference RNA and then regulate ascospore discharge.

Functional characterization of the developmental genes asm2, asm3, and spt3 required for fruiting body formation in the filamentous ascomycete Sordaria macrospora.

The formation of fruiting bodies is one of the most complex developmental processes in filamentous ascomycetes. It requires the development of sexual structures that give rise to meiosporangia (asci) and meiotic spores (ascospores) as well as surrounding structures for protection and dispersal of the spores. Previous studies have shown that these developmental processes are accompanied by significant changes of the transcriptome, and comparative transcriptomics of different fungi as well as the analysis of transcriptome changes in developmental mutants have aided in the identification of differentially regulated genes that are themselves involved in regulating fruiting body development. In previous analyses, we used transcriptomics to identify the genes asm2 and spt3, which result in developmental phenotypes when deleted in Sordaria macrospora. In this study, we identified another gene, asm3, required for fruiting body formation, and performed transcriptomics analyses of Δasm2, Δasm3, and Δspt3. Deletion of spt3, which encodes a subunit of the SAGA complex, results in a block at an early stage of development and drastic changes in the transcriptome. Deletion mutants of asm2 and asm3 are able to form fruiting bodies, but have defects in ascospore maturation. Transcriptomics analysis of fruiting bodies revealed a large overlap in differentially regulated genes in Δasm2 and Δasm3 compared to the wild type. Analysis of nuclear distribution during ascus development showed that both mutants undergo meiosis and postmeiotic divisions, suggesting that the transcriptomic and morphological changes might be related to defects in the morphogenesis of structural features of the developing asci and ascospores.

Pathogenic Fitness of Pyrenophora tritici-repentis Isolates Causing Tan Spot of Winter Wheat in Oklahoma

Tan spot of wheat (Triticum aestivum L.) caused by the fungus Pyrenophora tritici-repentis can be a significant disease, and thus developing resistant varieties using fit and virulent isolates is critical to wheat breeding programs. Hence, variability in fitness characters and virulence was determined for P. tritici-repentis isolates collected in three different decades (1980s, 1990s, and 2000s) from Oklahoma wheat fields. Growth (hyphal extension on media), conidia production on media, and conidia production on wheat leaves of a susceptible (TAM 105), an intermediate (Deliver) and a resistant (Red Chief) variety were determined for 17 isolates. Isolates also were evaluated for pseudothecia production and ascospore maturity on wheat straw, and isolate virulence was determined on all three wheat varieties. Isolates differed significantly (p<0.01) for growth on media, and for conidia production on agar and on wheat leaves. Isolates varied significantly in pseudothecia production and ascospore maturity on wheat straw. Isolates differed significantly (p<0.01) in virulence (percent leaf area infection) on the three wheat cultivars. Isolates collected from the 2000s were more virulent than those collected in the 1980s and 1990s, and also were deemed to be more fit than isolates collected in the 1980s and 1990s in terms of traits such as growth, conidia production, pseudothecia formation, mature ascospore production, and virulence. This fitness could affect the competitiveness of these isolates in the field, as well as facilitate selection of isolates used to test wheat germplasm lines for reaction to tan spot.

Methods for observing, culturing, and studying living ascospores

Ascospore morphologies provide important characters with which to diagnose and describe taxa in Ascomycota. Ascospore features such as size, shape, color, septation, wall thickness, and guttulation, among others, are provided in identification manuals and descriptions of new species. Yet, by tradition, ascospores are usually described from dead fungarium material, and unfortunately, occasionally from immature or overmature ones. However, living, mature ascospores display a wealth of taxonomically informative morphological features that are lost or obscured when they die. Examples of the severe morphological changes that ascospores undergo when they die are provided here. Data from living ascospores may not be observed and recorded by mycologists because field and laboratory practices do not prioritize the study of freshly collected specimens. In this review, we discuss how to assess ascospore maturity and describe methods to produce an ascospore deposit for the purpose of obtaining living, mature ascospores. Ascospores are ejected from living, mature asci onto a cover glass or growth medium. The ascospores collected on these surfaces can be used in microscopy and culture studies. Notes on a method for isolating conidia on growth medium are also provided. This guide is aimed at those who have a basic understanding of ascomycetes, including the various types of ascomata and mechanisms of ascospore liberation. Methods given in this paper are primarily applied to ascomycete fungi that have active ascospore discharge. Some methods may be adapted for use with other groups that have passive discharge. Our purpose is to promote standardized, accurate, and thorough morphological characterization of living ascospores, as well as to encourage the routine employment of culture-based methods.

