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.     

Effects of humidity and temperature on ascospore discharge of <Emphasis Type="Italic">Graphostroma platystoma</Emphasis> in the laboratory and in the field

The effects of air humidity and temperature on the ascospore discharge of Graphostroma platystoma were experimentally investigated. The ascospores were not discharged from the stromata in air at 100% relative humidity (RH). However, they were discharged from the wetted stromata at 3°, 10°, and 24°C under 100% RH or nearly so. The amount of the discharged ascospore was large at 24°C, medium at 10°C, and small at 3°C. The ascospores in the rainwater that washed down the stromata were counted after rainfall in the field. The discharge was observed from September to the following May.     

Ascospore discharge in Pleospora herbarutn (Pers.) Rabenh. (Dothideales) stimulated by near-ultraviolet radiation

An isolate of P. herbarum from beet seed failed to discharge ascospores in darkness but did so when exposed to light either continuously or cyclically (12 h light/12 h dark). When colonies with mature asci were subjected to a regime of alternating light and darkness for 54 days at a constant temperature of 20^°C, ascospores were discharged over the entire period. Maximum discharge occurred on the 23rd day; few spores were liberated towards the end of the period. Light-induced spore discharge occurred over a wide temperature range (10–30^°C) with the optimum being approximately 14–23^°C. When light of different wavelengths (300 nm-infrared) was tested, only near-ultraviolet (310–330 nm) radiation stimulated ascospore discharge. Vertical height of ascospore discharge was also determined. When ascospores were trapped above colonies over a range of heights (2–80 mm), most spores were caught at 2 mm; none was caught at heights above 30 mm. The number of spores trapped at 30 mm was only 1.3% of the capture at 2 mm.     

Effect of Trichoderma harzianum on perithecial production of Gibberella zeae on wheat straw

Conidial suspensions and cell-free filtrates of Trichoderma harzianum isolates were evaluated for their effectiveness in reducing perithecial and ascospore production of Gibberella zeae on wheat straw. Isolate T-22, which is registered in the US as a biological control agent (Plant Shield™), was included in the study as a positive control. When co-inoculated with G. zeae all 11 isolates of T. harzianum significantly reduced perithecial numbers on wheat straw. Five T. harzianum isolates, including T-22, reduced perithecial formation by 70% or greater. All isolates of G. zeae, varied in their ability to produce perithecia. Isolate 192132 produced the greatest number of perithecia and was used to further evaluate the effect of application time of the T. harzianum isolates. Perithecial reduction was highest (96-99%) when T. harzianum conidial suspension or cell-free filtrate was applied to straw 24 h prior to inoculation with G. zeae. Control was less effective when T. harzianum was applied at the same time (co-inoculated) or 24 h after G. zeae. Treatments which reduced perithecial numbers also reduced ascospore numbers; however, the average numbers of ascospores per perithecia were not significantly lowered. Field trials showed significant reduction of perithecia on residues treated with T. harzianum prior to placement on the soil surface. Both T. harzianum and G. zeae were re-isolated from residues sampled in July and August after 30 and 60 days of exposure to the environment.     

Environmental Factors Influencing the Dispersal of Venturia inaequalis Ascospores in the Orchard Air

A 6-year study was carried out in an apple-growing region of North Italy by trapping airborne ascospores of Venturia inaequalis with a volumetric spore trap operated continuously during the ascospore season, with the aim of better defining the weather conditions that allow ascospores both to discharge and to disperse into the orchard air. A total of more than 60 ascospore trapping events occurred. Rain events were the only occurrences allowing ascospores to become airborne (a rain event is a period with measurable rainfall ≥0.2 mm/h – lasting one to several hours, uninterrupted or interrupted by a maximum of two dry hours); on the contrary, dew was always insufficient to allow ascospores to disperse into the air at a measurable rate, in the absence of rain. In some cases, rain events did not cause ascospore dispersal; this occurred when: (i) rain fell within 4–5 h after the beginning of a previous ascospore trapping; (ii) rain fell at night but the leaf litter dried rapidly; (iii) nightly rainfalls were followed by heavy dew deposition that persisted some hours after sunrise. Daytime rain events caused the instantaneous discharge and dispersal of mature ascospores so that they became airborne immediately; for night-time rainfall there was a delay, so that ascospores became airborne during the first 2 h after sunrise. This delay did not always occur, and consequently the ascospore trapping began in the dark, when: (i) the cumulative proportion of ascospores already trapped was greater than 80% of the total season's ascospores; (ii) more than one-third of the total season's ascospores was mature inside pseudothecia and ready to be discharged.     

