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Fluorescent microscopic technique for viewing fungi in soil and its application to studies of a fusarium-suppressive soil
Soil Bid.
Biochem. Vol. 15, No. 6, PP. 715-718, 1983 Printed in Great Britain. All rights reserved
0038-0717/83 $3.00 + 0.00 Copyright 0 1983 Pergamon Press Ltd
SHORT COMMUNICATION FLUORESCENT MICROSCOPIC TECHNIQUE FUNGI IN SOIL AND ITS APPLICATION OF A FUSARIUM-SUPPRESSIVE FRAN M. Department
of Botany
and Plant Pathology,
FOR VIEWING TO STUDIES SOIL
SCHER* and R. BAKER Colorado
State University,
Fort Collins,
CO 80523, U.S.A.
(Accepted 20 July 1983)
Calcofluor solution (Calcofluor White M2R, Polysciences, Inc., Warrington, PA). The soil was held at 25°C for 1 to 24 h after staining. Four soil smears from each of the subsamples were prepared and viewed with an Olympus BH Microscope (Olympus Optical Co., Tokyo, Japan) equipped with epiifluorescent illuminator, blue excitor filter (BG-12, transmitting 400 nm light) and a 530 nm barrier filter. Slides with soil smears were made at 1, 2, 3, 5, IO and 24 h after addition of Calcoflour to the subsamples in order to determine the optimum staining time. When soil smears were viewed microscopically under fluorescent light, the chlamydospores fluoresced yellow-green. They were discernible after 1 h staining but optimum staining time was 3-10 h for maximum brightness. After 24 h, germ tubes remained weakly fluorescent, with a concentration of brightness at their tips, as described by Darken (1962). The number of germinated and non-germinated chlamydospores in five random areas on each slide was recorded and percentage germination determined. A chlamydospore was deemed germinated if its germ tube was as least as long as the diameter of the spore. Percentage germination was significantly less in the Fusarium-suppressive soil than theconducive soil (Table 1, Fig. I), confirming the results of Smith and Snyder (1971) and germ tube lengths were approx. 2&100 times greater in conducive than suppressive soil. Addition of chelates had no significant effect on germination in either soil, although previous addition of FeEDDHA controlled and FeEDTA increased Fusarium wilt diseases when added to conducive soil (Scher and Baker, 1983). No significant effect on chlamydospore germination was noted here, suggesting that these chelates influenced the development of Fusarium wilt disease by interactions that occurred in the rhizosphere of plants, not in bulk nonrhizosphere soil. Since the fluorescent light source used originated from above the soil smears, visibility of germinating chlamydospores was much greater than with phase-contrast micros-
The study of fungal propagules in soil by conventional light microscopy and staining precedures is difficult due to failure to detect propagules obscured by soil particles. Fluorescence microscopy has been used to view fungal spores in soil (Eren and Pramer, 1967: Tsao, 1970) but treatment and staining of the spores with a fluorescent brightener (FB) was required before their addition to soil. Darken (1964) demonstrated that FB increased the germination of fungal spores and, thus, such treatment could substantially alter subsequent fungal development in soil. We describe a simple technique for staining fungal propagules that, unlike the earlier methods, allows the spores to germinate in soil without the influence of FB. The usefulness of our technique was demonstrated by comparing germination of Fusarium oxysporum Schlecht f. sp. hi (Bolley) Snyd. and Hans. chlamydospores in Fusarium wiltconducive and -suppressive soils with and without iron chelates. We previously suggested (Scher and Baker, 1983) that addition of the chelate ethylenediamine di-ohydroxyphenyl-acetic acid (EDDHA) to conducive soil induced suppressiveness to Fusarium wilt diseases because of the high iron-binding ability of EDDHA, which rendered iron unavailable to the pathogen (Kloepper et al., 1980). In contrast, addition of ethylenediamine tetraacetic acid (EDTA) to suppressive soil nullified suppressiveness by releasing loosely-held iron and thereby making iron more available to the pathogen (Kloepper ef al., 1980; Scher and Baker, 1983). F. oxysporum f. sp. lini was grown on potato dextrose agar for 1 week. Conidia and hyphae were scraped from the surface of the agar, homogenized with water in a Waring Blendor (Model 501 I, Waring Products, New Hartford, CT) for 5 min and sieved (< I mm). Ten ml were mixed into each of three 50 g samples of Fusarium wilt-conducive (Colorado sandy loam) and -suppressive (Salinas Valley, California sandy loam) soils. In some treatments, the chelates EDDHA, EDTA, or their ferric complexes, were added (I mg g-t). The soil was air dried for 2 weeks, moistened with IO ml water 50 gg’ soil and air dried for 2 weeks. At this time most of the hyphal fragments and conidia had lysed or converted to chlamydospores. This procedure resulted in approximately IO6 Fusarium propagules gg’ soil. To study chlamydospore germination, 0.2 g soil subsamples were placed in wells in a plastic multi-well tissue-culture plate. Chlamydospores were stimulated to germinate by the addition of 0.1 ml of an aqueous 0.1% glucose, 0.1% asparagine solution to each subsample (Smith and Snyder, 1971). After 16 h at 25°C chlamydospores were stained for viewing by the addition to each subsample of 0.1 ml of a 0.3% *Present address: Allelix Inc., 6850 Goreway sissauga, Ontario, Canada L4V lP1.
