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Applied and Environmental Microbiology, December 1998, p. 5004-5007, Vol. 64, No. 12
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Ink and Vinegar, a Simple Staining Technique for
Arbuscular-Mycorrhizal Fungi
Horst
Vierheilig,1,2,*
Andrew
P.
Coughlan,1
Urs
Wyss,2 and
Yves
Piché1
Centre de Recherche en Biologie
Forestiére, Université Laval, Québec G1K 7P4,
Canada,1 and
Institut für
Phytopathologie, Christian-Albrecht-Universität Kiel, D-24118
Kiel, Germany2
Received 17 June 1998/Accepted 6 October 1998
 |
ABSTRACT |
We developed a reliable, inexpensive, and simple method for
staining arbuscular-mycorrhizal fungal colonizations in root tissues. Apart from applications in research, this nontoxic, high-quality staining method also could be of great utility in teaching exercises. After adequate clearing with KOH, an ink-vinegar solution successfully stained all fungal structures, rendering them clearly visible.
| |
TEXT |
Healthy, fertile soils are
characterized by the presence of a diverse population of
microorganisms, an important component of which are
arbuscular-mycorrhizal (AM) fungi (12). The arbuscular mycorrhiza is a symbiotic association formed between the roots of
members of over 80% of all families of land plants and a small group
of common soil-borne zygomycetic fungi (Glomales). In
general, this association is beneficial for both partners. The host
plant receives mineral nutrients from outside the root's depletion
zone via the extraradical fungal mycelium, while the heterotrophic mycobiont obtains photosynthetically produced carbon compounds from the
host (18). Research into the establishment and role of
mycorrhizal associations in natural ecosystems is of fundamental importance. Although data are available from several North American and
European ecosystems, few data have been obtained from developing countries where the maintenance of AM fungal populations could be
essential for sustainable agriculture. The availability and cost of
chemicals used for staining of AM fungi within root tissues, a basic
technique in AM research, can be constraints in some countries.
Phillips and Hayman (15) developed a method of staining
AM fungal structures in roots that uses trypan blue. Trypan blue is listed by the International Agency for Research on Cancer as a
possible carcinogen (9). Another frequently
applied technique (3) uses the possibly carcinogenic
dye chlorazol black E (10). Acid fuchsin,
which also is used to stain AM fungi in roots (7), is also a
suspected carcinogen (5). In addition, HCl, although used at
a low concentration, is frequently applied for the acidification of
roots after clearing with KOH (7, 15).
The use of such chemicals should be reduced for health and safety
reasons. Contact with caustic chemicals may cause skin irritation (2), and their vapors may irritate the eyes, nose, throat, and lungs (16, 17). For environmental reasons it is
preferable, wherever possible, to find substitutes for harmful
chemicals. The "International Directory of Mycorrhizologists" lists
more than 1,000 mycorrhizologists in 77 countries worldwide
(6); thus, we estimate that tens of thousands of root
samples are stained per year. In an attempt to eliminate some of the
hazardous compounds, a modified procedure for staining of AM fungi in
roots has been proposed (11); however, the carcinogenic dye
trypan blue is still used. Recently, a simple staining technique with
an ink-25% acetic acid solution for screening of
Pseudocercosporella herpotrichoides infection in wheat
leaves was developed (14). Our objective was to determine
whether this technique can be adapted for staining of AM fungi in
roots, thus replacing toxic chemicals with nontoxic but equally
effective products.
Biological materials.
Seeds of plants from different families
having differing root characteristics (bean [Phaseolus
vulgaris L.], soybean [Glycine max L.], cucumber
[Cucumis sativus L.], maize [Zea mays L.],
wheat [Tritium aestivum L.], barley [Hordeum
vulgare L.], and ryegrass [Lolium perenne L.]) were
surface sterilized by soaking in 0.75% sodium hypochlorite for
5 min, rinsed with tap water, and germinated in vermiculite.
