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Some aspects on the biotrophy of VAM fungi
29 Agriculture, Ecosystems and Environment, 29 (1989) 29-33 Elsevier Science Publishers B.V., Amsterdam Printed in Czechoslovakia
SOME ASPECTS ON THE BIOTROPHY OF VAM FUNGI G. B~ard, Y. Pich~ and J.A. Fortin Centre de Recherche en Biologie Foresti~re, Facult~ de Foresterie et de G~d~sie, Universit~ Lavai, GIK 7P4, Quebec, Canada ABSTRACT Hyphal growth from germinating spores of Gigaspora margarifawas monitored u n d e r several conditions: with or without roots, root colonization, or removal of the spore. The results indicate that roots stimulate fungal growth by different mechanisms. Before colonization, roots provide inductive factors which promote fungal growth when the nutrients in spores are available. When colonization is .completed, the roots also provide nutrients. The inductive action of roots probably involves root exudates and particularly gas balance, in which CO2 appears to play key role. Any attempts to cultivate VAM fungi in pure c.lture should consider the nutritional and inductive role of roots. INTRODUCTION The pure culture of YAM fungi would be achieved when hyphae, severed from fungal propagules (spores or vesicules), can be grown on a medium free of plant hosts. However, the relative contributions of fungal propagules and plant hosts must be considered if we are to replace these biotic factors by abiotic factors. In this study, we used a simple dual culture system where one Ri TDNA transformed root was introduced to one germinating spore of Gigas'pora margarita under standardized conditions. We manipulated this system to decipher mechanisms determining fungal growth during mycorrhiza establishment. MATERIALS AND METHODS Details on materials and methods have been described by l~card & Pich~ (1988). In the dual culture system, a single germinating spore of G. margarita and a single carrot root grew interactively on a common medium (M). Fungal colonization of the root was initiated by directing the
30
germ tube to a pre-selected point of the. root. It was prevented by using a dialysis membrane as a physical barrier between the two partners. Elongation of extraradical hyphae was recorded daily under a binocular lens. The presence of roots alters both the gas and agar composition of the "dual culture system. A bi-compartmental system was used to separate effects of these alterations on hyphal g~;owth. In one compartment, a germinating spore was placed on fresh medium (M) or on used medium (M') on which roots had grown for 3 weeks. Roots were or were not placed in the second compartment. Hyphal elongation from germinated spores was also recorded in M or M' in non sealed Petri dishes placed inside sealed chamber streamed with atmospheric air containing 0.03% CO2 or with air supplemented with o,s%co2. RESULTS AND DISCTJSSION In absence of roots, hyphal elongation was limited to 10 days growth and to 30 nun. The introduction of roots in the proximity of germinating spores considerably stimulated the growth of germ tubes and hyphae, with fungal growth continuing for 28 days and yielding 400ram of hyphae (Fig. 1). This stimulated growth occured without root colonization. When root colonization was permitted, hyphai growth was not limited (for up to 7 months) even when the spores were removed 8 days after root introduction (Fig. 2). Roots, therefore, promote hyphal growth by 2 mechanisms, one which acts at distance and is spore dependent and the other which begins during arbuscule formation (data not shown) and does not need the spore. We postulate that the first mechanism is an induction which activates fungal growth ~,hen a nutritional source is available. The second mechanism is the biotrophic state where the root is the nutritional source but where the inductive mechanism may still be essential. The inductive contribution of roots could be divided into a volatile component and a component related to a change in agar medium composition after root growth (Fig. 3). These two components had a negligible effect on hyphai growth when acting separately, but they acted synergistically in the stimulation of hyphal growth.
31
400 with root but no colonization
300
ooO o°°uooo°°° u o
~nJ
o I]
~200'
o D O
.~10O'
0 without root aa j,z j ~ t Q Q w m o u m u s e n m J * w a s a e a a e m m a o
0
@
|
0
|
10 20 days of dual culture
30
Fig. I. Hyphal growth from germinating spores of G. margarita in absence of root (u) or in presence of root without root colonization (,',). Each point of the curves is the mean of I0 replicates.
700
|=:
g m
4oo~ ]a, 200"
'~a QB
witho,,~t root
mu
~' 100"
o"~__L----. a u. u.
@o
with root
e0 colonization
4
.
