Agricultural and Food Science in Finland, Vol.10 (2001):113 –119

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Vol. 10 (2001): 113–119.

Research Note

Mycorrhizal colonisation of highbush blueberry and
its native relatives in central Finland

Anne Kasurinen, Toini Holopainen
Department of Ecology and Environmental Science, University of Kuopio, PO Box 1627,

FIN-70211 Kuopio, Finland, e-mail: anne.kasurinen@uku.fi

Seija Anttonen
The Finnish Forest Research Institute, Suonenjoki Research Station, FIN-77600 Suonenjoki, Finland

Transmission electromicroscopy, trypan blue staining in combination with stereomicroscope analy-
sis and biochemical ergosterol assay were used to study the mycorrhizal symbionts in wild bilberry
(Vaccinium myrtillus), bog whortleberry (Vaccinium uliginosum) and highbush blueberry (Vaccinium
corymbosum) roots. TEM-analysis showed that in all species ericoid mycorrhizas formed hyphae coil
inside the epidermal root cells. In stereomicroscopic viewing the highest mycorrhizal colonisation
was observed in the roots of wild bilberries (51%), whereas according to the ergosterol assay the
highest total fungal biomass of roots was found in bog whortleberries (209 µg g–1 of root d. wt). Both
ergosterol and microscopical method showed that the mycorrhizal associations in blueberry cultivars
and their wild relatives growing on natural soil medium are frequent, although ericoid mycorrhiza
formation of highbush blueberries was somewhat weaker than that of wild bilberries and bog whortle-
berries.

Key words: ergosterol, ericoid mycorrhiza, highbush blueberry, wild bilberry, bog whortleberry, Vac-
cinium sp.

Introduction

The first highbush blueberry cultivars were hy-
bridized from naturally occurring Vaccinium aus-
trale, V. corymbosum and V. angustifolium blue-
berry species in North America at the beginning
of this century. Since then several new blueber-

ry hybrids have been developed for commercial
as well as home garden use (Luby et al. 1986)
and commercial cultivation of highbush blueber-
ries has spread from North America to Europe.
In Finland, the closest relative species of high-
bush blueberry cultivars (V. corymbosum) are
bog whortleberry (V. uliginosum) and wild bil-
berry (V. myrtillus). All of these species can be

mailto:anne.kasurinen@uku.fi

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Kasurinen, A. et al. Mycorrhizas of wild and cultivated Vaccinium sp.

classified as typical calcifuges, since they thrive
in nutrient poor soils with pH about 5.5 or be-
low (Korcak 1989).

Both cultivated and wild Vaccinium-species
have special symbiotic mycorrhizal associations
(ericoid mycorrhiza) in their root systems (Jac-
quemart 1996, Straker 1996). Mycorrhizas are
considered mutualistic fungus-root associations
(Bouchér et al. 1982), when their effect on both
host plant and fungi fitness is positive and the
net benefits of mycorrhizal symbiosis are great-
er than the net costs (Johnson et al. 1997). In
ericoid mycorrhizas the mycorrhizal hyphae
form coils in the epidermal root cells of host
plant but do not penetrate the root endodermis
or ensheath hair roots with mycorrhizal mantle
(Smith and Read 1997, Deacon 1998). Benefits
of ericoid mycorrhizas to host plant include in-
creased nutrient and mineral uptake and toler-
ance to toxic substances like aluminium (Allen
1991, Read 1991).

Ericoid mycorrhizas are commonly found in
roots of dwarf shrubs throughout temperate and
boreal ecosystems (Bledsoe et al. 1990, Gardes
and Dahlberg 1996), but there are still only few
studies performed to investigate the intensity of
ericoid mycorrhiza formation of plants growing
in forests or plantings. The aim of the present
study was to examine the mycorrhizal status of
cultivated Vaccinium corymbosum as well as
V. uliginosum and V. myrtillus growing in natu-
ral soils and to study especially the intensity of
mycorrhizal development in V. corymbosum un-
der agricultural field conditions. To study the
exact localization and intracellular structure of
the mycorrhizas in epidermal cells we used trans-
mission electron microscopy (Duddridge and
Read 1982, Smith and Read 1997). The mycor-
rhizal colonisation level of roots was estimated
with two different methods, by biochemical er-
gosterol analysis and trypan blue staining com-
bined with stereomicroscopy viewing. Ergosterol
is a principal component of fungal membranes,
and ergosterol assay is a general method used to
determine the total fungal biomass in roots or
soil (Wallander 1992). The root clearing and
staining methods were originally developed for

the assessment of vesicular-arbuscular mycor-
rhizas and parasitic fungi (Phillips and Hayman
1970), but with slight modifications these pro-
cedures are suitable for identification of ericoid
mycorrhizas as well.

