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Departments of Plant Biology and Plant Pathology, Michigan State University, East Lansing, Michigan 48824
David Gadoury
Department of Plant Pathology, Cornell University, New York State Agricultural Experiment Station, Geneva, New York 14456
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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We investigated ascospore discharge in the perithecial fungus, Gibberella zeae. In a wind tunnel study that simulated constant rain and varying day and night lengths, the rate of ascospore release was approximately 8–30% greater under light than in complete darkness. Under constant light, ascospore discharge occurred at maximal rates at relative humidity levels greater than 92%. When perithecia were placed under conditions of high external osmolarity, ascospore discharge was significantly reduced. Ascospores were discharged from asci along with droplets of fluid, the epiplasm, from within the ascus. Analysis of discharged epiplasmic fluid by GC-MASS Spectrometry revealed that mannitol was the major simple sugar component of the fluid. Activity of mannitol dehydrogenase, which catalyzes the conversion of fructose to mannitol, was higher in protein extracts from mature perithecia than in extracts from vegetative tissue. Several inhibitors of K+ and Ca++ ion channels inhibited ascospore discharge, which suggested that ascospore discharge resulted from the buildup of turgor pressure generated by ion fluxes and mannitol accumulation.
Key words: ascus, ion channels, mannitol dehydrogenase, mannitol-1-phosphate dehydrogenase, perithecia, turgor pressure
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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).
For head blight, airborne ascospores of G. zeae are considered to be the primary inoculum. Macroconidia may be dispersed by wind and rain-splash (Sutton 1982
, Parry et al 1995
, Fernando et al 1997
), but are recovered from air samples in lower levels than ascospores (Fernando et al 2000)
. Several field and lab studies have explored the effects of environmental parameters on discharge of ascospores of G. zeae. Paulitz (1996)
studied the pattern of spore release in inoculated field plots and reported that high humidity is not necessary for discharge, and that ascospore release is reduced at relative humidity greater than 80%, and when daily rainfall exceeds 5 mm. Fernando et al (2000)
showed ascospore release, but not macroconidia release, follows a daily periodicity. Tschanz et al (1975)
reported that ascospore release follows drying of previously wetted ascocarps, and that a drop in relative humidity is required for spore release. Others have reported that ascospore release in the field is associated with high relative humidity or rainfall (Chen and Yuan 1984
, Reis 1990
). Thus, a review of previous studies presents a confusing array of environmental stimuli that appear to simultaneously stimulate and inhibit ascospore release.
It is generally assumed that forcible ascospore discharge is driven by hydrostatic pressure buildup within the mature ascus (Ingold 1966, 1971
, Burnett 1976
, Beckett 1981
, Read and Beckett 1996
). In several Pyrenomycetes species, mature asci, just prior to discharge, have been observed to be swollen (reviewed by Read and Beckett 1996
). In G. zeae, we have observed individual mature asci extending up through the ostiole (Trail and Common 2000)
. Ingold (1966)
identified glucose as the predominant osmoticum in the ascus fluid Sordaria fimicola, although no supporting data were published. A role for ions in the generation of turgor pressure has also been suggested (Ingold 1971
, Minter and Cannon 1984
).
Our goal is to understand the mechanism of forcible discharge of ascospores. To establish the optimal discharge conditions for our investigation, we examined environmental parameters affecting discharge in G. zeae. We have analyzed epiplasmic fluid from mature asci to identify components that may be responsible for the generation of turgor pressure within the ascus. A preliminary report of these studies was published previously (Trail et al 1998
).
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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) and strain PH-1 (NRRL 31084; Trail and Common 2000)
. Both strains were used for the relative humidity studies, only PH-1 was used for all other studies. Strains were maintained as soil stocks at -20 C. Nearly synchronous lawns of perithecia were produced on carrot agar as previously described (Klittich and Leslie 1988
). After 2–4 d of growth on carrot agar in 60 mm Petri dishes, perithecia were induced from vegetative hyphae with the addition of 1 mL of 2.5% aqueous Tween 60 (Sigma Company, St. Louis, Missouri). For ascospore discharge assays, agar blocks (1 cm x 1 cm) covered with perithecia were placed on the end of a glass microscope slide and oriented so the perithecium-bearing surface was perpendicular to the surface of the slide. Slides were placed on a platform in a transparent humidity chamber under continuous light. Discharged spores were collected on the slide. Unless otherwise stated, spores were quantified as follows: spores were collected by washing each slide with 1 mL sterile, distilled water, removed by centrifugation, resuspended in 1 mL distilled water and spore concentration was determined using a hemocytometer.
