T H E FUNGAL ENDOPHYTES O F DIPODIUM V A R I E G A T U M (ORCHIDACEAE)
J.J. Bougoure
1
School
2
1
and J.D.W. D e a r n a l e y
2 3
of Plant Biology,
Faculty of Natural and Agricultural
Sciences,
The University of Western
Australia,
Crawley 6009,
Australia.
Department
of Biological and Physical Sciences,
The University of Southern
Queensland,
Toowoomba
4350,
Australia.
Author for correspondence:
Fax +617 4631 1530; Email:
[email protected]
3
Abstract
W e h a v e a t t e m p t e d to g r o w fungi from p e l o t o n s isolated from roots o f t h e m y c o - h e t e r o t r o p h i c
orchid species Dipodium variegation o n 3 different g r o w t h m e d i a . A s w e l l , w e h a v e a n a l y s e d t h e
fungal D N A within roots o f the orchid u s i n g I T S - P C R analysis, cloning and m o l e c u l a r s e q u e n c i n g .
Fungi failed to g r o w out from p e l o t o n s isolated from plant roots. ITS r D N A s e q u e n c e s w e r e
successfully amplified and c l o n e d from roots o f three orchid plants. C o m p a r i s o n o f these
sequences w i t h I T S r D N A in G e n B a n k r e v e a l e d that the fungal c o m m u n i t y o f D. variegatum
roots
consists of Russula spp. and n o n - m y c o r r h i z a l soil D e u t e r o m y c e t e s .
J.J. B o u g o u r e & J . D . W . D e a r n a l e y ( 2 0 0 5 ) . T h e fungal e n d o p h y t e s o f Dipodium
Australasian
Mycologist 2 4 (1): 1 5 - 1 9 .
variegatum
(Orchidaceae).
Introduction
M y c o - h e t e r o t r o p h s are plant species that obtain a
carbon source from an associated fungus. M y c o heterotrophic plants exist as both m o n o c o t s and
dicots, w i t h m o s t being m o n o c o t y l e d o n o u s . T h e
largest g r o u p o f such plants is the O r c h i d a l e s , 2 8 0
species o f w h i c h are entirely m y c o - h e t e r o t r o p h i c
(Leake
1994). A l t h o u g h m y c o - h e t e r o t r o p h y
is
generally linked w i t h a c h l o r o p h y l l o u s plants, a
n u m b e r o f species exist that obtain carbon b o t h
autotrophically a n d heterotrophically. F o r e x a m p l e ,
m o s t m e m b e r s o f t h e F a m i l y O r c h i d a c e a e , w h i c h are
generally autotrophic w h e n m a t u r e , pass t h r o u g h a
heterotrophic p h a s e during their d e v e l o p m e n t , often
associated w i t h t h e limited food reserves o f t h e
generally m i n u t e seeds ( S m i t h & R e a d 1997).
F u n g i are generally quite easy to isolate from
p e l o t o n s (hyphal coils) in orchid roots a n d are
a m e n a b l e to g r o w t h in p u r e culture. A l t h o u g h a
diversity o f orchid species h a v e b e e n s a m p l e d across
different g e o g r a p h i c a l areas o f the w o r l d , t h e fungal
e n d o p h y t e s are largely similar ( R a s m u s s e n 2 0 0 2 ,
Smith
&
Read
1997). T h e
fungi
are
all
b a s i d i o m y c e t e s w i t h t h e majority b e l o n g i n g to t h e
form
genus
Rhizoctonia,
within
the
h e t e r o b a s i d i o m y c e t e s (Table 1). A few orchid m y c o -
rrhizal fungi, associated w i t h m y c o - h e t e r o t r o p h i c
species, are representatives o f t h e h o m o b a s i d i o m y c e t e s (Table 1).
R e s e a r c h into the fungal e n d o p h y t e s o f A u s t r a l i a n
terrestrial orchids has m o s t l y focussed o n t h e
autotrophic orchid species (e.g. H u y n h et al. 2 0 0 4 ,
M i l l i g a n & W i l l i a m s 1988, P e r k i n s & M c G e e 1995,
P e r k i n s et al. 1995, P o p e & Carter 2 0 0 1 , W a r c u p
1 9 7 1 , 1973, 1981) w h i l e t h e m y c o - h e t e r o t r o p h i c
species h a v e largely e s c a p e d attention. W a r c u p
( 1 9 8 5 , 1991) successfully isolated
Rhizoctonia-like
fungal e n d o p h y t e s from t w o species o f t h e r a r e
subterranean orchid Rhizanthella.