Material and methods

The two highbush blueberry (V. corymbosum)
varieties investigated in this study were ‘North-
country’ and ‘Northblue’, the most common cul-
tivated highbush blueberries in Finland. Roots
of wild bilberries (V. myrtillus) and bog whortle-
berries (V. uliginosum) grown under natural con-
ditions were also studied. Root samples were
collected twice, in September 1997 and early
June 1998. ‘Northcountry’ and ‘Northblue’ root
samples were collected from two different blue-
berry plantings near Muuruvesi (sites A and B,
63°00'N, 28°15'E) and from Kuopio University
Garden (site C, 62°53'N, 27°37'E) in central Fin-
land. Wild bilberries were sampled from natu-
rally regenerated and spruce-dominated forests
(podzol soil) in Kuopio and Muuruvesi, where-
as root samples of bog whortleberries were col-
lected only from a small Sphagnum bog in Kuo-
pio. All sampled wild bilberries and bog whortle-
berries were growing in the vicinity of plantings.

In site A (farm Huumonen, Muuruvesi) the
soil (fine sand) was treated with 80% sulphuric
acid (sulphur dose 3.5 kg per are) one year be-
fore planting the blueberries in 1994, the ob-
tained pH level being approximately 5.3 after this
artificial acidification. In site B (farm Alatalo,
Muuruvesi), pH level of the soil (fine sand +
chips as a surface layer) was approximately 6.5
near the sampled plants, and the field established
in 1992 was fertilised with Biolan Extra (NPK-
ratio 4:1:2, dose 4 dl per plant) annually. In site
C (Kuopio University Garden) cultivation had
started already in 1987 and soil was adjusted for
cultivars by applying peat and coarse sand to soil
(loamy fine sand). Mean pH level of the soil was
5.5 and field was fertilised with Herkkuperunan

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lannos (NPK-ratio 8:10:12, dose 3–6 litres per
are) annually from year 1995 onwards.

Highbush blueberry root samples were col-
lected systematically, i.e. from every fifth plant
in every second row, whereas wild bilberries and
bog whortleberries were collected randomly
from their growing sites. Roots were excavated
from 0–20 centimetres depth with a small scoop,
highbush blueberry roots being collected only
from the part of the plant that faces south. After
collecting, root samples were immediately rinsed
under running tap water over a sieve. Then the
hair root tips (length 2–3 mm) of washed roots
were collected for ultrastructural study. Root
samples for ergosterol analysis were frozen in
liquid nitrogen prior storing them at –80°C, and
the rest of the washed roots were stored at –20°C
for the stereomicroscopy viewing.

The root tips collected for ultrastructural
studies were placed immediately in 2.5% glu-
taraldehyde fixative in Eppendorf tubes. Prefix-
ation was carried out in 2.5% glutaraldehyde
made in 0.1 M phosphate buffer (pH 7.0) for 16 h
at 4°C and postfixation in buffered 1% OsO

solution for 3 h at 4°C. The samples were dehy-
drated in graded ethanol series, infiltrated and
finally embedded in Ladd’s LX 112 resin. Then
thin root sections were stained with uranyl ace-
tate and lead citrate and mycorrhizal status of
these sections was studied with a JEOL 1200 S
electron microscope.

The gridline intersect method (Giovannetti
and Mosse 1980) was used for visual estimation
of the mycorrhizal infection in the hair roots.
Approximately 50 milligrams (f. wt) of the fin-
est hair roots (∅ < 100 µm) cleared in hot 10%
KOH and acidified with 1% HCl were stained
with 0.05% trypan blue in lactophenol (Phillips
and Hayman 1970). After staining, the roots were
dispersed as evenly as possible on a round Petri
dish with help of glycerol. On the bottom of the
Petri dish was a gridline (1 cm x 1 cm) and my-
corrhizal colonisation status was studied from
all the intersections of roots and gridlines.

