Content uploaded by Francis Brearley
Author content
All content in this area was uploaded by Francis Brearley on Feb 13, 2014
Content may be subject to copyright.
Chapter 1
The Importance of Ectomycorrhizas for the
Growth of Dipterocarps and the Efficacy
of Ectomycorrhizal Inoculation Schemes
Francis Q. Brearley
1.1 Introduction
The Dipterocarpaceae is the most important tree family in the tropical forests of
South-east Asia as they are the ecosystem dominants, especially in lowland forests
of this region, where they often contribute up to a quarter of all stems and half of the
above-ground biomass (Proctor et al. 1983; Davies et al. 2003; Brearley et al.
2004). They also play major role in timber production from this area as they are
favoured for their fast growth rates, tall cylindrical boles and high quality wood.
In addition, they are a source of non-timber forest products such as oils and
resins (Shiva and Jantan 1998). Given the continued exploitation and degra dation
of lowland tropical forest habitats worldwide, there is interest in reforestation
programmes to maintain forest cover and to provide a sustained supply of wood
products. Many of these programmes have focussed on fast-growing exotic tree
species but, to provide the highest quality timber, reforestation efforts now need to
focus on the Dipterocarpaceae.
The majority of trees in tropical forests form arbuscular mycorrhizas but it was
first noted by Singh in 1966 that dipterocarps, like many temperate forest trees,
formed ectomycorrhizas (EcMs). Since then, many subsequent studies have exam-
ined the roots of numerous dipterocarps and found them to be colonised by EcM
fungi (Alexander and H
€
ogberg 1986; Chalermpongse 1987; Smits 1992; Lee 1998;
Hoang and Tuan 2008). There are also minor reports of arbuscular mycorrhizal
(Shamsuddin 1979; Ibrahim et al. 1995; Tawaraya et al. 2003) and ectendomy-
corrhizal (Tupas and Sajise 1976) fungi in dipterocarp roots, but more details
are needed on this potential dual colonisation before anything more concrete can
be written.
In this chapter, I have (1) given a brief overview of mycorrhizal symbioses in
dipterocarps, I now plan to (2) note which are the important fungal species involved
F.Q. Brearley
School of Science and the Environment, Manchester Metropolitan University, Chester Street,
Manchester M1 5GD, UK
e-mail: f.q.brearley@mmu.ac.uk
M. Rai and A. Varma (eds.), Diversity and Biotechnology of Ectomycorrhizae,
Soil Biology 25, DOI 10.1007/978-3-642-15196-5_1,
#
Springer-Verlag Berlin Heidelberg 2011
3
in the Ec M symbiosis and then (3) focus on the role of this symbiosis in improving
plant nutrition, growth and performance, and complete (4) with a special focus on
how we might critically use this knowledge to determine if we need to apply EcM
inoculation in reforestation programmes.
1.2 Fungal Species Associated with Dipterocarps
We know, from fruit body surveys, that the most speciose groups of fungi found in
dipterocarp-dominated forests are the families of Amanitaceae, Boletaceae and
Russulaceae (Watling and Lee 1995, 1998, 2007; Watling et al. 1998, 2002; Lee
et al. 2002, 2003), and these appear to form a reasonable proportion of the EcM root
tips belowground (Lee et al. 1997; Ingleby et al. 1998). However, it is known that
above-ground and below-ground views of the EcM community rarely show close
concordance (Gardes and Bruns 1996; Yamada and Katsuya 2001), and it has
recently been noted that fungi with cryptic fruiting bodies such as members of
the Thelephoraceae, which appear to have been overlooked in fruiting body
surveys, are also important EcM formers. For example, Ingleby et al. (2000)
found a Thelephoraceae species to be common on seedlings planted in soil from
Vietnamese forest and I have found two Thelephoraceae species common on
nursery-grown seedlings in Malaysia (Brearley et al. 2003, 2007; Brearley 2006).
Furthermore, seque nces from this group dominated the molecular studies on
dipterocarp EcM communitie s conduc ted by Sirikantaramas et al. (2003) and
Yuwa-Amornpitak et al. (2006). As an example, half of the sequences generated
by Sirikantaramas et al. (2003) were from this family. In this regard, the use of
molecular identification techniques has shown us that the EcM community
in dipterocarp-dominated forests is more similar to many boreo -temperate EcM
communities (e.g. Richard et al. 2005; Ishida et al. 2007; Morris et al. 2008) than
originally thought. As molecular work continues on the EcMs communities of
dipterocarps, it will reveal a much clearer below-ground picture of the dominant
species involved in this symbiosis.
1.3 Nutrient Relationships
There are numerous experiments showing that EcMs can improve plant nutrient
uptake (Smith and Read 2008) and, in the Dipterocarpaceae, this has also been
shown to be the case. For example, Lee and Lim (1989) correlated percentage EcM
colonisation (% EcM) with foliar phosphorus (P) in dipterocarp seedlings they
studied. Seedlings from a site with lower available soil P showed a positive
correlation between foliar P and % EcM suggesting that EcMs enhanced uptake
of P at this site. Hadi and Santoso (1988, 1989) presented data suggesting inocula-
tion with EcM fungi increased foliar concentrations of nitrogen (N), P, potassium
4 F.Q. Brearley
(K), calcium (Ca) and magnesium (Mg) of five dipterocarp species (although it is
not entirely clear if their data are for nutrient concentrations or total nutrient content
in the seedlings). The first experiments to show an unequivocal increase in foliar
nutrients in response to EcM colonisation were those of Lee and Alexander (1994)
and Yazid et al. (1994) who clearly showed increased P concentrations in response
to EcM colonisation in two species of Hopea studied. Additionally, in the experi-
ments of Lee and Alexander (1994), whilst there were no suggestions of EcM
colonisation increasing shoot N, K or Mg concentrations, there was some indication
of improved Ca nutritio n. These differential responses may be due to the experi-
mental conditions used but may also represent a certain degree of inter-specific
difference in fungal benefits to the host plant or may even be due to an EcM
diversity effect on nutrient uptake (Baxter and Dighton 2005).
