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Impact of osmotic and matric water stress on germination, growth, mycelial water potentials and endogenous accumulation of sugars and sugar alcohols in Fusarium graminearum

     Departamento de Microbiologia e Inmunologia, Facultad de Ciencias Exactas Fco-Qcas y Naturles, Universidad Nacional de Rio Cuarto, Ruta Nacional 36 Km 601(5800), Rio Cuarto, Cordoba, Argentina

N. Magan ¹

     Applied Mycology Group, Cranfield Biotechnology Centre, Cranfield University, Silsoe, Bedford MK45 4DT, UK

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 

Studies were conducted to determine the effect of osmotic (NaCl, glycerol) and matric (PEG 8000) water stress on temporal germination and growth of two F. graminearum strains over the water potential range of –0.7 to –14.0 MPa at 15 and 25 C. The effect on endogenous water potentials and accumulation of sugars and sugar alcohols also were measured. For both strains, germination occurred rapidly over the same range of osmotic or matric potential of –0.7 to –5.6 MPa after 4–6 h incubation. At lower osmotic and matric potentials (–7.0 to –8.4 MPa), there was a lag of up to 24 h before germination. Optimum germ-tube extension occurred between –0.7 and –1.4 MPa for both strains but varied with the solute used. Growth was optimal at –1.4 MPa and 25 C in response to matric stress, with the minimum being about –8.0 and –11.2 MPa at 15 and 25 C, respectively. In contrast, F. graminearum grew fastest at –0.7 MPa and was more tolerant of solute stress modified with either glycerol or NaCl with a minimum of about –14.0 MPa at 15 and 25 C. A decrease in the osmotic/matric water potential of the media caused a large decrease in the mycelial water potential (_c) as measured by thermocouple psychrometry. In general, the concentration of total sugar alcohols in mycelia increased as osmotic and matric potential were reduced to –1.2 MPa. However, this increase was more evident in mycelia from glycerol-amended media. The quality of the major sugar alcohol accumulated depended on the solute used to generate the water stress. The major compounds accumulated were glycerol and arabitol on osmotically modified media and arabitol on matrically modified media. In response to matric stress, the concentration of trehalose in colonies generally was higher in the case of osmotic stress. In each water-stress treatment there was a good correlation between _c and total sugar alcohol content.

Key words: Fusarium graminearum, germination, glucose, growth, matric, osmotic, sugar alcohols, trehalose, water stress

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Fusarium graminearum Schw. (teleomorph is Gibberella zeae [Schw.] Petch.) in Argentina predominates among several Fusarium species causing Fusarium head blight of wheat. F. graminearum also is associated with stalk and ear rot of maize and may cause cereal root rot. The fungus persists and multiplies on infected crop residues of small grains and maize. The chaff, light-weight kernels and other infected head debris of wheat and barley are returned to the soil surface during harvest and serve as important sites for overwintering of the fungus. Continued moist weather during the crop-growing season favors development of the fungus, and spores are windblown or water-splashed onto heads of cereal crops. Wheat and barley are susceptible to head infection from anthesis to the soft-dough stage of kernel development. Spores of the causal fungus may land on the exposed anthers of the flower and grow into kernels, glumes or other head parts (McMullen et al 1997). Parry et al (1994) considered that soil and crop debris infected with F. graminearum serve as the main reservoir of inoculum leading to wheat ear infection. In most cases, inoculum may take the form of conidia, chlamydospores, hyphal fragments or ascospores (Parry et al 1995).

Numerous studies have been carried out to investigate the influence of water potential on spore germination, growth and sporulation of F. graminearum, but much of the work has been done using osmotically controlled systems with salts, sugars or glycerol (Cook and Christen 1976, Sung and Cook 1981). Fusarium species survive on crop residue. In soil and cereal crop residue, matric potential is the major component of the total water potential (Magan and Lynch 1986). Indeed, Griffin (1981) suggested that matric potential would affect growth of soil fungi more than osmotic potential. However, little information is available on the comparison of the response of F. graminearum to osmotic and matric potential with regard to the different phases of growth, which is relevant to colonization of natural substrate such as crop residue. Work by Willcock and Magan (2001) showed that fungal colonization of crop residues, including that by Fusarium culmorum (W.G. Smith) Sacc., is rapid over a range of moisture and temperature regimes.