Mycorrhizal occurrence in the roots was es-
timated also with ergosterol analysis (Salmano-
wicz and Nylund 1988, Nylund and Wallander

1992). Prior to the assay, the roots were freeze-
dried and ground in liquid nitrogen. Ergosterol
was extracted with ethanol containing pyrogal-
lic acid from approximately 25–50 milligrams
of root (d. wt). Saponification with 60% KOH
released root ergosterol in free form, which was
then extracted with pentane. Finally, pentane
phase was left to evaporate to dryness and dis-
solved to methanol. HPLC-analysis was done
using reverse-phase column (Hewlett-Packard,
LiChrospher 100 RP-18) and 100% methanol as
an eluent. Samples of 20 µl were injected and
run at 1.6 ml per minute. Ergosterol peaks were
detected with UV-detector at 280 nm, peaks ap-
pearing after 7–8 minutes. At the beginning and
end of every sample sequence, internal ergos-
terol standards were run and regression formula
for the standard curve was determined.

Mycorrhizal infection level data assessed
both stereo- and electron microscopically was
arcsin-transformed prior to the statistical tests
(SPSS-PC-Windows programmes). One-way
ANOVA combined with Tukey’s test was used
to analyse the differences between the mean in-
fection levels and ergosterol concentrations of
different groups. In addition, correlations be-
tween the root ergosterol concentration and my-
corrhizal colonisation level data obtained from
stereomicroscopical studies were tested with
Pearson’s correlation coefficient. In stereomicro-
scopical and ergosterol analyses the number of
replicates was 10–25 (N = 107) and in ultrastruc-
tural analysis n = 5–10 (N = 45).

Results and discussion

In ultrastructural samples, mycorrhizal hyphae
colonised epidermal root cells (Fig. 1) in all stud-
ied species. Although cultivars from site A
(3.6%) and C (5.4%) had clearly the lowest eri-
coid mycorrhizal hyphae formation in epidermal
root cells, the hyphae growth in cultivars from
site B (27%) did not significantly differ from that
of wild bilberries (47.9%) or bog whortleberries

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Kasurinen, A. et al. Mycorrhizas of wild and cultivated Vaccinium sp.

(20.8%). Previous transmission electron micro-
scopic studies have revealed that in ericoid my-
corrhizas each epidermal cell is an infection unit

(Smith and Read 1997) and therefore even adja-
cent cells may have mycorrhizal complexes of
different developmental stage. This pattern was

Fig. 1. Electron micrographs of ericoid mycorrhiza in highbush blueberry (a–b) and in wild bilberry (c–d)
root epidermal cells. Well-developed mycorrhizal infection can be seen in the root epidermal cells, F =
mycorrhizal cells. Magnification 7200x (a, c–d) or 5300x (b). Photo: Seija Anttonen and Toini Holopainen.

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observed also in the present material and the
studied Vaccinium mycorrhizas represented the
typical ericoid mycorrhiza structure (Smith and
Read 1997).

Stereomicroscopical analysis showed that the
mycorrhizal colonisation rate of roots was quite
high in all studied groups in general, mean
colonisation values ranging from 40% to 50.8%
(Fig. 2a). This result is in accordance with the
findings made by Johansson (2000), who discov-
ered that naturally occurring ericoid mycorrhiz-
al heather (Calluna vulgaris) had relatively high
mycorrhizal infection level (approximately 40%
of roots infected) during growing season. Total
mycorrhizal infection percentage was lowest in
bog whortleberries and cultivars from site C,
whereas the highest total infection percentage
was observed in wild bilberries (Fig. 2a). One
explanation for the observed discrepancy be-
tween the TEM and stereomicroscopical data is
that in ultrastuctural analysis only a small area
of a two dimensional root section can be studied
by TEM. Thus, in these small root sections the
presence of infected cells is sporadic and there-
fore the results of TEM analysis can be regard-
ed as more qualitative than quantitative. It is also
important to remember that the visual assessment
of mycorrhizas by stereomicroscope is a subjec-
tive method, in which some over- or underesti-
mation of mycorrhizal status might easily occur,
especially if the studied root sample is strongly
pigmented.

The mean ergosterol content of roots in dif-
ferent study groups ranged from 71.3 µg g–1 to
153.7 µg g–1 (Fig. 2b). Contrary to the mycor-
rhizal infection data (Fig. 2a), bog whortleber-
ries had clearly the highest amount of ergosterol
in their roots. In addition, the cultivars from sites
A and B as well as wild Vaccinium-species had
clearly more ergosterol in their roots than the
highbush blueberry cultivars in site C. In gener-
al, ergosterol assay revealed that there is a high
total amount of vital mycorrhizal biomass in the
roots of all investigated species. The ergosterol
values observed in this study were approximately
of the same magnitude or sometimes even greater
than those found from ectomycorrhizal tree roots