Improved mineral nutrition has also been shown under field conditions where
reduction of EcM colonisation (by Mancozeb fungicide) led to reduced foliar N and
P in both Hopea nervosa and Parashorea tomentel la in addition to reduced Ca and
Mg in Hopea nervosa (Brearley 2003). I have also shown, through stable isotope
analysis, that EcM dipterocarps can also obtain more N from organic material
(Brearley et al. 2003) with a positive correlation found between % EcM colonisa-
tion and uptake of organic N.
An inoculation experiment that purports to show increases in nutrient uptake of
Shorea seminis when inoculated with two EcM species is that of Turjaman et al.
(2006). Total shoot N and P contents were indeed greater in the EcM inoculated
seedlings but, surprisingly, on a dry-weight basis, inoculation actually led to a
decrease in concentrations of these elements in the shoot (up to 55% in the most
extreme case). Smits (1983) also considers that dipterocarp EcMs may be providing
thiamin to plants although, in the absence of a strong presentation of data, we
should discount this hypothesis for now.
Increased nutrient concentrations within plants are generally likely to lead to
improved growth, and the role of foliar nutrients in determining seedli ng perf or-
mance is also important during out-planting as it has been shown that higher levels
of foliar N allow dipterocarp seedlings to better avoid photodamage when trans-
ferred to high irradiance conditions and to allow more rapid acclimation to these
conditions (Bungard et al. 2000).
1.4 Inoculation Experiments
Many of the inoculation experiments reported are in the grey literature and have a
number of shortcomings. The most serious of these is that they are poorly reported
and do not provide sufficient detail for the experiments to be evaluated fully nor
repeated by other researchers. Pseudoreplication or the lack of statistical analyses in
many cases also makes evaluation of the results problematic. In this chapter, I will
focus on papers which, I feel, have mostly overcome these shortcomings or are
otherwise noteworthy.
1 The Importance of Ectomycorrhizas for the Growth of Dipterocarps and the Efficacy 5
The first reported experiment attempting to inoculate dipterocarp seedlings
with EcM fungi and determine seedling responses was that of Hadi and Santoso
(1988). They inoculated species of Boletus, Russula and Scleroderma using
pieces of chopped fruiting body on the roots of five dipterocarp seedling species.
A shortcoming of this experiment was that the roots were not examined to deter-
mine the extent of EcM formation by any contaminant fungi. Nevertheless, after
6 months growth, inoculation appeared to at least double seedling height in all
fungus/seedling combinations. Furthermore, their approach to inoculation is rarely
used as it is difficult to control the inoculum viability and potential supplied to the
roots with this method.
Initial experiments, using chopped root inoculum (Lee and Alexander 1994),
found that EcM colonisation increased plant dry weights in Hopea odorata and
Hopea helferi. This increase was generally greatest in the absence of additi onal
nutrients supplied to the soil. However, the problem with experiments using root
inoculum is that the species of fungi on the inoculant roots cannot be determined
and ther efore controlled experiments using defined EcM species are needed. In
Malaysia, inoculation experiments have focussed on strains of Pisolithus species
and a member of the Thelephorales (FP160: Lee et al. 2008). In Indonesia, the use
of Scleroderma for inoculation appears more popular although the range of species
being used has increased recently (Turjaman et al. 2007).
1.4.1 Inoculation with Single Species
1.4.1.1 Malaysian Inoculation Experiments
In Malaysia, exotic Pisolithus isolates from Brazil (Pt 441 originally from under
Eucalyptus citriodora) and Thailand (Pt msn) were effective at forming EcMs
on four dipterocarp seedling species (although there was a certain degree of host-
fungal compatibility with one or other Pisolithus isolate having a greater % EcM on
each of the seedling species; Lee et al. 1995). Using Chil vers et al.’s (1986)
cardboard inoculum technique, Yazid et al. (1994) showed that Pt 441 formed a
high percentage of functional EcM colonisation (c. 80%) on Hopea odorata and
Hopea helferi and that this increased the growth (dry weights increased by 7.3 and
3.6 times, respectively) and foliar P concentrations (by at least 40%) after 9 months
growth. Similar results in terms of the growth of Hopea odorata were seen
(although with a lesser growth response) when coconut husk:vermiculite inoculum
was added in a ratio of 1:4 to a sterilised soil:sand mix (Yazid et al. 1996). In
contrast, problems were found when trying to inoculate a local species: Pisolithus
aurantioscabrosus (Lee et al. 1995). Why this is, is not clear but may simply
reflect the ability of Pisolithus tinctorius to form EcMs with a wide host range
(Martin et al. 2002), whereas Pisolithus aurantioscabrosus has only been reported
to be associated with Shorea parvifolia and Shorea macroptera to date (Watling
et al. 1995a, b; Martin et al. 2002).
6 F.Q. Brearley
Initial tests of successful inoculation between Hopea odorata and Hebeloma
crustulinifome (Lapeyrie et al. 1993) were reported, but growth responses were not
shown and this exotic European strain does not appear to be used any more. Recent
work has focussed on Thelephoraceae FP1 60 (Lee et al. 2008), which significantly
increased stem height, root length and biomass of Hopea odorata after 6 months
growth in the nursery by 30%, 62% and 40%, respectively (Lee et al. 2008).
It currently appears very difficult to bring further tropical dipter ocarp-associated
EcM species into culture (S.S. Lee, pers. comm.), and the species that are being
used form a very limited subset of those available. The best approach here would be
a wide-ranging screening using a variety of fungal structures, media and growth
conditions although, of course, this will be very labour intensive with potentially
little reward. Perhaps, the floating culture technique of Sangtiean and Schmidt
(2002) may help South-east Asian researchers to culture some of the later stage
EcM species found in these forests. This technique allowed Sangtiean and Schmidt
(2002) to carry out culture experiments on Amanita, Lactarius and Russula species ,
which are common in South-east Asian forests (see above).