The velvet Regulator VosA Governs Survival and Secondary Metabolism of Sexual Spores in Aspergillus nidulans.

The velvet regulator VosA plays a pivotal role in asexual sporulation in the model filamentous fungus Aspergillus nidulans. In the present study, we characterize the roles of VosA in sexual spores (ascospores) in A. nidulans. During ascospore maturation, the deletion of vosA causes a rapid decrease in spore viability. The absence of vosA also results in a lack of trehalose biogenesis and decreased tolerance of ascospores to thermal and oxidative stresses. RNA-seq-based genome-wide expression analysis demonstrated that the loss of vosA leads to elevated expression of sterigmatocystin (ST) biosynthetic genes and a slight increase in ST production in ascospores. Moreover, the deletion of vosA causes upregulation of additional gene clusters associated with the biosynthesis of other secondary metabolites, including asperthecin, microperfuranone, and monodictyphenone. On the other hand, the lack of vosA results in the downregulation of various genes involved in primary metabolism. In addition, vosA deletion alters mRNA levels of genes associated with the cell wall integrity and trehalose biosynthesis. Overall, these results demonstrate that the velvet regulator VosA plays a key role in the maturation and the cellular and metabolic integrity of sexual spores in A. nidulans.

Apc.LaeA and Apc.VeA of the velvet complex govern secondary metabolism and morphological development in the echinocandin-producing fungus Aspergillus pachycristatus.

The impact of the global secondary metabolite regulators LaeA and VeA on echinocandin B production and morphological development was evaluated in the industrial production strain Aspergillus pachycristatus NRRL 11440. Other representative secondary metabolites were examined as well to determine if the velvet complex functions as in A. nidulans and other species of fungi. Genetic methods used for gene manipulations in A. nidulans were applied to A. pachycristatus. Separate deletions of genes Apc.laeA and Apc.veA resulted in similar yet differing phenotypes in strain NRRL 11440. Disruption of Apc.laeA and Apc.veA significantly reduced, but did not eliminate, the production of echinocandin B. Similar to what has been observed in A. nidulans, the production of sterigmatocystin was nearly eliminated in both mutants. Quantitative reverse transcription PCR analyses confirmed that selected genes of both the echinocandin B and sterigmatocystin gene clusters were down-regulated in both mutant types. The two mutants differed with respect to growth of aerial hyphae, pigmentation, development of conidiophores, conidial germination rate, and ascospore maturation. Further functional annotation of key regulatory genes in A. pachycristatus and related Aspergillus species will improve our understanding of regulation of echinocandin production and co-produced metabolites.

Comparison of mathematical models of maturation rate of the airborne Venturia inaequalis (Cooke) Wint. ascospores in central Poland

Models of maturation rate of the airborne Venturia inaequalis (Cooke) Wint. ascospores were compared using field data collected from spore traps in Skierniewice, Poland, during 2005–2008 and 2010–2014. Two sets of models describing ascospores maturation rate as a function of cumulative temperature were compared. The first set consisted of the “Stensvand” model with degrees summed during and 147 h after the rainfall, and the “Only” type models accumulating temperature only during hours with observed high moisture level indicated by rain, leaf wetness or high humidity. The second set consisted of more complex “All” type models proposed in the study, with degrees summed each hour, but differently weighted for periods with various sources of moisture such as rain, leaf wetness (LW) and relative air humidity (RH). The “All” type models proved to be superior over the “Only” type models. The “All Rain + LW + RH,” “All Rain + LW,” “All LW” and the “Stensvand” models gave the best match with the data. The final and most important result of the study is such that all of the examined models had high and unsatisfactory range of models’ uncertainty for the data from central Poland. Therefore, further improvements in modeling of ascospore maturation rate are necessary. Low accuracy of the models limits their practical application at the current stage of development.