Drive,
Table 1. Mean percent germination of i%sarium o.x~~s~orum f. sp. lini chlamydospores in conducive and suppressive soil with and without iron chelates Soil type’ Conducive Suppressive
Mis-
Control
78”
45b
FeEDDHA EDDHA FeEDTA
74” 80” 72”
47b 4lb 49b
‘Treatments with like letters are not significantly different 715
(P = 0.05).
Short communications
716
copy (Fig. 2). where light from below was blocked due to soil particles. Fluorescent staining caused the spores to glow brightly against the dark background of soil and allowed for easy spore location and assessment of germination. Addition of FB after spore germination eliminated the influence of the stain on the germination process. Use of fluorescence microscopy and the fluorescent brightener Calcofluor increased visibility of spores in soil, thereby improving precision in assessment of germination. and the viability of the fungal propagules in soil was retained as well. With this method, it was shown that Fusarium oxysporum f. sp. lini chlamydospores germinated less in suppressive soil than in conducive soil and that the action of iron chelates in bulk soil did not overcome this suppression.
REFERENCES
Darken M. A. (1962) Absorption and fluorescent brighteners by microorganisms. crobiology 10, 387-393.
transport of Applied Mi-
Darken M. A. (1964) Effect of brightener on spore germination. Mycologia 56, 1588162. Eren J. and Pramer D. (1967) Use of a fluorescent btightener as aid to studies of fungistasis and nematophagous fungi in soil. Phytopafhology 58, 644-646. Kloepper J. W., Leonr J.. Teintze M. and Scroth M. N. (1980) Pseudomonas
Fig. 1. Fusarium oxysporum f. sp. lini chlamydospores germinating in conducive (a) and suppressive (b) soil. (Mag. 10 mm = 20 pm).
717
Fig. 2. Fusarium oxysporum f. sp. hi chlamydospores germinating in conducive soil as viewed by fluorescence (a) and phase-contrast (b) microscopy. (Mag. 10 mm = 20 pm). 718
Biochem. Vol. 15, No. 6, PP. 715-718, 1983 Printed in Great Britain. All rights reserved
0038-0717/83 $3.00 + 0.00 Copyright 0 1983 Pergamon Press Ltd
SHORT COMMUNICATION FLUORESCENT MICROSCOPIC TECHNIQUE FUNGI IN SOIL AND ITS APPLICATION OF A FUSARIUM-SUPPRESSIVE FRAN M. Department
of Botany
and Plant Pathology,
FOR VIEWING TO STUDIES SOIL
SCHER* and R. BAKER Colorado
State University,
Fort Collins,
CO 80523, U.S.A.
(Accepted 20 July 1983)
Calcofluor solution (Calcofluor White M2R, Polysciences, Inc., Warrington, PA). The soil was held at 25°C for 1 to 24 h after staining. Four soil smears from each of the subsamples were prepared and viewed with an Olympus BH Microscope (Olympus Optical Co., Tokyo, Japan) equipped with epiifluorescent illuminator, blue excitor filter (BG-12, transmitting 400 nm light) and a 530 nm barrier filter. Slides with soil smears were made at 1, 2, 3, 5, IO and 24 h after addition of Calcoflour to the subsamples in order to determine the optimum staining time. When soil smears were viewed microscopically under fluorescent light, the chlamydospores fluoresced yellow-green. They were discernible after 1 h staining but optimum staining time was 3-10 h for maximum brightness. After 24 h, germ tubes remained weakly fluorescent, with a concentration of brightness at their tips, as described by Darken (1962). The number of germinated and non-germinated chlamydospores in five random areas on each slide was recorded and percentage germination determined. A chlamydospore was deemed germinated if its germ tube was as least as long as the diameter of the spore. Percentage germination was significantly less in the Fusarium-suppressive soil than theconducive soil (Table 1, Fig. I), confirming the results of Smith and Snyder (1971) and germ tube lengths were approx. 2&100 times greater in conducive than suppressive soil. Addition of chelates had no significant effect on germination in either soil, although previous addition of FeEDDHA controlled and FeEDTA increased Fusarium wilt diseases when added to conducive soil (Scher and Baker, 1983). No significant effect on chlamydospore germination was noted here, suggesting that these chelates influenced the development of Fusarium wilt disease by interactions that occurred in the rhizosphere of plants, not in bulk nonrhizosphere soil. Since the fluorescent light source used originated from above the soil smears, visibility of germinating chlamydospores was much greater than with phase-contrast micros-