1.4.1.2 Indonesian Inoculation Experiments
In Indonesia, the use of Scleroderma species (and especially Scleroderma colum-
nare) appears to be favoured, probably from the initial work of Ogawa (1993,
2006) in the early 1990s. Sadly, much of this early work is difficult to evaluate as
it is not clearly reported (e.g. Supriyanto et al. 1993; Kikuchi 1997) but, more
recently, a number of much better controlled inoculation experiments have been
conducted by Turjaman et al. (2005, 2006, 2007). They showed that the growth of
Shorea pinanga was improved by the addition of spore tablets of Pisolithus
tinctorius (aka Pisolithus arhizus) and Scleroderma columnare species. Both
fungal species improved the growth of Shorea pinan ga (150% increase in dry
weight after 7 months) although there was some EcM colonisation of the controls.
Survival rates (86–87%) were also much higher than the control (16%), which is
an equally important factor to take into consideration when planning reforestation
schemes. In a follow-up experiment (Turjaman et al. 2006), tabletted spore
inoculum was compared with alginated bead mycelial inoculum of Pisolithus
tinctorius and Scleroderma columnare. Percentage EcM colonisation was higher
(61–65%) when seedlings were inoculated with spores than with mycelium
(35–37%), and there was, again, at least a doubling of dry weight after 7 months
growth. In the most recently reported experiment (Turjaman et al. 2007), inocula-
tion of four fungal species on the roots of Shorea balangeran increased seedling
growth. Whilst we might expect the use of Boletus sp., Scleroderma sp. and
Strobilomyes sp. to increase seedling growth as these are known to be EcM
forming fungi, the use of Calvatia sp. also increased seedling growth, which
was unexpected as this is not thought to be an EcM forming genus (Rinaldi
et al. 2008).
1 The Importance of Ectomycorrhizas for the Growth of Dipterocarps and the Efficacy 7
1.4.2 Other Inoculation Methods
1.4.2.1 Mycorrhizal Tablets
Where sterile facilities are not available to cultivat e species aseptically, researchers
have used “mycorrhizal tablets”. In this case, spores or mycelium are mixed with a
carrier (clay or alginate beads) and applied to seedlings’ rooting zones to allow
hyphal contact and subsequent EcM formation. The first record of this in the South-
east Asia region appears to be that of Ogawa (1993). Species used for this method
are often those such as Scleroderma species, which have the advantage that
their spores can be collected much more easily from their enclosed fruit bodies
than many other gilled or pored fruit bodied species. Clay tablets at 1:100 (crushed
fruit bodies:clay) were used in the experiments by Turjaman et al. (2005, 2006), and
these showed increased growth of Shorea pinanga and Shorea selanica when
inoculated as compared with uninoculated controls. However, in such experiments,
there is a need to confirm that the tablets do not contain additional nutr ients that
might improve seedling growth in the absence of a mycorrhizal effect.
1.4.2.2 Mother Tree Inoculation
Other inoculation methods include inoculation from a colonised mother tree in the
nursery – in other words, simply letting newly germinated seedlings’ roots contact
hyphae already radiating out in the soil around established, colonised trees. This
method was first used by Roeleffs (1930, in Nara et al. 1999) to inoculate seedlings
of Pinus species. The technique, also known as inoculation beds, is a low-tech
method that allows EcM inoculation before planting-out but it can be rather
haphazard in terms of the speed and reliability of inoculation (see Kikuchi 1997).
For example, Ogawa (2006) shows a diagram of the spread of Scleroderma colum-
nare fruiting bodies through a nursery containing seedlings of Shorea leprosula and
Shorea academica to be between 1 and 2 m per year.
1.4.3 Production of Inoculum
In order to produce inoculum rapidly, conditions for the optimum growth of fungi in
culture should be evaluated. For example, Patahayah et al. (2003a) showed that the
most rapid growth of Pisolithus albus (aka Pt Gemas) was obtained at 25
C when
grown on Oddo ux medium but at 30
C when grown on MMN or Pachlewski’s
medium (Patahayah et al. 2003b). We have also shown that this species grows
best when N is supplied in an organic form (BSA in the experiments conducted;
Brearley et al. 2005). Thelephoreaceae FP160 shows best growth at 25
C
(Patahayah et al. 2003b) but has minimal preferences for N source (Brearley
8 F.Q. Brearley
et al. 2005). In terms of efficient spread of EcM inoculum in the nursery, Nara et al.
(1999) considered that seedlings are often maintained under sub-optimal conditions
in potted soil with a high clay content and hence poor aeration, thereby slowi ng
growth of fungal hyphae. They found that, by using a growth medium with particles
of 2–4 mm diameter, the optimum growth of EcM mycelium (Th1 on Shorea
roxburghii) was obtained. It is also important to consider the longevity of the
different forms of inoculum. For example, Fakuara and Wilarso (1993) showed
that mycorrhizal tablets remained effective up to 4 months in storage (this was the
longest period tested). More experiments are clearly needed in this area, with longer
test periods, to gain a better idea of spore longevity.
1.4.4 Field Experiments
There is now a need to determine how well inoculated seedlings and their symbiotic
EcM species survive in the wild when seedlings are out-planted. This is important
as, if considerable effort is being put into inoc ulation programmes, this is simply
being wasted if seedlings or their inoculant fungal species are dying unnecessarily.
Furthermore, in terms of reforestation schemes, growth is not necessarily the mos t
important parameter, seedling survival is arguably equally as important.
Chang et al. (1994, 1995) showed that the species of Pisolithus in the Malaysian
inoculation experiments noted above did not remain competitive when colonised
seedlings of Shorea glauca were planted into the field; indeed Pisolithus had mostly
disappeared from the roots after 6 months suggesting that they are either early stage
fungi, or are poorly adapted to the biotic or abiotic environments of the Malaysian
forest soils. Using Thelephoraceae FP160, Lee et al. (2008) found it to remain
competitive on the roots of seedlings (Hope a odorata and Shorea leprosula) for up
to 23 months after out-planting in a sandy tin mine tailings site (after this time
contaminant EcM fungi had only colonised up to 15% of the root tips of less than
half the seedlings). However, the improved growth of Hopea odorata seen in
the nursery due to inoculation with this fungus (see above) was not seen in the
field (by measurement of root collar diameter) and improved growth of Shorea
leprosula was only seen for up to 3 months following out-planting. Under field
conditions, I found that the reduction in EcM colonisation by fungicide addition
to the roots of two species (Hopea nervosa and Parasho rea tomentella) did not
lead to changes in seedling growth but that foliar nutrient concentrations were
reduced (Brearley 2003). In field experiments in a degraded peat swamp forest in
Kalimantan, Turjaman et al. (2007) showed that a spore suspension of Boletus sp.
and Scleroderma sp. applied to the seedling rooting zone led to increased growth of
Shorea balangeran but application of Calvatia sp. and Strobilomyces sp. did not.