A Novel Gene,ROA, Is Required for Normal Morphogenesis and Discharge of Ascospores in Gibberella zeae

Head blight, caused by Gibberella zeae, is a significant disease among cereal crops, including wheat, barley, and rice, due to contamination of grain with mycotoxins. G. zeae is spread by ascospores forcibly discharged from sexual fruiting bodies forming on crop residues. In this study, we characterized a novel gene, ROA, which is required for normal sexual development. Deletion of ROA (Δroa) resulted in an abnormal size and shape of asci and ascospores but did not affect vegetative growth. The Δroa mutation triggered round ascospores and insufficient cell division after spore delimitation. The asci of the Δroa strain discharged fewer ascospores from the perithecia but achieved a greater dispersal distance than those of the wild-type strain. Turgor pressure within the asci was calculated through the analysis of osmolytes in the epiplasmic fluid. Deletion of the ROA gene appeared to increase turgor pressure in the mutant asci. The higher turgor pressure of the Δroa mutant asci and the mutant spore shape contributed to the longer distance dispersal. When the Δroa mutant was outcrossed with a Δmat1-2 mutant, a strain that contains a green fluorescence protein (GFP) marker in place of the MAT1-2 gene, unusual phenotypic segregation occurred. The ratio of GFP to non-GFP segregation was 1:1; however, all eight spores had the same shape. Taken together, the results of this study suggest that ROA plays multiple roles in maintaining the proper morphology and discharge of ascospores in G. zeae.Gibberella zeae (anamorph: Fusarium graminearum) causes Fusarium head blight in wheat, barley, and rice, as well as ear rot and stalk rot in maize (20, 23). The infected grains are frequently contaminated by mycotoxins, such as trichothecenes and zearalenone, which are harmful to humans and animals (6). The fungus overwinters in crop debris in the form of storage hyphae and develops ephemeral fruiting bodies (perithecia) at warmer temperatures. Ascospores formed within the perithecia are forcibly discharged into the air and are believed to serve as the primary inoculum of the disease (7, 27, 37, 39,–42). Therefore, sexual development and ascospore discharge are important factors in fungal survival and disease initiation.In fungi of the phylum Ascomycota, the sexual cycle is initiated when two genetically distinct nuclei combine to form a binucleate cell (31). As a homothallic fungus, G. zeae possesses the two mating type genes MAT1-1 and MAT1-2 in the haploid genome and therefore does not require a mating partner for sexual development (22, 46). Perithecium initials give rise to small, coiled initials that develop into perithecia filled with asci, tubular sacs of ascospores, which are the products of meiosis. Mature asci extend through the ostiole of perithecia and discharge their ascospores (40).Unique features of cell differentiation are involved in ascus and ascospore morphogenesis. Ascospore delimitation within the ascus and the development of a cell wall between the ascus and ascospore membranes are unique features of the process (31). Most studies of morphogenesis have described these changes in detail; however, much of these data have been limited to microscopic observations. Several genes involved in ascospore morphogenesis have been identified in Neurospora crassa (30), but the detailed mechanisms and genes involved in ascus and ascospore morphogenesis remain to be elucidated. The Round spore (R) mutant of N. crassa was shown to have round ascospores (24), and the gene responsible for this phenotype, rsp, was subsequently cloned (28). However, in G. zeae, no genes have been identified that are involved in ascus and ascospore morphogenesis.Although recent research has shed light on the physiological basis of ascospore discharge, the genetic basis remains largely unknown (38). The main force responsible for the observed shooting is turgor pressure within the extended asci. In G. zeae, a buildup of K⁺ and Cl⁻ ions drives the influx of water and causes turgor pressure that stretches the asci (41). Asci can accumulate polyols as well as ions. In a previous study, it was shown that the polyols are comprised mainly of mannitol and glucose; however, the concentration of these polyols is too low to make a significant contribution to turgor pressure (42). When the turgor pressure exceeds the threshold of the asci, apical pores rupture and ascospores are forcibly discharged (38). Trail et al. (41) estimated that the acceleration of ascospores in G. zeae is 8,500,000 m s⁻² using an iterative model to predict initial velocity. Recently, Yafetto et al. (44) used high-speed video photography to examine several large-spore fungi, including Ascobolus immerses, and to predict acceleration during dispersal. The asci of A. immerses are more than 12-fold larger in diameter than the asci of G. zeae (38). The size difference between these fungi greatly affects the behavior of their projectiles and results in an initial speed for G. zeae that is too great for application of the video photography method (for further discussion, see the supplemental material).To date, only one gene from G. zeae, the calcium ion channel gene cch1, has been shown to be involved in ascospore discharge (12). Deletion of this gene was shown to arrest ascospore discharge without affecting spore and ascus morphology. Since the genomic sequence of G. zeae is now available, the functional analysis of genes involved in sexual development has been accelerated. Random insertional mutagenesis is one strategy that has been used to identify novel genes associated with sexual development (13, 34). Previously, we produced a collection of more than 20,000 mutants from G. zeae by using the restriction enzyme-mediated integration (REMI) transformation procedure (13). In this study, the G. zeae mutant Z43R9901, which was isolated from a screening of REMI transformants, showed an unusual phenotype during sexual development. Further analysis demonstrated that the novel gene ROA is involved in ascospore morphogenesis and discharge in G. zeae. The results of this study increase our understanding of sexual development in the fungus.     