Seedlings (7 days old) were transferred to a steam-sterilized (40 min,
120°C) mixture of usual silicate sand (article number 142, 265;
Bourbeau et Fils Inc., Charlesbourg, Québec, Canada), TurFace
(baked clay substrate mechanically broken into pieces with diameters of
approximately 5 mm) (Applied Industrial Materials, Corp., Buffalo
Grove, Ill.), and soil (2:2:1, vol/vol/vol) (from the tree nursery of
the Université Laval, Québec, Canada) containing an
inoculum of one of the three AM fungi (Glomus mosseae [BEG 12; La Banque Européenne des Glomales, International
Institute of Biotechnology, Kent, England], Glomus
intraradices [DAOM 197198; Biosystemic Research Centre,
Ottawa, Canada], or Gigaspora margarita [DAOM 194757]).
Plants grown in a growth chamber (day/night cycle of 16 h,
22°C/8 h, 20°C; relative humidity, 50%) were harvested 7 weeks
after inoculation. After removal of the growth medium, roots were
rinsed with tap water.
For each of the plant species, roots from at least five replicate
plants per treatment were stained. When inks successfully stained the
fungi in the roots, root samples from at least another 20 replicates
were performed.
For comparisons of results obtained with an ink to those by a standard
method using a dye, trypan blue (
15), 7-day-old cucumber,
bean, and wheat seedlings were inoculated with
G. mosseae.
Four
weeks after inoculation, plants were harvested and their roots
were stained. The percentage of root colonization was determined
by the
line-intersect method (
1), with the colonization in
root
systems of five plants per treatment
quantified.
Staining.
Roots were cleared by boiling in 10% (wt/vol) KOH
(15) (boiling times differed according to the type of plant
[see Table 3]) and then rinsed several times with tap water. Cleared
roots were boiled for 3 min in a 5% ink-vinegar solution with pure
white household vinegar (5% acetic acid). Roots were destained by
rinsing in tap water (acidified with a few drops of vinegar) or by
rinsing in pure vinegar (destaining times differed according to the
type of ink [see Table 3]). The following inks were tested: purple (Waterman, Paris, France), green (Reynolds, Valence, France), red
(Parker, Boston, Mass.; Lamy, Heidelberg, Germany; and Pelikan, Hannover, Germany), blue (Pelikan; Kreuzer, Hannover, Germany; and
Shaeffer, Ft. Madison, Iowa), and black (Pelikan; Reform, Heidelberg,
Germany; Carrefour, Paris, France; Shaeffer; and Cross, Lincoln, R.I.).
Black ink (Shaeffer) was also tested with a 10% ink solution in 25%
acetic acid, as described by Mauler-Machnik and Nass (
14).
After being destained, roots were kept in tap water at room temperature
in the
laboratory.
Clearing of bean, soybean, and maize roots for 5 min and of cucumber,
wheat, barley, and ryegrass roots for 3 min in 10% KOH
provided
transparent roots suitable for staining. The green, the
purple, two
each of the blue and red, and one of the black inks
did not stain the
fungal tissue. However, one blue, one red and
the other black inks all
stained intraradical and extraradical
AM fungal structures (Table
1; Fig.
1),
allowing observation
of these structures within the roots. Colonized
roots were easily
distinguished from noncolonized roots (Fig.
1a).
Individual hyphae
in heavily and partially colonized sections of roots
were clearly
visible (Fig.
1b). Arbuscules, vesicles, intraradical and
extraradical
hyphae, and penetration units were all stained by the inks
(Fig.
1c to f). For photographic and assessment purposes, the best
contrast
was achieved with Shaeffer black ink. After destaining, roots
were pale brownish red, whereas fungal structures remained black
(Table
1; Fig.
1a to e). Staining with the 10% black ink-25%
acetic acid
solution, as proposed by Mauler-Machnik and Nass (
14),
gave
no better results than staining with a 5% black ink-vinegar
(= 5%
acetic acid) solution. No differences in the degree of root
colonization were observed when roots of plants of different plant
families were stained with trypan blue (standard method
[
15])
or an ink (Table
2).
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FIG. 1.