A New Phytosanitary Method to Reduce the Ascospore Potential of Venturia inaequalis.

Ascospores of Venturia inaequalis, released from pseudothecia in overwintered, infected apple leaves, serve as the primary inoculum for apple scab. In this study, we tested a new sanitation strategy to reduce ascospore inoculum under orchard conditions over three overwintering periods. After leaf fall, nutrient media containing different concentrations of degraded casein or a yeast extract from Saccharomyces cerivisiae were applied to leaf litter infected with apple scab. The application of 30 and 60% yeast extract showed the greatest efficacy, and significantly reduced ascospore discharge by 99% (P < 0.01) in 2013 and 2014. The efficacy of the treatments did not differ from treatment with 5% urea (P > 0.05). Leaf litter decay was accelerated in the plots treated with yeast extract compared with untreated control plots. Moreover, apple leaves treated with yeast extract had completely decayed due to earthworm activity before ascospore maturity. In comparison, up to 26% of the leaves in untreated control plots had not decayed. These results suggest that the treatment of leaf litter with yeast extract can almost completely eliminate apple scab inoculum in the course of the whole primary season. These sanitation practices may be beneficial for both organic and conventional cultivation. The reduced infection pressure may allow growers the usage of fungicides with lower efficacy or to reduce the number of applications needed to manage apple scab in spring.

Crosses Heterozygous for Hybrid Neurospora Translocation Strains Show Transmission Ratio Distortion Disfavoring Homokaryotic Ascospores Made Following Alternate Segregation.

By introgressing Neurospora crassa translocations into N. tetrasperma, we constructed heterokaryons bearing haploid nuclei of opposite mating types, and either the translocation and normal sequence chromosomes (i.e., [T + N]) or a duplication and its complementary deficiency (i.e., [Dp + Df]). The [T + N] heterokaryons result from alternate segregation of homologous centromeres, whereas adjacent-1 segregation generates [Dp + Df]. Self-cross of either heterokaryon produces [T + N] and [Dp + Df] progeny. Occasionally during N. tetrasperma ascus development, a pair of smaller homokaryotic ascospores replaces a heterokaryotic ascospore. Crosses with the Eight-spore mutant increase such replacement, and can generate asci with eight homokaryotic ascospores, either 4T + 4N from alternate segregation, or 4Dp + 4Df from adjacent-1 segregation. Crosses of some of the introgressed translocation strains with normal sequence N. tetrasperma produced more Dp than T or N homokaryotic progeny. We suggest this is due to an insufficiency for a presumptive ascospore maturation factor, which increases the chance that, in asci with > 4 viable ascospores, none properly mature. Since only four viable ascospores (Dp or [Dp + Df]) share the limiting factor following adjacent-1 segregation, whereas four to eight ascospores compete for it following alternate segregation, this would explain why Dp homokaryons outnumber T and N types, whereas the heterokaryons are not as affected. We believe that this novel form of transmission ratio distortion is caused by a Bateson–Dobzhansky–Muller Incompatibility (BDMI) triggered by an N. crassa gene in the N. tetrasperma background. Heterokaryons tend not to out-cross, and crosses of Dp strains are barren, thus the BDMI impedes interspecies gene flow.