8
colonization
. . . . 12
16
20
24
28
days of dual culture
Fig. 2. Hyphal growth from germinating spores of G. margarita in presence of root: with root colonization (88) or without root colonization (c3), with the spore removed (-S) after 8 days of dual culture in both cases. Each point of the curves is the mean of 6 replicates.
32
300
jill
II
Bit
i m
E
~200"
o t~
100" n.
.',~,C °'''q
me
®
0
l
0
!0
" .0"..... aa
o
a a a l
oa
aan
l
20 30 days of dual culture
M' M i
40
Fig. 3. Hyphal growth from germinating spores of G. margarita on M or M' medium, in presence (u) or absence (a) of gaseous components from roots. Each point of the curves is the m e a n of at least 8 replicates.
250"
C O 2 (0.5'~)
"~ 200"
a
.I 150'
i
I C O 2 (0.o3~) g - -
O_
@
M
m~m
M'
M
M'
Fig. 4. Hyphal g r o w t h from germinating spores of G. margarita after 4 weeks on M or M' medium under a stream of atmospheric air (CO2, 0.03~) or of air with additional CO2 (0.$%). Each histogram value is a mean of, at least, 13 replicates.
33
It was possible to replace the gaseous component from root by air supplemented with 0.5% CO2 (Fig. 4). The M' medium acted synergistically with 0.5% CO2 in stimulating hyphal growth but had little effect in atmospheric air containing CO2 0.03%. Similarly, the CO2 factor alone only had a small positive effect on hyphal growth. It appears that the presence of roots provided appropriate gaseous conditions in term of CO2 concentration. It also appears that roots provided exudates which probably acted as plant signals to stimulate fungal growth. The two inductive factors may represent the first barrier to pure culture of VAM fungi. An understanding of their action would facilitate investigation of the second barrier consisting of trophic conditions. REFERENCES
B]~CARD, G., PICH~, Y.: New aspects on the acquisition of biotrophic status by a VAM fungus,
Gigc~oramargarita.-NewPhytologist, submitted, 1988.
B6card, G., P i c h ~ , Y. and F o r t i n J . A.w 1989: Some a s p e c t s o f t h e b i o t r o p h y o f VAM f u n g i : & g r t c . Ecosystems E n v i r o n . , 29: 29-33.
SOME ASPECTS ON THE BIOTROPHY OF VAM FUNGI G. B~ard, Y. Pich~ and J.A. Fortin Centre de Recherche en Biologie Foresti~re, Facult~ de Foresterie et de G~d~sie, Universit~ Lavai, GIK 7P4, Quebec, Canada ABSTRACT Hyphal growth from germinating spores of Gigaspora margarifawas monitored u n d e r several conditions: with or without roots, root colonization, or removal of the spore. The results indicate that roots stimulate fungal growth by different mechanisms. Before colonization, roots provide inductive factors which promote fungal growth when the nutrients in spores are available. When colonization is .completed, the roots also provide nutrients. The inductive action of roots probably involves root exudates and particularly gas balance, in which CO2 appears to play key role. Any attempts to cultivate VAM fungi in pure c.lture should consider the nutritional and inductive role of roots. INTRODUCTION The pure culture of YAM fungi would be achieved when hyphae, severed from fungal propagules (spores or vesicules), can be grown on a medium free of plant hosts. However, the relative contributions of fungal propagules and plant hosts must be considered if we are to replace these biotic factors by abiotic factors. In this study, we used a simple dual culture system where one Ri TDNA transformed root was introduced to one germinating spore of Gigas'pora margarita under standardized conditions. We manipulated this system to decipher mechanisms determining fungal growth during mycorrhiza establishment. MATERIALS AND METHODS Details on materials and methods have been described by l~card & Pich~ (1988). In the dual culture system, a single germinating spore of G. margarita and a single carrot root grew interactively on a common medium (M). Fungal colonization of the root was initiated by directing the
30