H e named the
e n d o p h y t e o f R. gardneri,
Thanatephorus
gardneri
sp. n o v . after s u c c e e d i n g in p r o d u c i n g its t e l e o m o r p h
( W a r c u p 1988).
T h e orchid g e n u s Dipodium
or 'hyacinth orchids',
contains a n u m b e r o f leafless m y c o - h e t e r o t r o p h i c
species that appear to rely h e a v i l y o n c a r b o n
p r o v i d e d b y e n d o p h y t i c fungi ( W a r c u p 1990). T h e
identity o f these fungal e n d o p h y t e s is u n k n o w n .
W a r c u p ( 1 9 8 1 , 1991) s u c c e e d e d in isolating a fine,
w h i t e , u n d a m p e d a n d slow g r o w i n g e n d o p h y t e
from roots o f D. punctatum J.E. S m . b u t w a s u n a b l e t o
Table 1 . A list o f f u n g a l g e n e r a c a p a b l e o f f o r m i n g o r c h i d m y c o r r h i z a s . A n a m o r p h i c a n d t e l e o m o r p h i c g e n e r a a r e
included for the Heterobasidiomycetes. Adapted from R a s m u s s e n (2002) a n d the recent systematic overview of
B a s i d i o m y c e t e s a c c o r d i n g t o J e n s H. P e t e r s e n , h t t p : / / w w w . m y c o k e y . c o m / s y s t e m a t i c s . h t m l .
Heterobasidiomycetes
Order
Order
Genera
Ceratobasidiwn
Gloeocystidiales
Russula
Moniliopsis
Oliveonia
Thanatephorus
Hymenochaetales
Epulorhiza
Tulasnella
Thelephorales
Erythromyces
Thelephora,
Tomentella
Sebacina
Agaricales
Armillaria,
Anamorph
Teleomorph
Ceratorhiza
Ceratobasidiales
Tulasnellales
Homobasidiomycetes
Genera
Auriculariales
Mycena
Figure 1 . C r o s s s e c t i o n o f r o o t o f D.
variegatum
s h o w i n g l a r g e n u m b e r s of b r o w n - c o l o u r e d f u n g a l
structures. Scale bar = 100 u m .
assign t h e fungus a t a x o n o m i c identity other than
' n o t a r h i z o c t o n i a ' . W a r c u p (1981) isolated a
Tulasnella-Uke
fungal e n d o p h y t e from the epiphytic
D. pandanum
F . M . Bail. In this study w e h a v e used
t w o a p p r o a c h e s to identify t h e fungal e n d o p h y t e o f
the m y c o - h e t e r o t r o p h i c orchid Dipodium
variegatum
R. Br., a w i d e s p r e a d w o o d l a n d species in South-east
Q u e e n s l a n d (Stanley & Ross 1989). W e h a v e isolated
single p e l o t o n s from roots o f t h e orchid using
m i c r o t e c h n i q u e s and attempted to g r o w these on a
n u m b e r o f different media. A s well, w e h a v e used
PCR-amplification of r D N A I T S regions, c l o n i n g and
s e q u e n c e c o m p a r i s o n of fungi from
colonised
D. variegatum
roots.
The
results
of
these
investigations are presented here.
Materials and Methods
A c q u i s i t i o n of o r c h i d a n d f u n g a l m a t e r i a l
Roots w e r e collected from t w o Dipodium
variegatum
plants at T o o h e y State Forest, B r i s b a n e , Q L D ,
Australia ( 2 7 ° 3 3 ' S , 1 5 3 ° 0 2 ' E ) and t w o plants from
Duggan
Park,
Toowoomba,
QLD,
Australia
( 2 7 ° 3 5 ' S , 1 5 1 ° 5 9 ' E ) . O n e c m long root portions
w e r e cut and rinsed under tap water. T h e s e portions
w e r e then soaked in 1 0 0 % bleach for 3 0 s e c o n d s a n d
rinsed with sterile dFLO three t i m e s . Thin, transverse
sections w e r e then cut from t h e root portions a n d
these w e r e then crushed to release individual
p e l o t o n s from cortical cells ( s o m e u n c r u s h e d sections
w e r e kept and p h o t o g r a p h e d using a N i k o n E 6 0 0
upright
photomicroscope
(Nikon
Corporation,
T o k y o , Japan)). W h i l e v i e w i n g u n d e r a c o m p o u n d
microscope,
healthy
(non-collapsed)
individual
p e l o t o n s w e r e r e m o v e d with a 2.5 ul v o l u m e
m i c r o p i p e t t e to m a l t extract agar ( M E A ) o r o n e
sixth strength neutral d o x yeast ( N D Y 6 ) agar plates
containing 15 m g 1" streptomycin a n d 15 m g
1" tetracycline
or potato
dextrose
( P D ) agar
plates containing 100 m g 1" streptomycin and 50 m g 1"'
1
tetracycline. A t least six individual p e l o t o n s w e r e
r e m o v e d from each plant. Culture plates w e r e sealed
and incubated in t h e dark at 2 0 ° C for at least three
weeks.