Peroxisome dynamics during development of the fungus Podospora anserina

Peroxisomes are versatile and dynamic organelles that are required for the development of diverse eukaryotic organisms. We demonstrated previously that in the fungus Podospora anserina different peroxisomal functions are required at distinct stages of sexual development, including the initiation and progression of meiocyte (ascus) development and the differentiation and germination of sexual spores (ascospores). Peroxisome assembly during these processes relies on the differential activity of the protein machinery that drives the import of proteins into the organelle, indicating a complex developmental regulation of peroxisome formation and activity. Here we demonstrate that peroxisome dynamics is also highly regulated during development. We show that peroxisomes in P. anserina are highly dynamic and respond to metabolic and environmental cues by undergoing changes in size, morphology and number. In addition, peroxisomes of vegetative and sexual cell types are structurally different. During sexual development peroxisome number increases at two stages: at early ascus differentiation and during ascospore formation. These processes are accompanied by changes in peroxisome structure and distribution, which include a cell-polarized concentration of peroxisomes at the beginning of ascus development, as well as a morphological transition from predominantly spherical to elongated shapes at the end of the first meiotic division. Further, the mostly tubular peroxisomes present from second meiotic division to early ascospore formation again become rounded during ascospore differentiation. Ultimately the number of peroxisomes dramatically decreases upon ascospore maturation. Our results reveal a precise regulation of peroxisome dynamics during sexual development and suggest that peroxisome constitution and function during development is defined by the coordinated regulation of the proteins that control peroxisome assembly and dynamics.

Effects of Weather Variables on Ascospore Discharge from Fusarium graminearum Perithecia.

Fusarium graminearum is a predominant component of the Fusarium head blight (FHB) complex of small grain cereals. Ascosporic infection plays a relevant role in the spread of the disease. A 3-year study was conducted on ascospore discharge. To separate the effect of weather on discharge from the effect of weather on the production and maturation of ascospores in perithecia, discharge was quantified with a volumetric spore sampler placed near maize stalk residues bearing perithecia with mature ascospores; the residues therefore served as a continuous source of ascospores. Ascospores were discharged from perithecia on 70% of 154 days. Rain (R) and vapor pressure deficit (VPD) were the variables that most affected ascospore discharge, with 84% of total discharges occurring on days with R≥0.2 mm or VPD≤11 hPa, and with 70% of total ascospore discharge peaks (≥ 30 ascospores/m3 air per day) occurring on days with R≥0.2 mm and VPD≤6.35 hPa. An ROC analysis using these criteria for R and VPD provided True Positive Proportion (TPP) = 0.84 and True Negative Proportion (TNP) = 0.63 for occurrence of ascospore discharge, and TPP = 0.70 and TNP = 0.89 for occurrence of peaks. Globally, 68 ascospores (2.5% of the total ascospores sampled) were trapped on the 17 days when no ascospores were erroneously predicted. When a discharge occurred, the numbers of F. graminearum ascospores sampled were predicted by a multiple regression model with R2 = 0.68. This model, which includes average and maximum temperature and VPD as predicting variables, slightly underestimated the real data and especially ascospore peaks. Numbers of ascospores in peaks were best predicted by wetness duration of the previous day, minimum temperature, and VPD, with R2 = 0.71. These results will help refine the epidemiological models used as decision aids in FHB management programs.

Functional Analysis of Developmentally Regulated Genes chs7 and sec22 in the Ascomycete Sordaria macrospora.

During sexual development, filamentous ascomycetes form complex, three-dimensional fruiting bodies for the generation and dispersal of spores. In previous studies, we identified genes with evolutionary conserved expression patterns during fruiting body formation in several fungal species. Here, we present the functional analysis of two developmentally up-regulated genes, chs7 and sec22, in the ascomycete Sordaria macrospora. The genes encode a class VII (division III) chitin synthase and a soluble N-ethylmaleimide-sensitive-factor attachment protein receptor (SNARE) protein, respectively. Deletion mutants of chs7 had normal vegetative growth and were fully fertile but showed sensitivity toward cell wall stress. Deletion of sec22 resulted in a reduced number of ascospores and in defects in ascospore pigmentation and germination, whereas vegetative growth was normal in the mutant. A SEC22-EGFP fusion construct under control of the native sec22 promoter and terminator regions was expressed during different stages of sexual development. Expression of several development-related genes was deregulated in the sec22 mutant, including three genes involved in melanin biosynthesis. Our data indicate that chs7 is dispensable for fruiting body formation in S. macrospora, whereas sec22 is required for ascospore maturation and germination and thus involved in late stages of sexual development.