To overcome water stress, most fungi produce compatible solutes. These have been defined by Jennings and Burke (1990) as compounds that are able to change in concentration in the cell in response to a change in external water potential, thus maintaining turgidity while having no significant effect on enzyme activity. They first are accumulated within the mycelium and then translocated to the conidia during conidiation. In general, when fungi grow under water stress, the amounts of sugar alcohols accumulated by mycelia change quantitatively but not qualitatively (Pfyffer and Rast 1988). In the higher fungi, low molecular weight sugar alcohols (glycerol and erythritol) are accumulated at the expense of high molecular weight mannitol under water stress ( Jennings 1995). This selective accumulation probably decreases the internal osmotic potential in conidia because glycerol and erythritol molecules are smaller and more polar than mannitol (Hallsworth and Magan 1994a, Jennings 1995).

Fungal germination and growth generally have been demonstrated to be more sensitive to matric than osmotic potential stress (Brownell and Schneider 1985, Magan 1988, Magan et al 1995). However, no attempt has been made to compare and quantify the accumulation of compatible solutes (particularly sugar alcohols) by F. graminearum under such modifications in water availability. Recent studies by Ramos et al (1999) on Aspergillus ochraceus Link suggested that for this xerotolerant mycotoxigenic species there was surprisingly little difference in sensitivity to osmotic and matric stress. Furthermore, there is no information on whether the compatible solutes synthesized or accumulated by F. graminearum may differ when overcoming osmotic or matric stress. Thus the objectives of this study were to compare the effect of osmotic and matric potential stress on (i) germination, (ii) germ-tube extension, (iii) growth, (iv) intracellular water potential of mycelial colonies and (v) endogenous accumulation of low and high molecular weight sugar alcohols in two F. graminearum strains.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Fungal isolates. – F. graminearum strains (RC17-2 and RC22-2) were isolated from Argentinian wheat. These strains are deposited in the collection of Universidad Nacional de Rio Cuarto, Argentina (RC). Cultures were maintained in 15% glycerol at –80 C.

Media. – A 2% white unbleached wheat flour agar (= 0.35 MPa) was modified osmotically by the addition of the ionic solute NaCl (Lang 1967) or the nonionic solute glycerol (Dallyn and Fox, 1980) to –0.7, –1.4, –2.8, –5.6, –8.4, –11.2 and –14.0 MPa (= 0.995, 0.99, 0.98, 0.96, 0.94, 0.92 and 0.90 water activity, respectively). Experiments were carried out by growing the fungus either directly on the agar media or after overlaying the media with a 8.5 cm diam sterile cellophane disks (P400, Cannings Ltd, Bristol, U.K.), used to facilitate removal of the whole colony for sugar alcohol and sugar analyses.

For modification of the matric potential, the agar was omitted and known amounts of PEG 8000 were used (Michel and Kaufmann 1973, Magan 1988), resulting in matric potentials of –0.7, –1.4, –2.8, –5.6 and –8.4 MPa. It previously has been shown that the water potential generated by PEG 8000 is predominantly (99%) due to matric forces (Steuter et al 1981). Sterile disks of capillary matting (8.5 cm diam, 1.5 mm thick, Gardman, Spalding, Lincolnshire, U.K.) were placed in sterile 9 cm Petri dishes to which approx. 15 mL of the cooled medium was added. The matting was overlaid with sterile disk of black polyester lining cloth (0.15 mm thick) and then a cellophane disk.

The water potential of representative samples of media was checked with an Aqualab Series 3 water activity meter (Labcell Ltd., Basingstoke, Hants, U.K.) and converted to water potential. Petri plates were inoculated centrally with 3 mm diam agar plug from the margin of 7 d old colonies on 2% malt-extract agar. Inoculated plates of the same water potential were sealed in polyethylene bags. Triplicate sets of each treatment (solute x water potential) were incubated at 15 and 25 C for 20 d and all experiments repeated once.

Two perpendicular diameters of the growing colonies were measured daily until the colony reached the edge of the plate. The radii of the colonies were plotted against time for each replicate, and linear regression was applied to obtain the growth rate (mm/day) as the slope of the line.

Spore germination and germ-tube growth studies. – F. graminearum strains were grown on synthetic nutrient agar (Gerlach and Nirenberg 1982) 14 d, resulting in heavily sporulating cultures, which were flooded with 10 mL sterile water, and the spores dislodged by gently rubbing the surface with a sterile glass spreader. Stock suspension (1 mL) was added to 25 mL Universal bottles containing 9 mL sterile water amended to the appropriate osmotic or matric potential (–0.7 to –14.0 MPa) with glycerol, NaCl or PEG 8000. The final concentration of spores was in the range of 1–5 x 10⁵ per mL.