M o l e c u l a r a n a l y s i s of Dipodium
endophytes
R o o t s w e r e collected from three flowering
Dipodium
variegatum
plants at D u g g a n Park, T o o w o o m b a .
D N A w a s extracted from these roots u s i n g a D N e a s y
Plant mini kit ( Q i a g e n , D o n c a s t e r , V i c , Australia)
following t h e m a n u f a c t u r e r ' s instructions.
T h e fungal ITS region o f each s a m p l e w a s amplified
in 50 p i reaction v o l u m e s , each containing 3 8 p i
sterile distilled H 0 , 5 p i 10X buffer (50 m M K C 1 ,
10 m M Tris-HCl, 0 . 1 % Triton X - 1 0 0 ; Invitrogen
Australia, M t W a v e r l e y , V i c , Australia), 2.5 p i
50 m M M g C l (Invitrogen Australia), 1 p i 10 m M
d N T P (Invitrogen Australia), 1 p i o f each o f the
fungal specific I T S I F p r i m e r ( G a r d e s & B r u n s 1993)
and I T S 4 ( W h i t e et al. 1990), 0.5 p i of Taq D N A
p o l y m e r a s e (Invitrogen Australia) a n d 1 p i o f
extracted g e n o m i c D N A . P C R amplifications w e r e
p e r f o r m e d in a T h e r m o H y b a i d P C R E x p r e s s
t h e r m o c y c l e r (Integrated Sciences,
Willoughby,
N S W , Australia) w i t h 35 cycles o f 9 5 ° C for 1 min.,
5 0 ° C for 1 m i n . and 7 2 ° C for 1 min., w i t h a final
incubation at 7 2 ° C for 10 min. T h e s e reactions w e r e
p e r f o r m e d in duplicate and a n e g a t i v e control w a s
included w i t h o u t D N A . T h e resulting amplification
p r o d u c t s w e r e electrophoresed in 2 % ( w / v ) a g a r o s e
gels with ethidium b r o m i d e , and visualized u n d e r U V
light.
2
2
I T S - P C R p r o d u c t s w e r e purified u s i n g a D N A
purification kit ( R o c h e Applied Science, Castle Hill,
N S W , Australia) prior t o cloning with t h e p G E M - T
E a s y vector s y s t e m ( P r o m e g a , A n n a n d a l e , N S W ,
Australia), both c o n d u c t e d as p e r t h e m a n u f a c t u r e r ' s
instructions. S e q u e n c i n g reactions w e r e p e r f o r m e d in
10 p i v o l u m e s c o n t a i n i n g 4 0 0 ng o f purified p l a s m i d
D N A , 6.4 p m o l e s o f T 7 p r o m o t o r p r i m e r at t h e
B r i s b a n e laboratory o f the Australian G e n o m e
R e s e a r c h Facility ( A G R F ) . I T S s e q u e n c e s w e r e
analysed
using
BLAST
searches
(http://www.ncbi.nlm.nih.gov/).
Results
F u n g a l isolation
F u n g a l colonisation w a s o b v i o u s within the roots o f
t h e Dipodium plants with the cortex containing large
n u m b e r s of b r o w n - c o l o u r e d structures (Fig. 1).
A t h i g h e r magnification these structures c o n t a i n e d
both
collapsed
fungal
material
and
intact
h y p h a e (Fig. 2). A l t h o u g h single fungal p e l o t o n s w e r e
successfully r e m o v e d from root cortical cells t h e s e
failed to g r o w o n M E , N D Y - 6 and P D agar.