(Ekblad et al. 1995, Manninen et al. 1998). The
contradiction between ergosterol and mycorrhiz-
al infection data might be partially due to the
fact that ergosterol assay measures the total
amount of living fungi (mycorrhizal plus non-
mycorrhizal fungi), whereas in clearing and mi-
croscopic viewing method both dead and alive
mycorrhizas are stained and counted. On the oth-
er hand, as Wallander et al. (1997) and Johnson
and McGill (1990) point out, small seasonal var-
iation in ergosterol amount and repeatability of

Fig. 2. a) Ericoid mycorrhizal colonisation levels (%, ster-
eomicroscopic data) and b) ergosterol concentrations
(µg g–1 of root d.wt.) in hair roots of wild bilberries, bog
whortleberries and highbush blueberries in sites A, B and C.
Site A = farm Huumonen Muuruvesi, site B = farm Alatalo
Muuruvesi and site C = Kuopio University Garden, Kuo-
pio. The values (mean ± SD) followed by the same letters
are not significantly different from each other (P < 0.05).

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Kasurinen, A. et al. Mycorrhizas of wild and cultivated Vaccinium sp.

procedure makes it a better estimation method
of vital mycorrhizal biomass than any other cur-
rently available analysis. Apparently because of
the relatively large heterogeneity of data and
possible above-mentioned inaccuracies caused
by the methods used in this study, the ergosterol
concentration and mycorrhizal colonisation of
roots did not correlate significantly in any of the
groups.

In conclusion, the highbush blueberry culti-
vars seem to obtain a substantial colonisation of
ericoid mycorrhizal fungi from their growth
medium. However, the infection level is some-
what lower compared to wild relative species and
is probably dependent on the edaphic factors
also. For instance, the soil medium in site C was
more compact and loamy than in the other study
areas and appeared to have a stunting effect on
the growth of aboveground parts of plants as
well. Previous studies have also shown that er-
gosterol concentrations can change according to
the soil conditions (pH, nutrient and water sta-

tus), fungus species, plant cultivars and age of
fungus (Ekblad et al. 1995, 1998, Möttönen et
al. 1999). Thus, it is possible that the ergosterol
content of different plant roots varied merely due
to the soil conditions and as mentioned earlier,
edaphic conditions in site C seemed to control
both mycorrhizal and plant growth. Moreover,
the observed relatively high mycorrhizal forma-
tion in the studied cultivars does not necessarily
mean greater benefits for the host plants. A fur-
ther step would be to test experimentally wheth-
er the mycorrhizas infecting the roots are truly
beneficial (mutualistic) to the highbush blueber-
ries growing under agricultural field conditions.

Acknowledgements. We are grateful to M.Sc Katri Helppi
and Mrs. Mirja Korhonen for skillful technical assistance.
We thank also M.Sc Harri Kokko and Lic.Phil Sirpa Paasi-
salo for help with planning this study and Mr. Reijo Huu-
monen, Mr. Toivo Laitinen and the Kuopio University Gar-
den for providing the study material. This study was fund-
ed by the Savo Foundation for Advanced Technology and
the Academy of Finland (project number 48798).

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SELOSTUS
Viljellyn pensasmustikan ja luonnonvaraisten mustikan ja juolukan sienijuuret

Anne Kasurinen, Toini Holopainen ja Seija Anttonen
Kuopion yliopisto ja Metsäntutkimuslaitos

Viljellyn pensasmustikan (Vaccinium corymbosum)
sekä luonnonvaraisten mustikan (Vaccinium myrtil-
lus) ja juolukan (Vaccinium uliginosum) sienijuuria
tutkittiin transmissioelektronimikroskoopilla (TEM),
stereomikroskooppisesti trypaanisinisellä värjätyistä
juurista ja biokemiallisella ergosterolianalyysillä.
Kaikilta tutkituilta lajeilta löytyi erikoidimykoritsoil-
le tyypillisiä rihmastokiehkuroita juuren pintakerrok-
sen soluista TEM-analyysissä. Stereomikroskoop-

pianalyysin perusteella luonnonvaraisilla mustikoil-
la oli korkeimmat mykoritsainfektiot juurissaan (hius-
juurista 51 % infektoitunut), kun taas suurimmat er-
gosterolipitoisuudet olivat juolukoilla (ergosterolia
209 µg/g juurta). Näiden tulosten perusteella voidaan
sanoa, että mykoritsasymbioosit juurissa ovat yleisiä
kaikilla tutkituilla Vaccinium-lajeilla, tosin viljellyillä
pensasmustikoilla mykoritsainfektioiden määrä on
luonnonvaraisia sukulaisiaan jonkin verran alempi.

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Title
Introduction
Material and methods
Results and discussion
References
SELOSTUS