To determine the pattern of ascospore release relative to perithecium maturity, this procedure was slightly modified. Disks (1 cm diam) were removed from induced cultures, cut in half and placed on slides in humidity chambers. The perithecia were incubated for 24 h and quantified as above.
Spore release in the wind tunnel –
A wind tunnel, previously described for use with Venturia inaequalis (Gadoury et al 1996
), was used to observe discharge under varying light regimes and constant rainfall. The tunnel simulated a steady supply of rain, constant temperature (20 C) and air flow. Light (daylight-balanced artificial light comparable to that reported at mid-day during rain events) and dark cycles varied according to the experiment. Two 1 cm-diameter disks of carrot agar were removed from a culture producing mature perithecia (d 7–9 after induction) and mounted in the apparatus for each trial. Spores were collected at the end of the wind tunnel on cellophane tape mounted on a clock cylinder and quantified at the end of each trial as previously described(Gadoury et al 1996
), and the ascospores of G. zeae were enumerated. Linear regression was performed on the data using Minitab 10.5 (Pennsylvania State University, State College, Pennsylvania).
Relative humidity studies –
Slides with agar blocks (1 cm x 1 cm) covered with mature perithecia (d 7–9 after induction) were placed on a platform in sealed humidity chambers above the surface of a solution of the appropriate concentration of glycerol to maintain constant relative humidity (Forney and Brandl 1992
). Solutions were adjusted to the correct relative humidity with a hygrometer (Traceable® Hygrometer, VWR, U.S.A.). After 24 h, slides were removed, and the discharged spores were quantified by counting the total numbers of spores deposited along a 1 mm wide transect running lengthwise through the center of the spore deposit. Five to six independent trials were conducted at each relative humidity. Mean counts from treatments were transformed prior to analysis with the log transformation to homogenize the variances. Treatments were compared using PROC GLM of SAS Version 8 (SAS Institute, Cary, North Carolina).
Light microscopy – Ascospore discharge was observed and photographed using a IMT-2 Olympus inverted microscope (Olympus Corporation, Lake Success, New York) equipped with Nomarski differential interference-contrast optics to visualize ascospores.
Analysis of ascus fluid –
Ascospores were discharged onto clean glass slides overnight. Spores were collected and quantified as described above, except the spore washes (supernatant), containing the ascus exudates, were retained following centrifugation to remove the spores. Spore washes were frozen at -20 C, then dried in a Speed Vac (Savant Instruments, Inc., Farmingdale, New York). Identification of simple sugars in the spore washes was accomplished by GC-MASS Spectrometry analysis of alditol acetate derivatives as previously described (Higgins et al 1994
). Variation in sugar reduction procedure and derivitization prior to analysis was used to distinguish various sugar and sugar alcohols as described by Higgins et al (1994)
. Each test was performed on at least 3 independent samples.
Enzyme assays – Perithecia were removed from the surface of carrot agar by gently scraping with a sterile scalpel. Mycelia were grown in liquid culture in YES (6% sucrose, 3% yeast extract) for 5 d at room temperature, shaking at 180 rpm. The mycelium was harvested by filtering through Miracloth (Calbiochem) and washed twice with distilled water. Mycelia were also grown on solid culture in carrot agar for 5 d, and scraped from the surface of the agar. Tissues were stored at -80 C prior to use.
Mannitol dehydrogenase (MTD) and mannitol-1-phosphate dehydrogenase (M1PD) assays were performed using modifications of a previously published procedure (Stoop et al 1995
). Harvested tissue was placed in liquid nitrogen and ground to a fine powder with a mortar and pestle. Buffer [50 mM MOPS (pH 4.5, 5 mM dithiothreitol, 1 mM EDTA, 5 mM phenylmethylsulfonyl fluoride]) was added in a tissue-to-buffer ratio of 1 to 4. For initial assays to detect enzyme activity, undissolved components were removed from the homogenate by centrifugation (20 000 g for 20 min) at 4 C. For quantification of MTD activity in different tissues, the supernatant was brought to 45% saturation with (NH4)2SO4, stirred for 1 h on ice, and centrifuged as above. The supernatant was retained, brought to 80% saturation with (NH4)2SO4, stirred for 1 h on ice, and centrifuged again as above. The pellet was dissolved in a minimal volume of 50 mM MOPS (pH 7.5), 1 mM dithiothreitol. Protein concentration of the enzyme extract was determined spectrophotometrically using the BioRad Protein Assay system (BioRad Laboratories, Hercules, California) with bovine serum albumin as a standard.