G. mosseae-colonized roots stained with an
ink-vinegar solution. Roots in panels a to e were stained with black
ink (Shaeffer). (a) Colonized (dark) and noncolonized ryegrass roots.
The colonized root is easily distinguishable from the noncolonized
root. Bar = 25 µm. (b) Individual hyphae in both heavily and
partially colonized sections of a bean root are clearly visible.
Bar = 20 µm. (c) Arbuscules in ryegrass root tissue. Bar = 20 µm. (d) Vesicles and internal hyphae in ryegrass root tissue.
Bar = 20 µm. (e) Arbuscules and internal hyphae in bean root
tissue. Bar = 5 µm. (f) Penetration unit of G. mosseae in a ryegrass root stained with blue ink (Pelikan).
Bar = 20 µm.
|
|
Destaining of the blue and the black inks was most successful when tap
water (with a few drops of vinegar) rather than vinegar
was used (Table
3).
Staining of all three AM fungi
G. mosseae,
G. intraradices, and
G. margaritaby the black
ink-vinegar solutions gave excellent
results. After storage for 6 weeks
in tap water at room temperature
in the laboratory, fungal structures
were as clearly visible as
immediately after completion of the staining
process.
Red ink also stained AM fungi (Table
1). To obtain the best results,
bean roots had to be boiled in KOH for 15 min and ryegrass
roots had to
be boiled for 5 min (Table
3). When roots were boiled
in KOH for
shorter periods of time, the plant tissue remained
heavily stained even
after destaining and the fungal structures
could not be differentiated.
Destaining in vinegar gave better
contrast than destaining in tap water
(Table
3).
Our results with ink-vinegar solutions show that after an adequate
clearing time, good staining and destaining results are
obtained by the
method presented. The time for clearing of root
tissues must be
adjusted depending upon the plant species studied.
This method gives
not only excellent results but also safe alternatives
to the hazardous,
toxic, and potentially carcinogenic chemicals
used in usual staining
techniques, as vinegar, which is used in
human nutrition, is obviously
not harmful and ink, because it
is used by children, is subject to
strict regulations and must
be nontoxic in every respect
(
4).
As not all inks tested were capable of staining AM fungal tissue, and
as it might not always be possible to obtain inks from
the companies we
used in our study, a preassay should be performed
with each specific
ink
considered.
Grace and Stribley (
8) and Koske and Gemma (
11)
proposed modification of the staining method for AM fungi developed by
Phillips and Hayman (
15) which eliminate as many toxic
compounds
as possible. Our method, with ink-vinegar solutions, reduces
still
further the list of potentially hazardous chemicals needed.
Moreover,
by our method, the acidification procedure that follows the
clearing
of roots with KOH (
15) can be
omitted.
Our method provides a simple and safe technique with easily obtainable
compounds and also may be applicable for staining of
other
root-colonizing fungi. For example, on roots of wheat plants
inoculated
with
Rhizoctonia cerealis, extensive mycelium was clearly
visible with some hyphae penetrating the root (results not shown).
This
inexpensive staining technique might stimulate AM research
in parts of
the world where financial resources for scientific
studies are highly
limited. Moreover, the nontoxic chemicals used
make it an excellent
technique for teaching
situations.
| |
ACKNOWLEDGMENTS |
Ryegrass seeds were provided by Yves Desjardins, Centre de
Recherche en Horticulture, Université Laval, Québec,
Canada. We thank Maria Gagyi, Bernhard Holtmann, and Holger Klink,
Department of Phytopathology, University of Kiel, Kiel, Germany, for
helpful advice.
This work was supported by grants from the Deutsche
Forschungsgemeinschaft (Germany) and the NSERC.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Centre de
Recherche en Biologie Forestiére, Université Laval,
Pavillon C.-E.-Marchand, Québec G1K 7P4, Canada. Phone: (418)
656-2131, ext. 8129. Fax: (418) 656-7493. E-mail:
nonhorst{at}rsvs.ulaval.ca.
| |
REFERENCES |
| 1.
|
Ambler, J. R., and J. L. Young.