A 100 µL spore suspension in osmotic solutions (glycerol and NaCl) was pipetted onto 2% wheat-flour agar plates of the same osmotic potential, spread with a glass spreader and incubated at 25 C in polyethylene bags for 24–48 h. Experiments were carried out with three replicates per treatment and repeated twice.

The system for testing the effect of matric potential on germination consisted of a 9 cm Petri dish containing sterile capillary matting. Loops of spore suspensions made up in appropriate PEG solutions were streaked across the surface of 13 mm membrane filters (Nucleopore, polycarbonate 0.2 µm membranes), which were placed carefully on the capillary matting previously soaked with about 15 mL of 2% wheat-flour suspension amended with PEG 8000 solution of the same matric potential. Three replicate membrane filters were used for each matric potential, and the experiments were repeated twice. Petri dishes were sealed in polyethylene bags and incubated at 25 C. Water potential of all media was checked with an Aqualab Series 3 water activity meter (Labcell Ltd., Basingstoke, Hants, U.K.).

Three agar plugs from each replicate (osmotic potential medium) were aseptically removed every hour from each treatment plate using a cork borer (10 mm diam) and placed on a slide. Replicate membrane filters from the matric potential plates were removed with forceps and placed on labelled slides. The agar plugs were stained with cotton blue/lactophenol and examined microscopically. A total of 50 spores per agar plug (150 per replicate plate, 450 per treatment) were counted. Spores were considered germinated when the germ-tube length was equal to or longer than the diameter of the spore. Germ-tube growth was measured for up to 25 spores for each osmotic and matric potential at random using an eyepiece micrometer.

Measurements of water potential in the fungal tissue. – Thermocouple psychrometry was applied to determine the water potential of fungal tissue. After 21 d subsamples of each solute and matric treatment were placed in the sample well. A HR-33T Dew Point Microvoltmeter coupled to a C-52 chamber was used (Wescor, Logan, Utah; Beecher et al 2001). All measurements were made in the dew-point mode after calibration with a series of NaCl solutions of known _s. Three measurements of each replicate were made and µV readings subsequently were transformed into MPa using a standard curve developed with know molar concentrations of NaCl.

Sugar alcohol and sugar analysis. – Fresh mycelia (10–50 mg) from all the above 21 d old treatments were weighed in 2 mL Eppendorf microtubes. One mL of HPLC-grade water was added to each sample. The mixture was sonicated 1 min at 28 µm (Fisons Soniprep 150, 3.5 mm diam exponential probe). The samples were boiled in a water bath 5.5 min and allowed to cool (Hallsworth and Magan 1994a). Acetonitrile:water (650 µL; 40:60) was added to the samples to maintain the same ratio as the mobile phase. Samples were shaken vigorously and then centrifuged at 13 000 rpm in a microfuge for 15 min. The resulting supernatant was taken up into a sterile 1 mL syringe and filtered through a 0.2 µm, 13 mm diam, nylon syringe filter (Whatman) directly into HPLC vials and sealed.

The sugar alcohol and sugar content were analyzed with a Gilson modular HPLC system, a Hamilton HC-75 Ca²⁺ column and a RI detector, using acetonitrile:water (40:60) as the mobile phase with a flow rate of 1 mL/min. The peak areas were integrated and compared with calibration curves constructed with standards of 100–1000 ppm of each analyzed sugar alcohol (glycerol, erythritol, arabitol and mannitol) and sugar (trehalose and glucose). Sugar alcohol and sugar contents were calculated as µg/mg fresh weight of mycelia. Three replicates of each treatment were analyzed, and the experiment was repeated twice.

Statistical treatment of the results. – The linear regression of increase in radius against time (in days) was used to obtain the growth rates (mm/day) under each set of treatment conditions. The percentage germination and germ-tube length after 4 and 6 h of incubation at different water potential values were logit (log x[x/(100 – x)]) and natural log (ln) transformed respectively to homogenize variance previous to analysis of variance (ANOVA). The growth rates, percentage of germination, germ-tube length (µm), mycelial water potential and sugars and sugar alcohol concentrations were evaluated by ANOVA for each experiment to determine effect of water potential, solute, isolate and two-and three-way interactions.