M o l e c u l a r a n a l y s i s o f Dipodium
endophytes
I T S - P C R amplification p r o d u c e d a n u m b e r o f b a n d s
b e t w e e n 6 0 0 and 8 0 0 b p for e a c h D N A s a m p l e (data
n o t s h o w n ) . Full l e n g t h I T S s e q u e n c e s w e r e o b t a i n e d
for t w o clones from each o f t h e three s a m p l e d
D. variegatum
p l a n t s a n d these h a v e b e e n d e p o s i t e d
in G e n B a n k w i t h t h e accession c o d e s listed in T a b l e
2. B o t h clones from P l a n t 1 s h o w e d h i g h identities
( 9 0 % o v e r 3 9 3 b p ) t o Russula occidentalis
Singer
( T a b l e 2). T h e first c l o n e from Plant 2 h a d a high
identity to Trichoderma
hamatum
(Bonorden)
B a i n i e r ( 9 9 % o v e r 6 0 7 - 6 1 1 b p ) w h i l e the s e c o n d
clone h a d 8 9 - 8 8 % identity (over 3 3 7 - 3 7 2 b p ) t o t w o
species o f Russula ( T a b l e 2 ) . T h e first clone from
P l a n t 3 h a d h i g h identity to a Verticillium
sp. ( 9 5 %
o v e r 585 bp) a n d an u n c u l t u r e d fungus ( 9 7 % o v e r
3 6 7 b p ) , w h i l e t h e s e c o n d clone h a d affinity (96 &
9 5 % o v e r 166-168 b p ) t o t w o h o m o b a s i d i o m y c e t e
fungi, Halocyphina
villosa K o h l m . et K o h l m and
Merisimodes fasciculata
( S c h w e i n . ) Earle ( T a b l e 2 ) .
Discussion
S e q u e n c e c o m p a r i s o n o f t h e c l o n e d fungal I T S r D N A
s a m p l e s with G e n B a n k r e v e a l e d that t h e fungal
c o m m u n i t y o f D. variegatum
roots consists o f a
number
of
species.
The
non-mycorrhizal
Deuteromycetes,
Trichoderma
hamatum
and
Verticillium
sp. a r e likely to b e c o n t a m i n a n t s o n t h e
orchid roots as t h e y a r e c o m m o n soil inhabiting fungi
( A l e x o p o u l o u s , M i m s & B l a c k w e l l 1996). A s
Halocyphina
villosa is a m a r i n e fungus ( H i b b e r t &
B i n d e r 2 0 0 1 ) a n d Merismodes
spp. have never been
r e c o r d e d as f o r m i n g orchid m y c o r r h i z a s
(see
R a s m u s s e n 2 0 0 2 ) t h e results strongly s u g g e s t that the
p r i m a r y peloton f o r m i n g fungi in D.
variegatum
roots are m e m b e r s o f t h e h o m o b a s i d i o m y c e t e g e n u s
Russula.
T h i s h a s g o o d correlation w i t h m o l e c u l a r
studies o f species o f N o r t h A m e r i c a n m y c o heterotrophic o r c h i d s w h i c h h a v e also s h o w n t o b e
c o l o n i s e d b y Russula s p p . ( T a y l o r & B r u n s 1999).
W h i l e t h e s a m p l e size h e r e is small, t h e s e
findings
also c o m p a r e favourably with other studies o f m y c o heterotrophic o r c h i d s w h i c h h a v e s h o w n quite
specific fungal associations (Selosse et al. 2 0 0 2 ,
T a y l o r & B r u n s 1997, 1999). I n d e e d , as m o r e
m o l e c u l a r analyses o f a u t o t r o p h i c orchid e n d o p h y t e s
are b e i n g c o n d u c t e d it a p p e a r s that t h e l a c k o f
specificity u s u a l l y attributed to these o r c h i d t y p e s
(e.g. W a r c u p 1 9 8 1 , Z e l m e r et al. 1986) m a y n o t b e
t h e case (see B o u g o u r e et al. 2 0 0 5 , M c C o r m i c k et al.
2004).
Table 2 . C l o s e s t t w o m a t c h e s f r o m B L A S T s e a r c h e s o f f u n g a l s e q u e n c e s a m p l i f i e d f r o m t h e t h r e e D.
variegatum
plants. Included are t h e t w o closest G e n B a n k matches a n d a c c e s s i o n c o d e s , s e q u e n c e identity a n d overlap of
each match.
Plant &
clone
no.
Plant 1
clone 1
Plant 1
clone 2
Plant 2
clone 1
Plant 2
clone 2
Plant 3
clone 1
Plant 3
clone 2
GenBank
Accession
Code
AY702070
AY702071
AY702072
Closest species match & accession code
Sequence
identity (%)
Sequence
overlap (bp)
Russula
Russula
90
90
90
90
99
99
89
88
95
97
96
95
393
393
393
393
607
611
337
372
585
367
166
168
occidentalis
occidentalis
Russula
occidentalis
Russula
occidentalis
Trichoderma
hamatum
Trichoderma
hamatum
AY702073
Russula
Russula
AY702074
Verticillium s p .