Enzyme activity (reactions in both directions) was assayed spectrophotometrically (340 nm) by monitoring the reduction of nicotinamide adenine dinucleotide phosphate (NADP) and the oxidation NADPH by MTD or the reduction of nicotinamide adenine diphosphate (NAD) and the oxidation of NADH by M1PD. The assay mixtures, in a total volume of 1 mL, contained: for oxidation of mannitol, 100 mM Tris buffer (pH 9.5), 2 mM NADP, 25 µL enzyme extract, and 200 mM of mannitol; for the reduction of fructose, 100 mM MOPS (pH 7.5), 2 mM NADPH, 25 µL enzyme extract and 800 mM fructose. Oxidation of mannitol-1-phosphate by M1PD was similarly assayed in 10 mM HEPES (pH 9.0), 0.5 mM NAD, 10 mM mannitol-1-phosphate. Reduction of fructose-6-phosphate by M1PD was assayed in 10 mM HEPES (pH 7.0), 0.5 mM NADH, 10 mM fructose-6-phosphate. One unit of MTD activity was defined as the amount of enzyme that catalyzed the oxidation of 1 nmol NADPH per min.
Ion channel inhibition assays – Ascospore discharge assays were set up from cultures containing perithecia 5 d postinduction with the following modifications: thickness of the agar blocks was reduced to approximately 2 mm and blocks were placed on a similarly sized 2% water agar block containing dissolved inhibitors. Spores were allowed to discharge from the stacked blocks for 24 h, and then collected and quantified as described above. The number of perithecia on each agar block was also recorded and data normalized to reflect numbers of ascospores discharged per perithecium. Water agar blocks were prepared from inhibitor stocks dissolved in water [cesium chloride (CsCl), 8-(N,N-Diethylamino)-octyl-3,4,5-trimethoxybenzoate (TMB8), verapamil], or dimethylsulfoxide (glyburide, tolazamide) and added to molten agarose at 55 C. Control treatments were prepared with appropriate amounts of dimethylsulfoxide. Inhibitors were purchased from Calbiochem-Novabiochem Corp. (La Jolla, California). Data were collected from 3 independent trials, each run in triplicate. Treatments were compared to controls using PROC GLM of SAS Version 8. For comparisons of different trials, data for particular inhibitors were combined and experiment-wise error rates were controlled with Tukey-Kramer adjustments.
Effects of specific inhibitors on growth of mycelia were also measured. Inhibitors were added to 2% water agar as described above. Plates were center inoculated and radial growth was measured daily until mycelia in control cultures reached the periphery of the petri dish (3–4 d). Trials were run in triplicate.
Effect of osmotic changes on discharge – To investigate the effect of increased osmotic potential outside the asci on discharge of ascospores, assays were set up and analyzed in a similar manner to the ion channel inhibitor assays. Inhibition of mycelial growth by increased levels of mannitol and glycerol was studied in a manner similar to the specific inhibitors, using water agar amended with the appropriate amounts of osmoticum.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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To investigate the temporal pattern of ascospore release, and how this pattern could be affected by selected environmental factors, we used a previously developed apparatus (Gadoury et al 1996
) in which we could precisely control temperature, humidity, rainfall rate, light intensity and quality, and wind speed. Ascospore release followed a sigmoid distribution during simulated rain at 20 C under constant illumination (Fig. 1A
). The pattern of release was linearized by probit transformation, and subjected to linear regression. The linear model explained over 90% of the variation in observed release (Fig. 1B
). There was a significant (P = 0.05) effect of light upon the rate of ascospore release. The slope coefficients of linear models fit to ascospore release during light during h 4–6 of a wetting period (Fig. 2
), or h 3–4 of a wetting period (Fig. 3
) were approximately 8.2% and 31.8% greater, respectively.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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High relative humidity may be sufficient for release of ascospores, but its effect is difficult to separate from that of free water in the field. Subtle changes in microclimates may occur that are not possible to measure, such as condensation due to minor temperature fluctuations in the presence of vegetation, small changes in terrain, and variations in soil type. Paulitz (1996)
studied the pattern of spore release in inoculated field plots over the course of 24 h intervals. Peak release occurred 2 to 4 d after a rainfall. He concluded that rainfall was needed for maturation of ascospores, but not release. Paulitz (1996)
suggested that reports that discharge is inhibited by free water may be due to discharged ascospores being retained in a layer of free water present on the surface of the perithecium (Paulitz 1996
). Indeed, we have observed this phenomenon in the laboratory (unpubl obs). Given the osmotic nature of the discharge mechanism, it would be difficult to reconcile the discharge of ascospores with an absence of free water.