For the two fungal species that were beneficial, it took around 8 months for growth
improvements to be seen, perhaps due to the very wet conditions at the start of the
experiment (Turjaman et al. 2007). However, it is difficult to determine if the
species applied were those that maintained improved seedling growth as the roots
1 The Importance of Ectomycorrhizas for the Growth of Dipterocarps and the Efficacy 9
at the end of this 40-month experiment were not examined to determine which EcM
fungi were present – it would have been a notable im provement to the study design
to do this. The study of Tata et al. (2009) did report this examination at the end of
their experiments with Shorea selanica and Shorea lamellata, which were inocu-
lated with spore tablets of Scleroderma columnare and planted in natural forests or
rubber agroforests in Sumatra. Their results were complex but did not show
consistent increases in growth, performance or survival of the two dipterocarp
species over a 2-year period. It is notable that, among the 19 EcM types they
identified using PCR-RFLP at the end of the experiment, none of them were
Scleroderma species indicating that the inoculated fungus did not remain competi-
tive on the roots for more than this length of time.
There is clearly a need to further evaluate the growth and survival of inoculated
seedlings in the field as positive responses to EcM inoculation in the ecologically
simplified, and somewhat benign, nursery environment are unlikely to be represen-
tative of that found at out-planting sites. There is an argument to be made to use
indigenous species for inoculation schemes as they are anticipated to be the most
effective, but we may also need to consider the potential impact of biological
invasions if using exotic fungal species (Vellinga et al., 2009).
1.5 Under What Conditions Will EcM Inoculation
Be Beneficial?
Whilst the body of this chapter thus far has outlined how inoculation with EcM fungi
may improve dipterocarp seedling growth, and the various methods to do so, it is
certainly worth considering whether inoculation should indeed be conducted at all. In
the first paper on dipterocarp EcMs, Singh (1966) noted that “mycorrhizal infection
should not be taken as the ‘cure of all ills’ in the establishment of trees in all sorts of
habitats”, and this warning still stands, more than 40 years later. I now pose three key
questions for consideration before starting to plan inoculation schemes.
It is often considered that there is a need to inoculate seedlings prior to out-
planting but, in fact, in most cases inoculation will occur naturally, and inoculation
schemes may not yield any major benefits for seedling growth or survival (although
we cannot be confident that the same species, or most beneficial species, of EcM
fungi will be formed on each seedlings’ roots every time). The first key question is,
therefore, will inoculation be of benefit to the seedlings? The major benefits of
inoculation are knowing that a seedling, at out-planting, is mycorrhizal with a
known species of fungus which is functi onally beneficial, and thus it will not
need to wait to form EcMs with an unknown group of fungi present in the soil
which may or may not promote seedling growth; this gives it something of an initial
advantage over any out-planted non-mycorrhizal seedlings. However, the main
benefits of inoculation are more likely to be shown under poor soil conditions, as
I outline below.
10 F.Q. Brearley
1.5.1 Successful Inoculatio n Schemes
For inoculation schemes to be successful, a series of well-defined and consistently
repeatable techniques is needed. In other words, a pure culture of inoculum is
needed to allow a regular supply, and currently there are very few fungal species
being maintained in pure culture in the South-east Asian region. Access to a
laboratory with sterile facilities is needed which may be problematic for a number
of sites. In the absence of this, the production of mycorrhizal tablets may be useful
although vagaries of fungal fruit body production and genetic variation between
individual genets will remain unaccounted for. Infrequent production of dipterocarp
seeds can make regular production of planting stock difficult although production
of cuttings from a selected number of dipterocarp species now appears fairly routine
(Moura-Costa and Lundoh 1994; Itoh et al. 2003; Haji Ahmad 2006). It must also
be shown that the inoculant fungus has the ability to improve seedling growth or
survival over that of non-inoculat ed seedlings under field conditions. It appears
much easier to culture species such as Pisolithus or Scleroderma but it must be
remembered that these species are not necessarily those which are most beneficial
to seedling growth or, indeed, are found commonly on dipterocarp seedling roots.
1.5.2 When and Where to Inoculate?
It is often suggested that inoculation may be beneficial for seedlings plan ted
following logging operations. However, in most cases after logging, there are still
a number of smaller dipterocarp trees which will have EcM fungi associated with
them and, as long as the light conditions are not detrimental to seedling growth, this
should allow the rapid formation of EcMs on seedlings by hyphae, sclerotia and
spores already present in the soil (Lee et al. 1996; Ingleby et al. 1998). There is little
evidence so far that selective logging seriously impoverishes the funga l flora
(Watling et al. 1998) although there is an indication of a loss of the some of the
rarer EcM species in logged forest (Lee et al. 1996). Out-planted dipterocarp
seedlings are almost certain to become colonised within a short period of time as
long as they have below-ground access to roots and mycelium radiating from adult
trees (Lee 1991; Alexander et al. 1992; Lee and Alexander 1996; Lee et al. 1996).
The second key question is, therefore, is inoculation beneficial under all situations?
If not, which situations or conditions are most likely to be improved by inoculating
seedlings prior to out-planting?
Inoculation is consider ed more likely to be of benefit when seedlings are planted
in areas where suitable EcM inoculum is not available. This may include severely
degraded areas such as mine tailings (Lee et al. 2008), burnt areas (Akema et al.