Unculturedfungus
Halocyphina
villosa
Merismodes
fasciculata
AY702075
Solaris
lepida
The endophytes of a number of European and North
A m e r i c a n m y c o - h e t e r o t r o p h i c orchids h a v e b e e n
recently identified v i a m o l e c u l a r biology t e c h n i q u e s .
T a y l o r & B r a n s ( 1 9 9 7 , 1999) a n d M c K e n d r i c k et al.
( 2 0 0 0 a ) found that Cephalanthera
austinae
and
Corallorhiza
trifida w e r e c o l o n i s e d b y m e m b e r s of
the T h e l e p h o r a c e a e , w h e r e a s Corallorhiza
maculata
and
C. mertensiana
w e r e colonised b y t h e
R u s s u l a c e a e . Selosse et al. ( 2 0 0 2 ) recently s h o w e d
that Neottia nidus-avis
w a s c o l o n i s e d b y sebacinoid
fungi, w h i l e T a y l o r et al. ( 2 0 0 3 ) s h o w e d that t h e
p r i m a r y fungal partner o f Hexalectris
spicata w a s
also a m e m b e r o f t h e S e b a c i n a c e a e . T o g e t h e r with
the p r e s e n t study, these a n a l y s e s show that both
h o m o b a s i d i o m y c e t e s a n d h e t e r o b a s i d i o m y c e t e s can
be e n d o p h y t e s o f m y c o - h e t e r o t r o p h i c orchids.
R e s e a r c h into m y c o - h e t e r o t r o p h i c orchids a n d their
fungal partners s u g g e s t s that t h e y are often i n v o l v e d
in tripartite interactions w i t h s u r r o u n d i n g tree a n d
s h r u b species, t h u s p r o v i d i n g t h e orchid w i t h a
greater
and
more
reliable
nutrient
source
( M c K e n d r i c k et al. 2 0 0 0 a , b , Selosse et al. 2 0 0 2 ,
W a r c u p 1985). T h e orchids u s e d in this study all
o c c u r r e d close t o species o f Eucalyptus
a n d it is
p o s s i b l e that t h e orchids a r e linked t o t h e s e tree
species v i a e c t o m y c o r r h i z a l associations. Support for
this c o m e s from t h e fact that Russula species, w h i c h
are well k n o w n e c t o m y c o r r h i z a l fungi in Australia
(Brundrett et al. 1996), h a v e b e e n implicated in
tripartite relationships b e t w e e n trees a n d m y c o heterotrophic orchids ( T a y l o r & B r a n s 1999).
T h e lack o f success in g r o w i n g extracted p e l o t o n s
m a y b e o w i n g t o t h e a b s e n c e o f specific cultural
r e q u i r e m e n t s in t h e m e d i a u s e d o r that there w e r e
p r o b l e m s w i t h t h e extraction m e t h o d o l o g y . T a y l o r &
B r a n s ( 1 9 9 7 ) a n d Z e l m e r et al. (1996) h a v e also
e n c o u n t e r e d difficulties in isolating fungi from vari­
AY228349.1
AY534206.1
AY228349.1
AY534206.1
AY154937.1
Z48816.1
AF418627.1
AY061686.1
A Y 172097.1
AF504849.1
AY571042.1
AY571052.1
ous species o f terrestrial orchids (including m e m b e r s
o f t h e R u s s u l a c e a e in the former study). T h e latter
authors s u g g e s t that s o m e orchid e n d o p h y t e s m a y
h a v e antibiotic sensitivity o r h a v e an obligate
d e p e n d e n c y on their plant hosts.
In c o n c l u s i o n , in the a b s e n c e o f p u r e culture
synthesis o f an association u n d e r e x p e r i m e n t a l
conditions, w e h a v e identified t h e p r i m a r y p e l o t o n f o r m i n g fungal e n d o p h y t e s o f D. variegatum
as
members of the Russulaceae using molecular-based
techniques. It w o u l d be interesting t o e x a m i n e t h e
fungal e n d o p h y t e s of other Dipodium
species to
d e t e r m i n e if t h e s e w e r e similarly colonised by these
homobasidiomycetes.
Acknowledgements
W e t h a n k t h e University o f S o u t h e r n Q u e e n s l a n d a n d
t h e C e n t r e for B i o m e d i c a l R e s e a r c h for their
financial s u p p o r t , t h e Q u e e n s l a n d P a r k s a n d Wildlife
Service for g r a n t i n g u s a p e r m i t t o collect orchids a n d
Mr Damian Bougoure, M r Austen Chen and Mrs
A d e l e J o n e s for advice and t e c h n i c a l assistance.
References
A l e x o p o u l o s C.J., M i m s C . W . & B l a c k w e l l M .
( 1 9 9 6 ) . Introductory
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