Applied Soil Ecology 15 2000 125–136
In vitro and post vitro inoculation of micropropagated Rhododendrons with ericoid mycorrhizal fungi
Jan Jansa
1
, Miroslav Vosátka
∗
Institute of Botany, Academy of Sciences of the Czech Republic, CZ-252 43 Pruhonice, Czech Republic Received 31 May 1999; received in revised form 25 November 1999; accepted 23 March 2000
Abstract
Isolation of more than 200 strains of endophytic fungi from the roots of several host plants belonging to order Ericales Vaccinium, Calluna, Rhododendron, Empetrum, etc. was followed by a successful attempt to verify ericoid mycorrhiza status
of some of these fungal isolates under axenic conditions. In two screening experiments, the most efficient ericoid mycorrhiza fungal strains were found beneficial for the growth of micropropagated Rhododendron plants when inoculated post vitro
after transplantation to peat-based substrate. No negative influence on the growth of host plants has been observed for any inoculated isolate, while about 10 of tested strains exhibited positive effects on the growth of Rhododendron microcuttings
grown in peat-based media. © 2000 Elsevier Science B.V. All rights reserved.
Keywords: Ericoid mycorrhiza; Ericaceae; Horticulture; Micropropagation
1. Introduction
Ericoid mycorrhiza ERM belong, together with orchidaceous and arbuscular mycorrhiza types, to the
group of endotrophic mycorrhizal associations. Eri- coid mycorrhizas are associations between ascomyce-
tous or rarely hyphomycetous fungi and plant species belonging to the families Ericaceae, Epacridaceae and
Empetraceae
Smith and Read, 1997. Also arbutoid and monotropoid mycorrhiza can be found within the
family Ericaceae but the most common ericoid my- corrhiza are found in genera such as Calluna, Erica,
∗
Corresponding author. Tel.fax: +42-2-67750022. E-mail addresses:
jan.jansaipw.agrl.ethz.ch J. Jansa, vosatkaibot.cas.cz M. Vos´atka
1
Present address: IPW-ETH Zürich, Eschikon 33, Postfach 185, CH-8315 Lindau ZH, Switzerland. Tel.: +41-52-3549216;
fax: +41-52-3549119.
Rhododendron , Vaccinium, and Empetrum Smith and
Read, 1997. The ERM is characterized by considerably uniform
structure, similar to those in arbuscular mycorrhizas, but usually more delicate Peterson et al., 1980; All-
away and Ashford, 1996. A range of differently sep- tated hyphae simple septal pores of ascomycete type
as well as dolipore septated fungi of basidiomyce- tous type can occur were found inside the corti-
cal cells of ericaceous plants Bonfante-Fasolo and Gianinazzi-Pearson, 1979; Bonfante-Fasolo, 1980.
The hyphae of ERM fungi penetrate a single layer of cortical cells of the roots and fill them with intracel-
lular hyphal coils. Cortical cells of ericaceous plants never form structures like root-hairs, well described
in other plant families.