2009), degraded peatlands (Turjaman et al. 2007) and areas previously used for
agriculture (Ingleby et al. 2000). For example, Ingleby et al. (2000) found that the
inoculation potential of soils which had been under agriculture for over 20 years
1 The Importance of Ectomycorrhizas for the Growth of Dipterocarps and the Efficacy 11
was essentially absent when compared with an undisturbed forest or plantation in
Vietnam. The work of Turjaman et al. (2007) in degraded peat swamp forest is also
of relevance here as they showed improv ed growth of inoculated dipterocarp
seedlings when out-planted in a degraded area.
The final key question is, is simply adding colonised soil appropriate as inocu-
lum? In many cases, local soil from the vicinity of dipterocarp trees may be equally
as effective as any inoculation schemes although these EcMs are not necessarily the
best species to promote seedling growth and there is no way to control which fungal
species successfully colonise the seedlings’ roots. Smits (1992) outlines a simple
method by which large numbers of seedlings can be inoculated by soil colonised by
EcM hyphae and spores. He advocates the use of soil collected from beneath an
adult tree of the same species, but this is based on his weak assertions (Smits 1983,
1985) of a high degree of host specificity. I suggest it is equally likely that host-
specific pathogens will be present (Packer and Clay 2000) and therefore suggest a
general soil inoculum but ensuring that it is collected in the vicinity of dipterocarps.
In the absence of any other schemes, the inclusion of forest soil should be seen as
the minimum to ensure early EcM colonisation of dipterocarp seedlings.
1.6 Conclusions
Whilst EcMs are often thought to be essential for the successful growth of diptero-
carp seedlings, there is surprisingly little evidence confirming this assertion under
natural conditions. In nursery experiments, mycorrhizal inocula tion has regularly
been shown to increase seedling growth and nutrient concentrations, but when
similar experiments have been conducted in the field, the results are much more
equivocal with inoculation often showing minimal improvements in growth if
seedlings are planted in natural forests (e.g. Tata et al. 2009). If inoculated seedlings
are planted in degraded soils, the improvement in growth is often more marked
although these improvements may not be maintained if the inoculated fungus does
not remain competively dominant on the seedlings’ roots. I therefore suggest that
researchers and forest restorationists carefully consider whether EcM inoculation is
of benefit in the areas they plan to re-plant.
Acknowledgements I thank Dr. Lee Su See and Dr. Robin Sen for helpful thoughts and com-
ments on the manuscript. My work on dipterocarp ectomycorrhizas was supported by the British
Ecological Society Overseas Research Programme.
References
Akema T, Nurhiftisni I, Suciatmih, Simbolon H (2009) The impact of the 1998 forest fire on
ectomycorrhizae of dipterocarp trees and their recovery in tropical rain forests of East
Kalimantan, Indonesia. JARQ 42:137–142
12 F.Q. Brearley
Alexander IJ, H
€
ogberg P (1986) Ectomycorrhizas of tropical angiospermous trees. New Phytol
102:541–549
Alexander IJ, Ahmad N, Lee SS (1992) The role of mycorrhizas in the regeneration of some
Malaysian forest trees. In: Marshall AG, Swaine MD (eds) Tropical rain forest: disturbance and
recovery. The Royal Society, London, UK, pp 357–367
Baxter JW, Dighton J (2005) Diversity–functioning relationships in ectomycorrhizal fungal com-
munities. In: Dighton J, White JF Jr, Oudemans P (eds) The fungal community: its organization
and role in the ecosystem, 3rd edn. CRC Press, Boca Raton, Florida, USA, pp 383–398
Brearley FQ (2003) The role of ectomycorrhizas in the regeneration of dipterocarp seedlings. PhD
Thesis, University of Sheffield, UK
Brearley FQ (2006) Differences in the growth and ectomycorrhizal community of Dryobalanops
lanceolata (Dipterocarpaceae) seedlings grown in ultramafic and non-ultramafic soils. Soil
Biol Biochem 38:3407–3410
Brearley FQ, Press MC, Scholes JD (2003) Nutrients obtained from leaf litter can improve the
growth of dipterocarp seedlings. New Phytol 160:101–110
Brearley FQ, Prajadinata S, Kidd PS, Proctor J, Suriantata (2004) Structure and floristics of an old
secondary rain forest in Central Kalimantan, Indonesia, and a comparison with adjacent
primary forest. For Ecol Manage 195:385–397
Brearley FQ, Scholes JD, Lee SS (2005) Nitrogen nutrition and isotopic discrimination in tropical
ectomycorrhizal fungi. Res Microbiol 156:184–190
Brearley FQ, Scholes JD, Press MC, Palfner G (2007) How does light and phosphorus fertilisation
affect the growth and ectomycorrhizal community of two contrasting dipterocarp species?
Plant Ecol 192:237–249
Bungard RA, Press MC, Scholes JD (2000) The influence of nitrogen on rain forest dipterocarp
seedlings exposed to a large increase in irradiance. Plant Cell Environ 23:1183–1194
Chalermpongse A (1987) Mycorrhizal survey of dry-deciduo us and semi-evergreen dipterocarp
forest ecosystems in Thailand. In: Kostermans AJCH (ed) Proceedings of the third round table
conference on dipterocarps. UNESCO Regional Office for Science and Technology, Jakarta,
Indonesia, pp 81–103
Chang YS, Lapeyrie FF, Lee SS (1994) The survival and competitiveness of Pisolithus tinctorius
on outplanted seedlings of Shorea glauca in Malaysia. In: Khoo KC, Appanah S (eds) Pro-
ceedings of the fifth round table conference on dipterocarps. Forest Research Institute of
Malaysia, Kepong, Malaysia, pp 165–169
Chang YS, Lee SS, Lapeyrie FF, Yazid SM (1995) The competitiveness of two strains of
Pisolithus tinctorius on seedlings of three species of dipterocarps under nursery and field
conditions: preliminary results. In: Wickneswari R, Yahya AZ, Shariff AHM, Haji Ahmad D,
Khoo KC, Suzuki K, Sakurai S, Ishii K (eds) Proceedings of the international workshop of
BIO-REFOR, Kangar, 1994. BIO-REFOR, IUFRO-SPDC, Tokyo, Japan & FRIM, Kepong,
Malaysia, pp 208–212
Chilvers GA, Douglas PA, Lapeyrie FF (1986) A paper-sandwich technique for rapid synthesis of
ectomycorrhizas. New Phytol 103:397–402
Davies SJ, Nur Supardi MN, LaFrankie JV Jr, Ashton PS (2003) The trees of Pasoh Forest: stand
structure and floristic composition of the 50-ha forest research plot. In: Okuda T, Manokaran N,
Matsumoto Y, Niiyama K, Thomas SC, Ashton PS (eds) Pasoh: ecology of a lowland rain
forest in Southeast Asia. Springer-Verlag, Tokyo, Japan, pp 35–50
Fakuara Y, Wilarso S (1993) Effect of mycorrhizal tablet storage on seedling growth of Shorea
pinanga Scheff. In: Anon. (ed) BIO-REFOR: proceedings of Tsukuba-workshop.