An improved method to estimate the uncertainty of models describing the seasonal maturation of Venturia inaequalis ascospores

A simple method for reducing the uncertainty intervals of models describing the maturation of Venturia inaequalis (Cooke) Wint ascospores is described. The number of trapped ascospores related to the current accumulated degree days of the season, and the confidence interval predicted with the historical ascospore release datasets, were used to re-calculate the confidence interval for the ascospore proportion for the rest of the season. The historical uncertainty in ascospore proportion was described with logistic functions fitted to upper and lower confidence limits of the mean of fitted values for several years of ascospore release data. The re-calculated confidence interval was derived by the determination of the total number of ascospores that would be trapped during the season, if the current day cumulative release were at either the upper or lower confidence limit of the historical data. The method was examined by applying three models of ascospore maturation and using ascospore trap and weather data collected in Skierniewice, Poland during the period from 2005 to 2009. The historical uncertainties were established by the calibration of models to the data from 2005–2007. The effectiveness of the method was simulated using data of 2008 and 2009. In these years, the re-calculated confidence interval was never greater than the historical confidence interval and was often 3–4 times smaller than it, even though ascospore release observations in 2009 strongly differed from those of the years 2005–2007. In conclusion, the proposed method may be an effective approach to a more precise description of V. inaequalis ascospores maturation.

A Putative Transcription Factor pcs1 Positively Regulates Both Conidiation and Sexual Reproduction in the Cereal Pathogen Fusarium graminearum.

The plant pathogen Fusarium graminearum causes Fusarium head blight in cereal crops and produces mycotoxins that are harmful to animals and humans. For the initiation and spread of disease, asexual and sexual reproduction is required. Therefore, studies on fungal reproduction contribute to the development of new methods to control and maintain the fungal population. Screening a previously generated transcription factor mutant collection, we identified one putative C2H2 zinc-finger transcription factor, pcs1, which is required for both sexual and asexual reproduction. Deleting pcs1 in F. graminearum resulted in a dramatic reduction in conidial production and a complete loss of sexual reproduction. The pathways and gene ontology of pcs1-dependent genes from microarray experiments showed that several G-protein related pathways, oxidase activity, ribosome biogenesis, and RNA binding and processing were highly enriched, suggesting that pcs1 is involved in several different biological processes. Further, overexpression of pcs1 increased conidial production and resulted in earlier maturation of ascospores compared to the wild-type strain. Additionally, the vegetative growth of the overexpression mutants was decreased in nutrient-rich conditions but was not different from the wild-type strain in nutrient-poor conditions. Overall, we discovered that the pcs1 transcription factor positively regulates both conidiation and sexual reproduction and confers nutrient condition-dependent vegetative growth.

The cAMP-PKA Pathway Regulates Growth, Sexual and Asexual Differentiation, and Pathogenesis in Fusarium graminearum

Like many other filamentous ascomycetes, Fusarium graminearum contains two genes named CPK1 and CPK2 that encode the catalytic subunits of cyclic AMP (cAMP)-dependent protein kinase A (PKA). To determine the role of cAMP signaling in pathogenesis and development in F. graminearum, we functionally characterized these two genes. In addition, we generated and characterized the cpk1 cpk2 double and fac1 adenylate cyclase gene deletion mutants. The cpk1 mutant was significantly reduced in vegetative growth, conidiation, and deoxynivalenol production but it had increased tolerance to elevated temperatures. It was defective in the production of penetration branches on plant surfaces, colonization of wheat rachises, and spreading in flowering wheat heads. Deletion of CPK1 had no effect on perithecium development but the cpk1 mutant was defective in ascospore maturation and releasing. In contrast, the cpk2 mutant had no detectable phenotypes, suggesting that CPK2 contributes minimally to PKA activities in F. graminearum. Nevertheless, the cpk1 cpk2 double mutant had more severe defects in vegetative growth and rarely produced morphologically abnormal conidia. The double mutant, unlike the cpk1 or cpk2 mutant, was nonpathogenic and failed to form perithecia on self-mating plates. Therefore, CPK1 and CPK2 must have overlapping functions in vegetative growth, differentiation, and plant infection in F. graminearum. The fac1 mutant was also nonpathogenic and had growth defects similar to those of the cpk1 cpk2 mutant. However, deletion of FAC1 had no effect on conidium morphology. These results indicated that CPK1 is the major PKA catalytic subunit gene and that the cAMP-PKA pathway plays critical roles in hyphal growth, conidiation, ascosporogenesis, and plant infection in F. graminearum.