When the analysis was statistically significant, the Tukey’s multiple-comparison procedure test was used for separation of the means. Statistical significance was determined at the level P < 0.05. Pearson correlation coefficients between endogenous reserves and mycelial water potential also were calculated. All the studies (ANOVA and correlation) were made by using SigmaStat for Windows version 2.03 (SPSS Inc.).

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Comparison between effects of osmotic and matric potential on germination. – The effect of osmotic and matric potential on macroconidial germination of two strains (4 and 6 h of incubation) are shown in FIG. 1. For both strains, germination occurred over the same range of osmotic or matric potential at both incubation times. The minimum water potential for germination at both incubation times was –8.4 MPa, although under this condition the lag before germination was 24 h and germination was slow. ANOVA showed that the effects of solute, water potential and two- and three-way interactions with the strains were significant statistically (P < 0.001, data not shown). However, there was no significant difference between the response of the two strains.

View larger version (20K): [in this window] [in a new window]   FIG. 1. Effect of water potential modified with glycerol (), NaCl () and PEG 8000 () on germination of F. graminearum spores after incubation at 25 C.

View larger version (17K): [in this window] [in a new window]   FIG. 2. Effect of water potential, modified with glycerol (), NaCl () and polyethylene glycol 8000 () on germ-tube length (µm) two strains of Fusarium graminearum (RC 17-2 and 22-2) after incubation at 25 C.

View larger version (27K): [in this window] [in a new window]   FIG. 3. Comparison of the effect of water potential modified with NaCl (), glycerol () and matrically modified with PEG 8000 (), on growth rates of F. graminearum RC 17-2 and RC 22-2 at 15 and 25 C.

View larger version (44K): [in this window] [in a new window]   FIG. 4. Effect of water potential, modified with glycerol (), NaCl () and PEG 8000 () on mycelial water potential in F. graminearum RC 17-2 (a) and RC 22-2 (b). Columns with the different letters indicate significant differences between solutes according to Tukey Test within each water potential treatment (P < 0.05).

View larger version (22K): [in this window] [in a new window]   FIG. 5. Effect of water potential modified with glycerol (), NaCRC 22-21 () and PEG 8000 (), on intracellular accumulation of sugars in mycelia of F. graminearum RC 17-2 (—) and RC 22-2 (- - -).

View this table: [in this window] [in a new window]   TABLE II. Correlation between sugar and sugar alcohol concentrations and mycelial water potential (c) for each solute used to modify water potential

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Sung and Cook (1981) investigated the influence of water potential on production of macroconidia and perithecia, and on germination of macroconidia, ascospores and chlamydospores of Fusarium roseum ‘‘Graminearum’’ Group II (F. graminearum), but this work was done using osmotically controlled systems with salts (NaCl and KCl) only. They found that sporulation was maximal at about –0.14 to –0.3 and perithecial production was maximal at –0.14 to –1.5 MPa. Spore germination was uniformly maximal at all the water potentials between –0.1 and –2.0 MPa.

Both strains of F. graminearum grew fastest at –1.4 MPa at both temperatures on matrically modified media. With nonionic and ionic solutes, both strains grew fastest at –0.7 MPa at both temperatures. Mycelial extension of other fungi, such as Alternaria alternata, Microdochium bolleyii, and a range of basidiomycetes previously have been found to be significantly more sensitive to matric than osmotic potential stress (Adebayo and Harris 1971, Douglas and Deacon 1994, Magan et al 1995, Mswaka and Magan 1999).

Sung and Cook (1981) also found that many kinds of living plant tissues, especially dryland wheat, have water potential between –1.0 and –5.0 MPa and are thus in the ideal range for sporulation, spore germination, perithecial production and hyphal extension. However, in soil and cereal crop residues, matric potential is the major component of the total water potential (Magan and Lynch 1986). Thus information on tolerance to matric stress in such systems may be more important. The present study suggests that there are some differences in tolerance with regard to both germination and mycelial extension in response to matric and osmotic imposed water stress.

The total cell water potential (_c) is the sum of the solute potential () and the turgor pressure (_p) of the cell wall. When cells are exposed to water stress, low molecular mass compounds often are synthesized or accumulated intracellularly to equilibrate the cytoplasm _c with that of the surrounding environment. This is the first detailed study and measurement of the _c levels of F. graminearum mycelia as effected by osmotic and matric water stress. Our results show that mycelial _c decreased with decreasing medium water potential, regardless whether osmotic or matric stress was imposed. The decrease in total cellular water potential is necessary for the extraction of water from the substrate and its translocation to the growing mycelial front. This can be done effectively only by maintaining a water potential gradient from the substrate into the hyphal cells, which also facilitates the functioning of enzyme systems ( Jennings 1995).