BIO-REFOR, IUFRO-SPDC, Tsukuba Science City, Japan, pp 174–179
Gardes M, Bruns TD (1996) Community structure of ectomycorrhizal fungi in a Pinus muricata
forest: above and below-ground views. Can J Bot 74:1572–1583
Hadi S, Santoso E (1988) Effect of Russula spp., Scleroderma sp. and Boletus sp. on the
mycorrhizal development and growth of five dipterocarp species. In: Mohinder Singh M (ed)
Agricultural and biological research priorities in Asia, Proceedings of the IFS symposium of
1 The Importance of Ectomycorrhizas for the Growth of Dipterocarps and the Efficacy 13
science Asia 87. International Foundation for Science & Malaysian Scientific Association,
Kuala Lumpur, Malaysia, pp 183–185
Hadi S, Santoso E (1989) Accumulation of macronutrients by five dipterocarp species inoculated
with different species of mycorrhizal fungi. In: Mahadevan A, Raman N, Natarajan K (eds)
Mycorrhizae for Green Asia: proceedings of the first Asian conference on mycorrhizae. Centre
for Advanced Studies on Botany, University of Madras, India, pp 139–141
Haji Ahmad D (2006) Vegetative propagation of dipterocarp species by stem cuttings using a very
simple technique. In: Suzuki K, Ishii K, Sakurai S, Sasaki S (eds) Plantation technology in
tropical forest science. Springer-Verlag, Tokyo, Japan, pp 69–77
Hoang PND, Tuan DLA (2008) Investigating the ectomycorrhizal appearance of seedlings in the
Tan Phu forest enterprise’s nursery, Dong Nai Province. J Sci Technol Dev 11(1):96–100
Ibrahim Z, Mahat MN, Lee SS (1995) Response of Hopea odorata seedlings to biological
soil conditioners. In: Wickneswari R, Yahya AZ, Shariff AHM, Haji Ahmad D, Khoo KC,
Suzuki K, Sakurai S, Ishii K (eds) Proceedings of the international workshop of BIO-REFOR,
Kangar, 1994. BIO-REFOR, IUFRO-SPDC, Tokyo, Japan & FRIM, Kepong, Malaysia,
pp 179–182
Ingleby K, Munro RC, Noor M, Mason PA, Clearwater MJ (1998) Ectomycorrhizal populations
and growth of Shorea parvifolia (Dipterocarpaceae) seedlings regenerating under three
different forest canopies following logging. For Ecol Manage 111:171–179
Ingleby K, Thuy LTT, Phong NT, Mason PA (2000) Ectomycorrhizal inoculum potential of soils
from forest restoration sites in South Vietnam. J Trop For Sci 12:418–422
Ishida TA, Nara K, Hogetsu T (2007) Host effects on ectomycorrhizal fungal communities: insight
from eight host species in mixed conifer-broadleaf forests. New Phytol 174:430–440
Itoh A, Yamakura T, Tan S, Kendawang JJ, Lee HS (2003) Effects of resource plant size on
rooting of Dryobalanops lanceolata cuttings. J For Res 8:117–121
Kikuchi J (1997) Ectomycorrhiza formation of dipterocarp seedlings. In: Sangwanit U, Thaiutsa B,
Puangchit L, Thammincha S, Ishii K, Sakurai S, Suzuki K (eds) Proceedings of the fifth
international workshop of BIO-REFOR, Bangkok, 1996. BIO-REFOR, IUFRO-SPDC,
Tokyo, Japan, pp 49–52
Lapeyrie FF, Lee SS, Yazid SM (1993) Controlled ectomycorrhizal inoculation of Hopea odorata
(Dipterocarpaceae) cuttings with Hebeloma crustuliniforme. In: Anon. (ed) BIO-REFOR:
proceedings of Tsukuba-workshop. BIO-REFOR, IUFRO-SPDC Tsukuba Science City,
Japan, pp 189–190
Lee SS (1991) Some views on dipterocarp mycorrhiza research in Malaysia. In: Anon. (ed)
BIO-REFOR: proceedings of pre-workshop. BIO-REFOR, IUFRO-SPDC, Bogor, Indonesia,
pp 66–70
Lee SS (1998) Root symbiosis and nutrition. In: Appanah S, Turnbull JM (eds) A review of
dipterocarps: taxonomy, ecology and silviculture. Center for International Forestry Research,
Bogor, Indonesia, pp 99–114
Lee SS, Alexander IJ (1994) The response of seedlings of two dipterocarp species to nutrient
additions and ectomycorrhizal infection. Plant Soil 163:299–306
Lee SS, Alexander IJ (1996) The dynamics of ectomycorrhizal infection of Shorea leprosula
seedlings in Malaysian rain forests. New Phytol 132:297–305
Lee SS, Lim KL (1989) Mycorrhizal infection and foliar phosphorus content of seedlings of three