Release of Erysiphe necator Ascospores and Impact of Early Season Disease Pressure on Vitis vinifera Fruit Infection

Populations of <i>Erysiphe necator</i> cleistothecia can dehisce and release ascospores over an extended period ranging from fall through late spring and are an important source of primary inoculum for grapevine powdery mildew epidemics. The temporal distribution of ascospore maturity was monitored and measured as ascospore release from field-stored samples in a controlled environment assay. Assays were conducted over multiple seasons, in multiple locations, using multiple source populations. The influence of primary inoculum quantity and how that influences subsequent fruit disease severity was assessed. Cumulative ascospore release was positively correlated with accumulated wetting events and heat units; however, in most situations, &gt;50% of the season-total ascospores were released before the date of local budbreak of <i>Vitis vinifera</i>. Both abbreviated and season-long fungicide programs suppressed mildew on fruit across a 100-fold gradient of inoculum dose when seasonal weather was relatively unfavorable for epidemic development. Suppression was degraded with progressive 10-fold increases of ascosporic inoculum dose when seasonal weather was more conducive to epidemic development. Combined, these findings suggest that differences between severe and mild years in grape powdery mildew can relate to the amount of primary inoculum present in the vineyard, that the levels of primary inoculum can be influenced by pre-budbreak weather conditions, and that effectiveness of spray programs at controlling primary infection events is related to the favorability of in-season weather conditions.

Role of urea in the reduction of apple primary infections by Venturia inaequalis (Cooke) Aderh

The effect of pre-leaf fall of 5% urea treatment on primary infection of apple by &lt;i&gt;Venturia inaequalis&lt;/i&gt;, the cause of scab, was investigated in commercial apple orchards of McIntosh cv. in different regions of Poland, at Miłobądz, Sinołęka and Dąbrowice. Additionally, the development of pseudothecia and maturation of ascospores were evaluated in the spring on apple leaves of two cultivars (McIntosh and Gloster) treated with 5% solution of urea. Microscopic observations of leaves showed that urea treatment significantly reduced the number of pseudothecia (by ca. 90%) and ascospore production. Low number of ascospores after urea treatment has strongly affected primary infection of apple by the fungus in all tested orchards. Reduction of leaf infection on control trees was usually more than 30%. Also efficacy of chemical control of apple scab was significantly higher on plots treated with urea.

The PaAlr1 magnesium transporter is required for ascospore development in Podospora anserina

The PaAlr1 gene encoding a putative plasma membrane magnesium (Mg) transporter in Podospora anserina was inactivated. The PaAlr1Δ mutants showed sensitivity to deprivation and excess Mg2+ and Ca2+. They also exhibited an autonomous ascospore maturation defect. Mutant ascospores were arrested at an early stage when they contained two nuclei. These data emphasize the role of Mg ions during sexual development in a filamentous fungus.

Evaluation of Six Models to Estimate Ascospore Maturation in Venturia pyrina.

Estimates of ascospore maturity generated by models developed for Venturia pyrina in Victoria, Australia (NV and SV), Oregon, United States (OR), and Italy (IT) or for V. inaequalis in New Hampshire, United States (NH-1) or modified in Norway (NH-2) were compared with observed field ascospore release of V. pyrina from 21 site-year combinations. The models were also compared with ascospore release data from laboratory assays. In the laboratory assays, the forecasts of the NH-1 and NH-2 models provided the best fit to observed spore release. Under field conditions, the lag phases and slope coefficients of all models differed from those of observed release of ascospores. Identifying the precise time of bud break of pear to initiate degree-day accumulation was problematic at both Australian sites. This resulted in a higher deviance between bud break and first released ascospore compared with the sites in Norway and Belgium. Linear regressions of observed release against forecasted maturity generated similarly high concordance correlation coefficients. However, where differences were noted, they most often favored models that included adjustment for dry periods. The NH-2, IT, and NV models using pooled data also provided the most accurate estimates of 95% ascospore depletion, a key event in many disease management programs.