germ tube to a pre-selected point of the. root. It was prevented by using a dialysis membrane as a physical barrier between the two partners. Elongation of extraradical hyphae was recorded daily under a binocular lens. The presence of roots alters both the gas and agar composition of the "dual culture system. A bi-compartmental system was used to separate effects of these alterations on hyphal g~;owth. In one compartment, a germinating spore was placed on fresh medium (M) or on used medium (M') on which roots had grown for 3 weeks. Roots were or were not placed in the second compartment. Hyphal elongation from germinated spores was also recorded in M or M' in non sealed Petri dishes placed inside sealed chamber streamed with atmospheric air containing 0.03% CO2 or with air supplemented with o,s%co2. RESULTS AND DISCTJSSION In absence of roots, hyphal elongation was limited to 10 days growth and to 30 nun. The introduction of roots in the proximity of germinating spores considerably stimulated the growth of germ tubes and hyphae, with fungal growth continuing for 28 days and yielding 400ram of hyphae (Fig. 1). This stimulated growth occured without root colonization. When root colonization was permitted, hyphai growth was not limited (for up to 7 months) even when the spores were removed 8 days after root introduction (Fig. 2). Roots, therefore, promote hyphal growth by 2 mechanisms, one which acts at distance and is spore dependent and the other which begins during arbuscule formation (data not shown) and does not need the spore. We postulate that the first mechanism is an induction which activates fungal growth ~,hen a nutritional source is available. The second mechanism is the biotrophic state where the root is the nutritional source but where the inductive mechanism may still be essential. The inductive contribution of roots could be divided into a volatile component and a component related to a change in agar medium composition after root growth (Fig. 3). These two components had a negligible effect on hyphai growth when acting separately, but they acted synergistically in the stimulation of hyphal growth.
31
400 with root but no colonization
300
ooO o°°uooo°°° u o
~nJ
o I]
~200'
o D O
.~10O'
0 without root aa j,z j ~ t Q Q w m o u m u s e n m J * w a s a e a a e m m a o
0
@
|
0
|
10 20 days of dual culture
30
Fig. I. Hyphal growth from germinating spores of G. margarita in absence of root (u) or in presence of root without root colonization (,',). Each point of the curves is the mean of I0 replicates.
700
|=:
g m
4oo~ ]a, 200"
'~a QB
witho,,~t root
mu
~' 100"
o"~__L----. a u. u.
@o
with root
e0 colonization
4
.
8
colonization
. . . . 12
16
20
24
28
days of dual culture
Fig. 2. Hyphal growth from germinating spores of G. margarita in presence of root: with root colonization (88) or without root colonization (c3), with the spore removed (-S) after 8 days of dual culture in both cases. Each point of the curves is the mean of 6 replicates.
32
300
jill
II
Bit
i m
E
~200"
o t~
100" n.
.',~,C °'''q
me
®
0
l
0
!0
" .0"..... aa
o
a a a l
oa
aan
l
20 30 days of dual culture
M' M i
40
Fig. 3. Hyphal growth from germinating spores of G. margarita on M or M' medium, in presence (u) or absence (a) of gaseous components from roots. Each point of the curves is the m e a n of at least 8 replicates.
250"
C O 2 (0.5'~)
"~ 200"
a
.I 150'
i
I C O 2 (0.o3~) g - -
O_
@
M
m~m
M'
M
M'
Fig. 4. Hyphal g r o w t h from germinating spores of G. margarita after 4 weeks on M or M' medium under a stream of atmospheric air (CO2, 0.03~) or of air with additional CO2 (0.$%). Each histogram value is a mean of, at least, 13 replicates.
33
It was possible to replace the gaseous component from root by air supplemented with 0.5% CO2 (Fig. 4). The M' medium acted synergistically with 0.5% CO2 in stimulating hyphal growth but had little effect in atmospheric air containing CO2 0.03%. Similarly, the CO2 factor alone only had a small positive effect on hyphal growth. It appears that the presence of roots provided appropriate gaseous conditions in term of CO2 concentration. It also appears that roots provided exudates which probably acted as plant signals to stimulate fungal growth. The two inductive factors may represent the first barrier to pure culture of VAM fungi. An understanding of their action would facilitate investigation of the second barrier consisting of trophic conditions. REFERENCES
B]~CARD, G., PICH~, Y.: New aspects on the acquisition of biotrophic status by a VAM fungus,
Gigc~oramargarita.-NewPhytologist, submitted, 1988.
B6card, G., P i c h ~ , Y. and F o r t i n J . A.w 1989: Some a s p e c t s o f t h e b i o t r o p h y o f VAM f u n g i : & g r t c . Ecosystems E n v i r o n . , 29: 29-33.