The study of fungal propagules in soil by conventional light microscopy and staining precedures is difficult due to failure to detect propagules obscured by soil particles. Fluorescence microscopy has been used to view fungal spores in soil (Eren and Pramer, 1967: Tsao, 1970) but treatment and staining of the spores with a fluorescent brightener (FB) was required before their addition to soil. Darken (1964) demonstrated that FB increased the germination of fungal spores and, thus, such treatment could substantially alter subsequent fungal development in soil. We describe a simple technique for staining fungal propagules that, unlike the earlier methods, allows the spores to germinate in soil without the influence of FB. The usefulness of our technique was demonstrated by comparing germination of Fusarium oxysporum Schlecht f. sp. hi (Bolley) Snyd. and Hans. chlamydospores in Fusarium wiltconducive and -suppressive soils with and without iron chelates. We previously suggested (Scher and Baker, 1983) that addition of the chelate ethylenediamine di-ohydroxyphenyl-acetic acid (EDDHA) to conducive soil induced suppressiveness to Fusarium wilt diseases because of the high iron-binding ability of EDDHA, which rendered iron unavailable to the pathogen (Kloepper et al., 1980). In contrast, addition of ethylenediamine tetraacetic acid (EDTA) to suppressive soil nullified suppressiveness by releasing loosely-held iron and thereby making iron more available to the pathogen (Kloepper ef al., 1980; Scher and Baker, 1983). F. oxysporum f. sp. lini was grown on potato dextrose agar for 1 week. Conidia and hyphae were scraped from the surface of the agar, homogenized with water in a Waring Blendor (Model 501 I, Waring Products, New Hartford, CT) for 5 min and sieved (< I mm). Ten ml were mixed into each of three 50 g samples of Fusarium wilt-conducive (Colorado sandy loam) and -suppressive (Salinas Valley, California sandy loam) soils. In some treatments, the chelates EDDHA, EDTA, or their ferric complexes, were added (I mg g-t). The soil was air dried for 2 weeks, moistened with IO ml water 50 gg’ soil and air dried for 2 weeks. At this time most of the hyphal fragments and conidia had lysed or converted to chlamydospores. This procedure resulted in approximately IO6 Fusarium propagules gg’ soil. To study chlamydospore germination, 0.2 g soil subsamples were placed in wells in a plastic multi-well tissue-culture plate. Chlamydospores were stimulated to germinate by the addition of 0.1 ml of an aqueous 0.1% glucose, 0.1% asparagine solution to each subsample (Smith and Snyder, 1971). After 16 h at 25°C chlamydospores were stained for viewing by the addition to each subsample of 0.1 ml of a 0.3% *Present address: Allelix Inc., 6850 Goreway sissauga, Ontario, Canada L4V lP1.
Drive,
Table 1. Mean percent germination of i%sarium o.x~~s~orum f. sp. lini chlamydospores in conducive and suppressive soil with and without iron chelates Soil type’ Conducive Suppressive
Mis-
Control
78”
45b
FeEDDHA EDDHA FeEDTA
74” 80” 72”
47b 4lb 49b
‘Treatments with like letters are not significantly different 715
(P = 0.05).
Short communications
716
copy (Fig. 2). where light from below was blocked due to soil particles. Fluorescent staining caused the spores to glow brightly against the dark background of soil and allowed for easy spore location and assessment of germination. Addition of FB after spore germination eliminated the influence of the stain on the germination process. Use of fluorescence microscopy and the fluorescent brightener Calcofluor increased visibility of spores in soil, thereby improving precision in assessment of germination. and the viability of the fungal propagules in soil was retained as well. With this method, it was shown that Fusarium oxysporum f. sp. lini chlamydospores germinated less in suppressive soil than in conducive soil and that the action of iron chelates in bulk soil did not overcome this suppression.
REFERENCES
Darken M. A. (1962) Absorption and fluorescent brighteners by microorganisms. crobiology 10, 387-393.
transport of Applied Mi-
Darken M. A. (1964) Effect of brightener on spore germination. Mycologia 56, 1588162. Eren J. and Pramer D. (1967) Use of a fluorescent btightener as aid to studies of fungistasis and nematophagous fungi in soil. Phytopafhology 58, 644-646. Kloepper J. W., Leonr J.. Teintze M. and Scroth M. N. (1980) Pseudomonas
Fig. 1. Fusarium oxysporum f. sp. lini chlamydospores germinating in conducive (a) and suppressive (b) soil. (Mag. 10 mm = 20 pm).
717
Fig. 2. Fusarium oxysporum f. sp. hi chlamydospores germinating in conducive soil as viewed by fluorescence (a) and phase-contrast (b) microscopy. (Mag. 10 mm = 20 pm). 718