dipterocarp species grown in selectively logged forest and a forest plantation. Plant Soil
117:237–241
Lee SS, Lapeyrie FF, Yazid SM (1995) Techniques for controlled ectomycorrhizal inoculation
of dipterocarp seedlings and cuttings. In: Supriyanto, Kartana JT (eds) Proceedings of the
second symposium on biology and biotechnology of mycorrhizae and third Asian conference
on mycorrhizae (ACOM III). BIOTROP Special Publication 56, SEAMEO BIOTROP, Bogor,
Indonesia, pp 217–221
Lee SS, Alexander IJ, Moura-Costa PH, Yap SW (1996) Mycorrhizal infection of dipterocarp
seedlings in logged and undisturbed forests. In: Appanah S, Khoo KC (eds) Proceedings of the
14 F.Q. Brearley
fifth round table conference on dipterocarps. Forest Research Institute of Malaysia, Kepong,
Malaysia, pp 157–164
Lee SS, Alexander IJ, Watling R (1997) Ectomycorrhizas and putative ectomycorrhizal fungi of
Shorea leprosula Miq. (Dipterocarpaceae). Mycorrhiza 7:63–81
Lee SS, Watling R, Noraini Sikin Y (2002) Ectomycorrhizal basidiomata fruiting in lowland rain
forests of peninsular Malaysia. Bois Fo
ˆ
r Trop 274(4):33–43
Lee SS, Watling R, Turnbull E (2003) Diversity of putative ectomycorrhizal fungi in Pasoh Forest
Reserve. In: Okuda T, Manokaran N, Matsumoto Y, Niiyama K, Thomas SC, Ashton PS (eds)
Pasoh: ecology of a lowland rain forest in Southeast Asia. Springer-Verlag, Tokyo, Japan,
pp 149–159
Lee SS, Patahayah M, Chong WS, Lapeyrie FF (2008) Successful ectomycorrhizal inoculation of
two dipterocarp species with a locally isolated fungus in Peninsular Malaysia. J Trop For Sci
20:237–247
Martin F, Dı
´
ez J, Dell B, Delaruelle C (2002) Phylogeography of the ectomycorrhizal
Pisolithus species as inferred from nuclear ribosomal DNA ITS sequences. New Phytol
153:345–357
Morris MH, Smith ME, Rizzo DM, Rejma
´
nek M, Bledsoe CS (2008) Contrasting ectomycorrhizal
fungal communities on the roots of co-occurring oaks (Quercus spp.) in a California woodland.
New Phytol 178:167–176
Moura-Costa PH, Lundoh L (1994) A method for vegetative propagation of Dryobalanops
lanceolata (Dipterocarpaceae) by cuttings. J Trop For Sci 6:533–541
Nara K, Kawahara M, Okamura K, Sakurai K, Hogetsu T (1999) Prospects and problems
pertaining to the application of ectomycorrhizal fungi to dipterocarp seedlings in tropical
nurseries. In: Anon. (ed) Proceedings of the international symposium “Can Biological Produc-
tion Harmonize with Environment?”. University of Tokyo, Japan, pp 151–154
Ogawa M (1993) Inoculation method of Scleroderma columnare to dipterocarp seedlings. In:
Anon. (ed) BIO-REFOR: proceedings of Tsukuba-workshop. BIO-REFOR, IUFRO-SPDC,
Tsukuba Science City, Japan, pp 185–188
Ogawa M (2006) Inoculation methods of Scleroderma columnare onto dipterocarps. In: Suzuki K,
Ishii K, Sakurai S, Sasaki S (eds) Plantation technology in tropical forest science. Springer-
Verlag, Tokyo, Japan, pp 185–197
Packer A, Clay K (2000) Soil pathogens and spatial patterns of seedling mortality in a temperate
tree. Nature 404:478–481
Patahayah M, Cynthia PC, Lee SS (2003a) Optimizing growth conditions for ectomycorhizal
inoculum production of the Malaysian strain of Pisolithus tinctorius. Tropical forestry research
in the new millenium: international conference on forestry and forest products research 2001,
pp 551–552
Patahayah M, Brearley FQ, Lee SS (2003b) Responses of three ectomycorrhizal fungi to different
temperatures and media in vitro. Poster presentation at conference on forestry and forest
products research 2003, 6–8 October 2003, Kuala Lumpur, Malaysia
Proctor J, Anderson JM, Chai P, Vallack HW (1983) Ecological studies in four contrasting
lowland rain forests in Gunung Mulu National Park, Sarawak. I. Forest environment, structure
and floristics. J Ecol 71:237–260
Richard F, Millot S, Gardes M, Selosse M-A (2005) Diversity and specificity of ectomycorrhizal
fungi retrieved from an old-growth Mediterranean forest dominated by Quercus ilex.