1977.
Techniques for determining root length infected by vesicular-arbuscular mycorrhizae.
Soil Sci. Soc. Am. J.
41:551-555.
[Abstract/Free Full Text] |
| 2.
|
Arena, J. M.
1986.
Poisoning, 5th ed.
Charles C. Thomas, Springfield, Ill.
|
| 3.
|
Brundrett, M. C.,
Y. Piché, and R. L. Peterson.
1984.
A new method for observing the morphology of vesicular-arbuscular mycorrhizae.
Can. J. Bot.
62:2128-2134.
|
| 4.
|
Colditz, A. W.,
E. Kunkel, and K. H. Bohne.
1987.
Drawing and writing materials, p. 37-47.
In
W. Gerhartz, Y. S. Yamamoto, L. Kaudy, J. F. Rounsaville, and G. Schulz (ed.), Ullmann's encyclopedia of industrial chemistry, vol. A9. VCH Verlagsgesellschaft, Weinheim, Germany.
|
| 5.
|
Combes, R. D., and R. B. Haveland-Smith.
1982.
A review of the genotoxicity of food, drugs and cosmetic colours and other azo, triphenylmethane and xanthene dyes.
Mutat. Res.
98:101-248[Medline].
|
| 6.
|
Furlan, V.
1996.
International directory of mycorrhizologists, 6th ed.
Mycorrhiza
6:279-402.
|
| 7.
|
Gerdemann, J. W.
1955.
Relation of a large soil-borne spore to phycomycetous mycorrhizal infections.
Mycologia
47:619-632.
|
| 8.
|
Grace, C., and P. Stribley.
1991.
A safer procedure for routine staining of vesicular-arbuscular mycorrhizal fungi.
Mycol. Res.
95:1160-1162.
|
| 9.
|
International Agency for Research on Cancer.
1975.
IARC Monogr.
Eval. Carcinog. Risks Man
29:295.
|
| 10.
|
International Agency for Research on Cancer.
1975.
IARC Monogr.
Eval. Carcinog. Risks Man
8:267.
|
| 11.
|
Koske, R. E., and J. N. Gemma.
1989.
A modified procedure for staining roots to detect VA mycorrhizas.
Mycol. Res.
92:486-489.
|
| 12.
|
Linderman, R. G.
1992.
Vesicular-arbuscular mycorrhizae and soil microbial interactions, p. 45-70.
In
G. J. Bethlenfalvay, and R. G. Linderman (ed.), Mycorrhizae in sustainable agriculture. ASA special publication no. 54. Soil Science Society of America, Madison, Wis.
|
| 13.
|
MacDonald, R. M., and M. Lewis.
1978.
The occurrences of some acid phosphatases and dehydrogenases in the vesicular-arbuscular mycorrhizal fungus Glomus mosseae.
New Phytol.
80:135-141.
|
| 14.
|
Mauler-Machnik, A., and P. Nass.
1990.
Einfache Methode zur Frühdiagnose von Pseudocercosporella herpotrichoides mit dem Bayer Getreide-Diagnose-System nach Verreet/Hoffmann.
Gesunde Pflanze
42:130-132.
|
| 15.
|
Phillips, J. M., and D. S. Hayman.
1970.
Improved procedures for clearing roots and staining parasitic and vesicular-arbuscular mycorrhizal fungi for rapid assessment of infection.
Trans. Br. Mycol. Soc.
55:158-161.
|
| 16.
|
Procter, N. H., and J. P. Hughes.
1978.
Chemical hazards in the workplace.
Lippincott, Philadelphia, Pa.
|
| 17.
|
Settig, M.
1979.
Hazardous and toxic effects of industrial chemicals.
Noyes Data Corporation, Park Ridge, Ill.
|
| 18.
|
Smith, S. E., and D. J. Read.
1997.
Mycorrhizal symbiosis.
Academic Press, London, England.
|
Applied and Environmental Microbiology, December 1998, p. 5004-5007, Vol. 64, No. 12
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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