The ericaceous plants occur in most climatically and edaphically stressed environments particularly
when soil acidity becomes extreme and the rate of
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126 J. Jansa, M. Vos´atka Applied Soil Ecology 15 2000 125–136
nutrient mineralization is low. The function of the ERM fungi is, most probably, to cover nutrient de-
mands of the plant under such stress conditions Harley, 1969. The ERM fungi in acidic heath soils
produce external mycelium that is supposed to have an active function in obtaining mineral nutrients: en-
zymatic release of nitrogen from predominant organic compounds, that would otherwise be unavailable for
the roots. Most remarkable is a high C:N ratio in these soils, which is overcome due to the activity of
enzymes produced by the ERM fungi. The ericoid mycorrhizal plants showed access to nitrogen sources
that are almost inaccessible for nonmycorrhizal plants Pearson and Read, 1975; Kerley and Read, 1998. En-
zymes hydrolyzing different carbon polymers oligo- and polysaccharides, including cellulose and pectins
were described from pure cultures of the ERM fungi Pearson and Read, 1975; Perotto et al., 1993; Varma
and Bonfante, 1994. Effective uptake of nitrogen by the ERM fungi from different sources in peat-based
soils was described — both free ammonia and nitrate fixing pathways are more efficient for fungi compared
to the plants, especially under acidic conditions Pear- son and Read, 1975. Organic polymers-bounded
nitrogen was utilized by the ERM fungi especially un- der low pH Leake and Read, 1990a and chitinolytic
activity of the fungi was proved as well Leake and Read, 1990b. Increase in phosphate uptake of erica-
ceous host plants due to ERM fungi was described by Read and Stribley 1973, mainly due to solubilization
of ferric or aluminium phytates Mitchell and Read, 1981. Moreover, a phosphodiesterase attacking nu-
cleic acid bound phosphate was described from fungal cultures by Leake and Miles 1996. A high affinity
of the ERM fungi for iron is probably important as maintained by production of siderophores Schuler
and Haselwandter, 1988. Some of the ericoid my- corrhizal host plants Calluna and Vaccinium were
reported for high tolerance to different environmen- tal stresses. This makes these ERM fungi practically
interesting for their potential to enhance plant fitness under unfavourable conditions Bradley et al., 1982;
Burt et al., 1986; Yang and Goulart, 1997. Attempts were made to find a proper fungal strain to decrease
losses in propagation of Rhododendrons at commer- cial level, which may reach up to 10 at weaning
stage Lemoine et al., 1992. This work showed com- plexity and probable strain-to-strain specificity of
plant and fungi with respect to positive plant growth reaction, which may depend also on substrate type.
The ERM fungi are characterized by a very slow development — first apparent colonization struc-
tures were observable after 3 weeks in g-irradiated reinoculated soil and after 4 weeks in horticultural
soil, collected under Rhododendrons Duddridge and Read, 1982. This documents possible role of
other microorganisms, which may slow down the development of the fungus to a certain extent. The
breakdown of some of the mycorrhizal structures was evident after 8 or 11 weeks in irradiated or unsterile
soil, respectively. The breakdown process starts by structural desintegration of plant organelles and cells,
followed by a loss of integrity of the fungal struc- tures Duddridge and Read, 1982. This means that
the fungus at least in part of its life span plays a role of a sapro-parasitic partner. Simply septated fungi,
supposed to be symbiotic ones, frequently observed within cortical cells, can be isolated from roots, but
they very scarcely form spores in culture, which hin- ders their classification. They are divided into two
groups — slow growing, dark-coloured and usually sterile mycelia McNabb, 1961; Pearson and Read,
1973a; Singh, 1974, and a group of Oidiodendron sp. observed mainly in isolations from Calluna or Vac-
cinium
Couture et al., 1983; Dalpe, 1986; Douglas et al., 1989. Considerable genetic diversity was found
amongst isolates that were superficially very similar, on both biochemical-isozymes Hutton et al., 1994
and molecular basis Perotto et al., 1995, as well as classical microbiological techniques Hambleton and
Currah, 1997. A question remains as to whether such diversity observed in root-associated fungi has any
impact upon performance of plants grown under con- trolled conditions Smith and Read, 1997. A demand
to extend the research observation beyond classical Calluna
–Vaccinium–Rhododendron model to include more taxonomically diverse host and fungal partners
has arisen in recent time Straker, 1996. The objective of our work was to isolate endophytic
fungi from different plants and to attempt the reinoc- ulation of micropropagated Rhododendron plantlets.
This was made in both in vitro system, accord- ing to Koch’s postulate Koch, 1912, to reveal
mycorrhizal status of these fungi, and in post vitro system, to check for any growth effect of mycorrhiza
on plants.
J. Jansa, M. Vos´atka Applied Soil Ecology 15 2000 125–136 127
2. Material and methods