New Phytol 166:1011–1023
Rinaldi AC, Comandini O, Kuyper TW (2008) Ectomycorrhizal fungal diversity: separating the
wheat from the chaff. Fungal Div 33:1–45
Roeleffs JW (1930) Over kunstmatige verjonging van Pinus merkusii Jungh. et de Vr. en Pinus
khasya Royle. Tectona 23:874–907
Sangtiean T, Schmidt S (2002) Growth of subtropical ECM fungi with different nitrogen sources
using a new floating culture technique. Mycol Res 106:74–85
1 The Importance of Ectomycorrhizas for the Growth of Dipterocarps and the Efficacy 15
Shamsuddin MN (1979) Mycorrhizas of tropical forest trees. In: Furtado JI (ed) Abstracts:
fifth international symposium of tropical ecology. University of Malaya, Kuala Lumpur,
Malaysia, p 173
Shiva MP, Jantan I (1998) Non-timber forest products from dipterocarps. In: Appanah S, Turnbull
JM (eds) A review of dipterocarps: taxonomy, ecology and silviculture. Center for Interna-
tional Forestry Research, Bogor, Indonesia, pp 187–197
Singh KG (1966) Ectotrophic mycorrhiza in equatorial rain forests. Malay For 29:13–18
Sirikantaramas S, Sugioka N, Lee SS, Mohamed LA, Lee HS, Szmidt AE, Yamazaki T (2003)
Molecular identification of ectomycorrhizal fungi associated with Dipterocarpaceae. Tropics
13:69–77
Smith SE, Read DJ (2008) Mycorrhizal symbiosis, 3rd edn. Academic Press, London, UK
Smits WTM (1983) Dipterocarps and mycorrhiza – an ecological adaptation and a factor in forest
regeneration. Flora Males Bull 36:3926–3937
Smits WTM (1985) Specificity of dipterocarp mycorrhiza. In: Molina R (ed) Proceedings of the
6th North American conference on mycorrhizae. Forest Research Laboratory, Corvallis,
Oregon, USA, p 364
Smits WTM (1992) Mycorrhizal studies in dipterocarp forests in Indonesia. In: Read DJ,
Lewis DH, Fitter AH, Alexander IJ (eds) Mycorrhizas in ecosystems. CAB International,
Wallingford, UK, pp 283–292
Supriyanto, Setiawan I, Omon RM (1994) Effect of Scleroderma sp. on the growth of Shorea
mecistopteryx Ridl. seedlings. In: Suzuki K, Sakurai S, Ishii K (eds) Proceedings of the
International Workshop of BIO-REFOR, Yogjakarta, 1993. BIO-REFOR, IUFRO-SPDC,
Tokyo, Japan, pp 186–188
Tata HL, van Noordwijk M, Summerbell R, Werger MJA (2009) Limited response to nursery-
stage mycorrhiza inoculation of Shorea seedlings planted in rubber agroforest in Jambi,
Indonesia. New For 39:51–74
Tawaraya K, Takaya Z, Turjaman M, Tuah SJ, Limin SH, Tamai Y, Cha JY, Wagatsuma T,
Osaki M (2003) Arbusc ular mycorrhizal colonization of tree species grown in peat swamp
forests of Central Kalimantan, Indonesia. For Ecol Manage 182:381–386
Tupas GL, Sajise PE (1976) Mycorrhizal associations in some savanna and reforestation trees.
Kalikasan 5:235–240
Turjaman M, Tamai Y, Segah H, Limin SH, Cha JY, Osaki M, Tawaraya K (2005) Inoculation
with the ectomycorrhizal fungi Pisolithus arhizus and Scleroderma sp. improves early growth
of Shorea pinanga nursery seedlings. New For 30:67–73
Turjaman M, Tamai Y, Segah H, Limin SH, Osaki M, Tawaraya K (2006) Increase in early growth
and nutrient uptake of Shorea seminis inoculated with two ectomycorrhizal fungi. J Trop For
Sci 18:243–249
Turjaman M, Saito H, Santoso E, Susanto A, Gaman S, Limin SH, Shibuya M, Takahashi K,
Tamai Y, Osaki M, Tawaraya K (2007) Effect of ectomycorrhizal fungi inoculated on
Shorea balangeran under field conditions in peat-swamp forests. In: Rieley JO, Banks CJ,
Radjagukguk B (eds) Proceedings of the international symposium and workshop on tropical
Peatland, Yogyakarta, 27–29 Aug 2007. CARBOPEAT, University of Leicester, UK,
pp 143–148
Vellinga EC, Wolfe BE, Pringle A (2009) Global patterns of ectomycorrhizal introductions. New
Phytol 181:960–973
Watling R, Lee SS (1995) Ectomycorrhizal fungi associated with members of the Dipterocarpa-
ceae in Peninsular Malaysia – I. J Trop For Sci 7:657–669
Watling R, Lee SS (1998) Ectomycorrhizal fungi associated with members of the Dipterocarpa-
ceae in Peninsular Malaysia – II. J Trop For Sci 10:421–430
Watling R, Lee SS (2007) Mycorrhizal mycodiversity in Malaysia. In: Jones EBG, Hyde KD,
Vikineswary S (eds) Malaysian fungal diversity. Mushroom Research Centre, University
of Malaya & Ministry of Natural Resources and Environment, Kuala Lumpur, Malaysia,
pp 201–219
16 F.Q. Brearley
Watling R, Taylor AFS, Lee SS, Sims K, Alexander IJ (1995a) A rainforest Pisolithus;
its taxonomy and ecology. Nova Hedwig 61:417–429
Watling R, Taylor AFS, Lee SS, Sims K, Alexander IJ (1995b) Pisolithus aurantioscabrosus.
In: Agerer R (ed) Colour atlas of ectomycorrhizae. Einhorn-Verlag, Schw
€
abisch Gm
€
und,
Germany, plate 85
Watling R, Lee SS, Turnbull E (1998) Putative ectomycorrhizal fungi of Pasoh Forest Reserve,
Negri Sembilan, Malaysia. In: Lee SS, Dan YM, Gauld ID, Bishop J (eds) Conservation,
management and development of forest resources. Forest Research Institute of Malaysia,
Kepong, Malaysia, pp 96–104
Watling R, Lee SS, Turnbull E (2002) The occurrence and distribution of putative ectomycorrhizal
basidiomycetes in a regenerating south-east Asian rainforest. In: Watling R, Frankland JC,
Ainsworth AM, Isaac S, Robinson CH (eds) Tropical mycology, vol 1, Macromycetes. CAB
International, Wallingford, UK, pp 25–43
Yamada A, Katsuya K (2001) The disparity between the number of ectomycorrhizal fungi and
those producing fruit bodies in a Pinus densiflora stand. Mycol Res 105:957–965
Yazid SM, Lee SS, Lapeyrie FF (1994) Growth stimulation of Hopea spp. (Dipterocarpaceae)
seedlings following mycorrhizal inoculation with an exotic strain of Pisolithus tinctorius.
For Ecol Manage 67:339–343
Yazid SM, Lee SS, Lapeyrie FF (1996) Mycorrhizal inoculation of Hopea odorata (Dipterocar-
paceae) in the nursery. J Trop For Sci 9:276–278
Yuwa-Amornpitak T, Vichitsoonthonkul T, Tanticharoen M, Cheevadhanarak S, Ratchadawong S
(2006) Diversity of ectomycorrhizal fungi on Dipterocarpaceae in Thailand. J Biol Sci
6:1059–1064
1 The Importance of Ectomycorrhizas for the Growth of Dipterocarps and the Efficacy 17