Our results support those of previous reports that turgor pressure is, at least in part, the driving force behind ascospore discharge. In Gibberella zeae, evidence for this is twofold: droplets are discharged along with the spores when an ascus fires (Fig. 4 ), and in the presence of high external osmotic conditions, discharge is inhibited (Table II ).
The presence of mannitol as the major simple sugar component of the ascus sap indicates this polyol is likely to be involved in generation of turgor pressure. In fungi, mannitol biosynthesis occurs by one of two pathways: the NADPH-dependent MTD-catalyzed conversion of fructose to mannitol, and the conversion of fructose-6-phosphate to mannitol-1-phosphate by mannitol-1-phosphate dehydrogenase (NAD-dependent), with the action of a phosphatase making the final conversion to mannitol (Jennings and Burke 1990
). We did not detect the M1PD activity in hyphae nor in perithecia. Hult et al (1980)
also reported that such activity was not detected in G. zeae, although cultural conditions were limited in both studies. Resolution of the role of mannitol in ascospore discharge necessitates the isolation of gene(s) involved in mannitol biosynthesis, such as the MTD gene and the M1PD gene, and the specific disruption of these gene(s) in G. zeae. This work is in progress.
Accumulation of a compatible osmolyte, such as mannitol, for the generation of osmotic pressure has obvious advantages for the preservation of enzyme activity (Jennings and Burke 1990
). Fungi are known to use glycerol, mannitol, sorbitol, trehalose and proline as compatible osmolytes, although other polyols may also be present in osmolyte mixtures (Jennings and Burke 1990
, Davis et al 2000
). A recent study shows that many of the compatible osmolytes do not differ in the osmotic effects at physiological concentrations and thus the evolutionary advantage of a particular one for a specific function is not clear. In Magnaporthe grisea, glycerol has been identified as the osmolyte used to buildup large levels of turgor pressure (De Jong et al 1997
) in the highly melanized appressoria used for leaf penetration. However, glycerol is known to pass through cell membranes at a higher rate than other compatible osmolytes (Davis et al 2000)
. In the absence of a physical barrier such as is found in these specialized appressoria, glycerol may not be as useful as the larger polyols in ascus turgor generation.
The source of mannitol within the ascus is not known. In fungi, lipids, glycogen and trehalose are known to be the main compounds for energy storage (Jennings 1995
). The conversion of glycogen to reducing sugars has been suggested as the general biochemical basis for ascus turgor and forcible discharge (Ingold 1933, 1965
, Jackson and Wheeler 1974
). However, Ingold (1966)
suggested that there is not sufficient glycogen in asci of Sordaria fimicola to account for the change in turgor pressure. Glycogen was not involved in forcible ascospore discharge in Uncinula necator (Gadoury and Pearson 1990
), which appeared to be related to lipid metabolism during ascus maturation. In Magnaporthe grisea, evidence was recently presented in support of lipids as the source of glycerol in appressoria (Weber et al 2001)
The potassium ion channel inhibitors did not inhibit discharge at levels greater than 50%, suggesting the presence of another mechanism also contributing to discharge (Table III ). It is likely that turgor pressure is generated through a combination of mannitol accumulation and K+ influx. Calcium ion channel inhibitors inhibited discharge nearly 100%. Calcium ion fluxes are likely to be involved in signalling discharge rather than direct generation of turgor pressure. Relatively high levels of some of the inhibitors were required to see the inhibitory effect. This may be due to the particular mode of absorption. To function, the inhibitors must be absorbed through the thick-walled, senescing perithecial wall cells into the centrum of the perithecium. We will pursue the identification and characterization of ion channels involved in ascospore discharge as part of an ongoing genomics project.
The present research may assist in the generation of novel control methods for controlling ascospore discharge in plant pathogenic fungi. In the G. zeae-wheat host-pathogen system, host susceptibility to ascospores is limited to the the time of wheat flowering—a relatively short time-period. Once the basic mechanism of discharge is understood on a physiological and genetic level, investigation into the control mechanisms for ascospore discharge and the environmental signals that trigger the mechanism will be pursued. These signals are probably complex—accounting for conflicting field observations.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Current address: Department of Biology, University of Maryland, Baltimore, Maryland
3 Current address: Biolex, Inc.,158 Credle Street, Pittsboro, North Carolina 27312
Accepted for publication July 17, 2001.