There is a wealth of information on the physiological adaptation of yeasts and some filamentous fungi to solute stress. However, few studies have examined the effects of matric stress on fungal activity (Blomberg and Adler 1992, Magan 1997). The present study shows that the patterns of accumulation of sugar alcohols and sugars by F. graminearum are modified significantly by osmotic and matric water stress. When glycerol was used to modify osmotic potential, there was a significant increase in glycerol content of the colonies, perhaps via passive diffusion and some endogenous synthesis. When NaCl was used to modify osmotic potential, there was a marked increase in glycerol and arabitol content in the mycelia, suggesting endogenous synthesis to overcome the imposed water stress. The increase in glycerol content is expected because it is a better internal water potential adaptation solute than other high molecular weight sugar alcohols (Magan 1997). It is interesting to note that, although Griffin (1981) suggested that matric imposed water stress is more difficult to overcome, F. graminearum accumulated increased levels of arabitol under extreme matric stress (–11.4 MPa), which is not as effective a compatible solute in allowing enzyme systems to function as glycerol or erythritol (Chirife et al 1984). This shows that matric stress may not be as imposing for pathogens such as F. graminearum, which are adapted for effective and efficient survival and growth on cereal residues. Similar results were obtained for A. ochraceus when studying partitioning of sugars and sugar alcohols into mycelium and conidia under osmotic and matric water stress (Ramos et al 1999).

The accumulation of progressively lower molecular weight polyols in fungi as water potential stress increased has been demonstrated previously for other fungal species (Brown 1978, Adler et al 1982, Luard 1982, Hocking and Norton 1983, Meikee et al 1991, van Eck et al 1993, Hallsworth and Magan 1994a, b, Ramos et al 1999). When considering the functions of the sugar alcohols in intracellular osmotic adjustment, the roles of individual compounds become more important because they are differentially effective as compatible solute. High molecular weight polyols (e.g., mannitol) cause slight inhibition of enzymatic activity compared to low molecular weight polyols (e.g., glycerol) at equivalent concentrations (Chirife et al 1984). Glycerol is more important relative to the other polyols at the same molar concentration, followed by arabitol, erythritol and then mannitol (Magan 1997). With regard to carbon energy, glycerol production represents an effective method of osmoregulation (Hocking 1993).

There were also marked changes in the content of trehalose and glucose in mycelia of F. graminearum in relation to osmotic and matric potential modification. With matrically imposed water stress, the concentration of trehalose in colonies generally was higher than on osmotically modified media. This is of interest as synthesis of higher trehalose concentrations improves desiccation tolerance of the conidia. This may contribute to survival in the environment and improve potential for subsequent infection. Trehalose has been shown to interact with hydrated cell components in relation to heat shock and desiccation (Crowe et al 1984). The osmotic effect of trehalose accumulation, therefore, may be of secondary value due to its other cellular functions (Davis et al 2000).

We have demonstrated that F. graminearum accumulates a combination of different sugar alcohols like many fungi in response to osmotic and matric stress. There are many reasons why an organism might produce a cocktail of osmolytes rather than a single compound (Davis et al 2000). For example, mixtures of sugar alcohols increase the water pressure in a more efficient way and also may reduce the toxicity associated with high concentrations of a single osmolyte and obviate feedback mechanisms that down regulate metabolic pathways in the presence of high concentrations of product. These studies enable a better understanding of the survival and growth strategy employed by F. graminearum and other similar pathogens for survival, growth and establishment in natural ecosystems.

	ACKNOWLEDGMENTS

 
Dr. M.L. Ramirez is grateful to Consejo Nacional de Investigaciones Cientificas y Tecnicas de la Republica Argentina (CONICET) for a postdoctoral fellowship held in the Applied Mycology Group of Cranfield Biotechnology Centre, Cranfield University, U.K.

	FOOTNOTES

 
Accepted for publication August 2, 2003.

¹ Corresponding author: Prof. N. Magan, Applied Mycology Group, Cranfield Biotechnology Centre, Cranfield University, Silsoe, Bedford MK45 4DT, UK. Tel: +44 1525 863539. Fax: +44 1525 863540. E-mail: n.magan{at}cranfield.ac.uk

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