Mid1, a mechanosensitive calcium ion channel, affects growth, development, and ascospore discharge in the filamentous fungus Gibberella zeae

The role of Mid1, a stretch-activated ion channel capable of being permeated by calcium, in ascospore development and forcible discharge from asci was examined in the pathogenic fungus Gibberella zeae (anamorph Fusarium graminearum). The Δmid1 mutants exhibited a >12-fold reduction in ascospore discharge activity and produced predominately abnormal two-celled ascospores with constricted and fragile septae. The vegetative growth rate of the mutants was ～50% of the wild-type rate, and production of macroconidia was >10-fold lower than in the wild type. To better understand the role of calcium flux, Δmid1 Δcch1 double mutants were also examined, as Cch1, an L-type calcium ion channel, is associated with Mid1 in Saccharomyces cerevisiae. The phenotype of the Δmid1 Δcch1 double mutants was similar to but more severe than the phenotype of the Δmid1 mutants for all categories. Potential and current-voltage measurements were taken in the vegetative hyphae of the Δmid1 and Δcch1 mutants and the wild type, and the measurements for all three strains were remarkably similar, indicating that neither protein contributes significantly to the overall electrical properties of the plasma membrane. Pathogenicity of the Δmid1 and Δmid1Δcch1 mutants on the host (wheat) was not affected by the mutations. Exogenous calcium supplementation partially restored the ascospore discharge and vegetative growth defects for all mutants, but abnormal ascospores were still produced. These results extend the known roles of Mid1 to ascospore development and forcible discharge. However, Neurospora crassa Δmid1 mutants were also examined and did not exhibit defects in ascospore development or in ascospore discharge. In comparison to ion channels in other ascomycetes, Mid1 shows remarkable adaptability of roles, particularly with regard to niche-specific adaptation.     

Phenotypic Diversity among Alleles at the PER-1 Locus of NEUROSPORA CRASSA

Comparison of 11 perithecial color mutants suggested that all were alleles at the per-1 locus but nonetheless separable into two groups because of phenotypic differences. Three of the mutant strains produced orange perithecia and black ascospores, and eight produced paler, yellow perithecia and white ascospores. Perithecial phenotype was dependent upon the genotype of the protoperithecial parent; ascospore phenotype, upon the genotype of the individual ascospore. No evidence was found that the white ascospores were due to chromosomal rearrangements. No separation of the perithecial and ascospore phenotypes by recombination was observed in a cross between one of the mutants and a per-1+ strain. However, apparent low levels of recombination in crosses between some of the mutants indicated possible genetic complexity at the per-1 locus. The phase specificity of the per-1 mutations and the possible nature and mode of expression of the orange and yellow perithecial pigments are discussed.     