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LITERATURE CITED |
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Beckett A., 1981 The ascus with an apical pore: development, composition, and function In: Reynolds DR, ed. Ascomycete systematics. The Luttrellian concept. New York: Springer-Verlag. p 7–26
Burnett JH., 1976 Fundamentals of mycology 2nd ed. London, England: Edward Arnold Publishers. 673 p
Chen XM, Yuan C., 1984 Application of microcomputer in studying wheat scab epidemiology and forecasting Zhejiang Agric Sci 2:55-60
Davis DJ, Burlak C, Money NP., 2000 Osmotic pressure of fungal compatible osmolytes Mycol Res 104:800-804
De Jong JC, McCormack BJ, Smirnoff N, Talbot NJ., 1997 Glycerol generates turgor in rice blast Nature 389:244-245
Fernando WGD, Miller JD, Seaman WL, Seifert K, Paulitz TC., 2000 Daily and seasonal dynamics of airborne spores of Fusarium graminearum and other Fusarium species sampled over wheat plots Can J Bot 78:497-505
———, Paulitz TC, Seaman WL, Dutilleul P, Miller JD., 1997 Head blight gradients caused by Gibberella zeae from area sources of inoculum in wheat field plots Phytopath 87:414-421
Forney CF, Brandl DG., 1992 Control of humidity in small controlled-environment chambers using glycerol-water solutions HortTechnology 2:52-54
Gadoury DM, Pearson RC., 1990 Ascocarp dehiscence and ascospore discharge in Uncinula necator Phytopath 80:393-401
Gadoury DM, Stensvand A, Seem RC., 1996 A wind tunnel for controlled-environment studies of ascospore release by Venturia inaequalis Phytopath 86:596-601
Higgins MK, Bly R, Morgan SL, Fox A., 1994 Differentiation of isomeric alditol hexaacetates and identification of aldohexoses by electron-impact mass-spectrometry Analytical Chemistry 66:2625-2668
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———. 1965 Spore liberation Oxford: Clarendon Press. 210 p
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———. 1971 Fungal spores: their liberation and dispersal Oxford: Clarendon Press. 302 p
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Jennings DH., 1995 The physiology of fungal nutrition Cambridge, England: Cambridge University Press. 622 p
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Klittich CJR, Leslie JF., 1988 Nitrate reduction mutants in Fusarium moniliforme (Gibberella fujikuroi) Genetics 118:417-423
McMullen M, Jones R, Gallenberg D., 1997 Scab of wheat and barley: a re-emerging disease of devastating impact Plant Dis 81:1340-1348
Minter DW, Cannon PF., 1984 Ascospore discharge in some members of the Rhytismataceae Trans Br Mycol Soc 83:65-92
Parry DW, Jenkinson P, McLeod L., 1995 Fusarium ear blight (scab) in small grain cereals—a review Plant-Pathology (UK) 44:207-238
Paulitz TC., 1996 Diurnal release of ascospores by Gibberella zeae in inoculated wheat plots Plant Dis 80:674-678
Read ND, Beckett A., 1996 Ascus and ascospore morphogenesis Mycol Res 100:1281-1314
Reis EM., 1990 Effects of rain and relative humidity on the release of ascospores on the infection of wheat heads by Gibberella zeae Fiopatol Bras 15:339-343
Stoop JMH, Williamson JD, Conkling MA, Pharr DM., 1995 Purification of NAD-dependent mannitol dehydrogenase from celery suspension cultures Plant Physiol 108:1219-1225[Abstract]
Sutton JC., 1982 Epidemiology of wheat head blight and maize ear rot caused by Fusarium graminearum Can J Plant Path 4:195-209
Trail F, Common R., 2000 Perithecium development in Gibberella zeae: a light microscopy study Mycologia 92:130-138
Trail F, Gadoury D, Loranger R., 1998 Environmental parameters of ascospore discharge in Gibberella zeae In: Proceedings of the National Fusarium Head Blight Forum. East Lansing, Michigan: Michigan State University. p 11–13
Tschanz AT, Horst RK, Nelson PE., 1975 Ecological aspects of ascospore discharge in Gibberella zeae Phytopath 65:597-599
Weber RWS, Wakley GE, Thines E, Talbot NJ., 2001 The vacuole as central element of the lytic system and sink for lipid droplets in maturing appressoria of Magnaporthe grisea Protoplasma 216:101-112[Medline]
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