Ejection mechanics and trajectory of the ascospores of Gibberella zeae (anamorph Fuarium graminearum)

Since wind speed drops to zero at a surface, forced ejection should facilitate spore dispersal. But for tiny spores, with low mass relative to surface area, high ejection speed yields only a short range trajectory, so pernicious is their drag. Thus, achieving high speeds requires prodigious accelerations. In the ascomycete Gibberella zeae, we determined the launch speed and kinetic energy of ascospores shot from perithecia, and the source and magnitude of the pressure driving the launch. We asked whether the pressure inside the ascus suffices to account for launch speed and energy. Launch speed was 34.5 ms-1, requiring a pressure of 1.54 MPa and an acceleration of 870,000 g--the highest acceleration reported in a biological system. This analysis allows us to discount the major sugar component of the epiplasmic fluid, mannitol, as having a key role in driving discharge, and supports the role of potassium ion flux in the mechanism.     

An Analysis of Spore Discharge in Sordaria

Sordaria fimicola can develop mature perithecia from which sporesare discharged when grown in darkness or in light. Under conditionsof alternating dark and light (12 hrs.: 12 hrs.) each day, sporedischarge is periodic with a low rate during the dark period,succeeded by a gradual rise to a relatively high rate in thelight period followed by a decline before the onset of the nextdark period. There is no trace of an endogenous rhythm. Transferfrom darkness to light always leads to an increase in the rateof discharge, and from light to dark to a decrease. The heightof the peak of discharge rate attained in light following adark period seems to be related to the length of the precedingdark period. Experiments with light of different colours but of roughly thesame energy value show that it is the blue rays that are mainlyeffective. From cultures of filter-paper yeast-extract mediuman orange pigment can be extracted with a maximum absorption,in the visible spectrum, at 470 mµ. It is possible thatthis is important in connexion with the sensitivity of the fungusto blue light.     

Gibberella zeae ascospore production and collection for microarray experiments

Fusarium graminearum Schwabe (teleomorph Gibberella zeae) is a plant pathogen causing scab disease on wheat and barley that reduces crop yield and grain quality. F. graminearum also causes stalk and ear rots of maize and is a producer of mycotoxins such as the trichothecenes that contaminate grain and are harmful to humans and livestock (Goswami and Kistler, 2004). The fungus produces two types of spores. Ascospores, the propagules resulting from sexual reproduction, are the main source of primary infection. These spores are forcibly discharged from mature perithecia and dispersed by wind (Francl et al 1999). Secondary infections are mainly caused by macroconidia which are produced by asexual means on the plant surface. To study the developmental processes of ascospores in this fungus, a procedure for their collection in large quantity under sterile conditions was required. Our protocol was filmed in order to generate the highest level of information for understanding and reproducibility; crucial aspects when full genome gene expression profiles are generated and interpreted. In particular, the variability of ascospore germination and biological activity are dependent on the prior manipulation of the material. The use of video for documenting every step in ascospore production is proposed in order to increase standardization, complying with the increasingly stringent requirements for microarray analysis. The procedure requires only standard laboratory equipment. Steps are shown to prevent contamination and favor time synchronization of ascospores.     

The soluble carbohydrates of Euglena gracilis—their identity and response to increased external solute concentration

Abstract Mannitol and trehalose were the predominant soluble carbohydrates in Euglena gracilis strain z growing heterotrophically in complete darkness or in light. The ratio of trehalose to mannitol was correlated with the water activity of the medium. That is, extracts of Euglena gracilis adapted to grow in media supplemented with either sodium chloride or glucose, thereby reducing the water activity, yielded molar ratios of trehalose to mannitol 10 times greater than extracts of cultures grown under conditions in common usage.     

Ascospore Discharge and Survival in Pseudopezicula tracheiphila, Causal Agent of Rotbrenner of Grape

A recording volumetric spore trap was operated continuously amidst overwintered grape leaves in a vineyard at Walenstadt, Switzerland from early May to mid-July 1988. Ascospores of Pseudopezicula tracheiphila were captured in the air beginning 11 May and 96 % of the total seasonal release occurred between 16 May and 2 June. Rain always preceded ascospore release. However, trap catches were associated with the simulataneous cessation of rainfall, decreased relative humidity (RH), increased temperature, and drying of foliage. Maximum ascospore release occurred in the second hour, following commencement of drying. Ascospores discharged dry onto glass coverslips survived with greater than 60 % viability after 1, 3, and 6 days exposure to 10, 15, 20, and 25°C at 70 % RH. Only at 30°C was viability reduced to slightly less than 50 % after 6 days.     

The use of heterokaryons to determine the origin of the ascogeneous nuclei in Neurospora crassa

Summary By crossing artificially produced heterokaryons with the wild type and recording the ascospore cultures coming from individual perithecia, it has been shown that the nuclei of the ascogeneous hyphae come from several initial pairs.     

Genes Influencing Selective Fertilization in NEUROSPORA CRASSA

The mutual attraction of conidia to protoperithecia of the opposite mating type was studied genetically in crosses where a mixture of conidia from two different strains, one of which was marked by an ascospore color mutant gene tan spore (ts), was applied to protoperithecia. Selective fertilization was measured as the frequency of perithecia fertilized by conidia from one strain in competition with conidia from another strain. Selective fertilization by a given strain varied throughout the range from 10 to 97% according to the strains of protoperithecial parent. The selective fertilization was revealed to be under the control of two or more loci, which appeared to have multiplicative action. No indication of a cytoplasmic effect on selective fertilization was obtained. The strength of the mutual attraction between conidia and protoperithecia decreased as genetic similarity increased.     

Life Cycle of Neurospora crassa Viewed by Scanning Electron Microscopy

Scanning electron microscopy was used to examine the major stages of the life cycle of two wild-type strains of Neurospora crassa Shear and Dodge (St. Lawrence 3.1a and 74A): mycelia, protoperithecium formation, perithecia, ascospores, ascospore germination and outgrowth, macro and microconidia, and germination and outgrowth of macroconidia. Structures seen at the limit of resolution of bright-field and phase-contrast microscopes, e.g., the ribbed surface of ascospores, are well resolved. New details of conidial development and surface structure are revealed. There appears to be only one distinguishable morphological difference between the two strains. The pattern of germination and outgrowth which seems relatively constant for strain 74A or strain 3.1a, appears to be different for each. Conidia from strain 3.1a almost always germinate from a site between interconidial attachment points; whereas the germ tubes of strain 74A usually emerge from or very near the interconidial attachment site. These germination patterns usually do not segregate 2:2 in asci dissected in order. This observation suggests that conidial germination pattern is not under the control of a single gene.     

Spore discharge in Entomophthora grylli

Entomophthora grylli Fres. has been found on larvae of Bradysia (Sciaridae, Diptera) on wood in Kansas State University greenhouses since November 1967. Infected larvae crawled to an exposed site in the night and by morning spores of E. grylli were being discharged. In the greenhouse and under controlled environments spore discharge showed a marked periodicity; spore discharge occurred in the light with peaks in the first 1–4 h; discharge then gradually declined but extended into the dark. On the second and third days peaks occurred in the light but were progressively smaller. In a greenhouse under fluctuating conditions twin peaks occurred at 10·00 and 22·00; in a growth chamber, at constant 21° C. 90% r.h., alternating 12 h light and dark periods, spore discharge was similar, with maximum numbers about 21/2 h after the onset of the photo-period. In continuous dark and continuous light no endogenous pattern was evident. Temperature range for spore discharge was 2–28·, with optimum at 15·. Decreasing humidities resulted in decreased spore production and cessation of discharge below 40% r.h.     

Effects of Temperature and Moisture on Development of Fusarium graminearum Perithecia in Maize Stalk Residues

Fusarium graminearum is the predominant component of the Fusarium head blight complex of wheat. F. graminearum ascospores, which initiate head infection, mature in perithecia on crop residues and become airborne. The effects of temperature (T) and moisture on perithecium production and maturation and on ascospore production on maize stalk residues were determined. In the laboratory, perithecia were produced at temperatures between 5 and 30°C (the optimum was 21.7°C) but matured only at 20 and 25°C. Perithecia were produced when relative humidity (RH) was ≥75% but matured only when RH was ≥85%; perithecium production and maturation increased with RH. Equations describing perithecium production and maturation over time as a function of T and RH (R² > 0.96) were developed. Maize stalks were also placed outdoors on three substrates: a grass lawn exposed to rain; a constantly wet, spongelike foam exposed to rain; and a grass lawn protected from rain. No perithecia were produced on stalks protected from rain. Perithecium production and maturation were significantly higher on the constantly wet foam than on the intermittently wet lawn (both exposed to rain). Ascospore numbers but not their dispersal patterns were also affected by the substrate.