Pertanika 10(3), 271 - 275(1987)

Propagation and Maintenance of VAM Cultures

H. AZIZAHCHULAN
Department of Soil Science,
Faculty of Agriculture,
Universiti Pertanian Malaysia,
43400 Serdang, Selangor Darul Ehsan, Malaysia.
MOHAMMAD OMAR
Ministry of Defence, Kuala Lumpur, Malaysia.
Key words: VAM species; Seteria grass, Allium ascalonicum
ABSTRAK
Kertas ini melaporkan perambatan dan pengkelasan lapan kultur kulat mikoriza vesikularbuskul (MVA). Kulat MVA dapat dibiak dan dikekalkan diatas rumput Setaria sebagaiperumah.
ABSTRACT
This paper reports the establishment of eight vesicular-arbuscular mycorrhizal (VAM) fungal
species under Malaysian conditions. VAM fungi can be propagated and maintained on Setaria as the
host plant.
INTRODUCTION
Mass production of VA mycorrhizal inoculum
has become technically feasible with the introduction of the pot culture technique of Mosse
and Gerdemann thirty years ago (Wood, 1985).
Since then, several attempts to culture the fungi
in axenic or soilless media have met with little
success (Hepper, 1984). This may well be
because VAM fungi are obligate symbionts and
could therefore only be expected to be propagated and maintained in association with
plant roots (Sylvia, 1984), and not isolated,
grown or increased on routine laboratory culture
media (Schenck, 1984).
Using techniques similar to those suggested
by Menge (1984), large quantities of inocula
have been successfully produced commercially
by the Native Plant Inc. (NPI) which can now produce contaminant-free inoculum in 2 - 4 months
consisting of 100 - 120g fresh weight of roots per
litre, with 50 - 75% VAM colonization and containing 100-1,000 VAM fungal spores per
milliliter (Wood, 1985). Ultimately, NPI hopes
to achieve a target of 80,000- 100,000 litres of
pot culture inoculum annually.

Similarly, Dehne (pers. comm. to I.R. Hall)

has produced a method of raising VAM
inoculum cheaply inside expanded clay. However, the current price of these inocula prohibits
their commercial use in developing countries.
Hence, techniques for propagating and maintaining VAM cultures under the local conditions
still have to be found.
Sylvia (1984) has listed several host plants
which are considered suitable for raising VAM
inoculum e.g. sudangrass (Sorghum bicolor, L.
Moench var. sundanese); guineagrass (Panzcum
maimum Jacq.) as recommended by Bagyaraj
and Manjunath (1980); bahiagrass (Paspalum
notatum Flugge) which can withstand high glasshouse temperature (Sylvia and Schenck, 1983)
and mixed host plant combinations such as soybean (Glycine max (L.) Merr.) and bahiagrass
(Schenck and Smith, 1981). To date, no report
has been made on the use of Setaria as a host
plant. The pot experiment reported here was an
attempt to find a suitable host plant for propagating the VAM inoculum under Malaysian
conditions.

H. AZIZAH CHULAN

MATERIALS AND METHODS

Source of Inoculum
Inoculum of eight endogonaceous species consisting of roots, mycelium and spores were
obtained from I.R. Hall in New Zealand. The
soil used by him is Warepa silt loam mixed 1:1
(v/v) with quartz sand and basal fertilizer added
(Hall, 1978). The soil : sand mixture was steamed overnight at 95C to kill all pathogens and
mycorrhizal propagules. The host plant used was
Trifolium repens (white clover) cultivar "Grasslands Huia'\ Table 1 gives the list of the eight
VAM species and their origin.
Growth Medium
Two soil types were tested for suitability as a
growth medium for the mycorrhizal fungi: Carey
series, a member of the fine loamy mixed
isohyperthermic family of the Sulfic Tropaquepts and an organic soil from the UKM area in
Bangi. Table 2 gives the physical and chemical
properties of these soils.
The soils were steam-sterilized for lhr at
100C in a steam sterilizer (to remove pathogens
and the indigenous VAM species) and incubated
for 2 weeks before use.
Experiment I: Multiplication of the Inoculum Soil The eight soil inocula were checked for purity of the individual species by examining both spores and roots of the individual soil
inoculum. When they were confirmed to be free

TABLE 2
Chemical properties of Carey (Expt. 1) and
organic soil (Expt. 2)
Carey series

Organic soil

Carbon (%)

1.61

2.72

Total N (%)
Available P ug g ~x

0.13

0.16

14.00
0.21

2.80
33.07

0.28

7.28

4.50

5.12

Soil properties

K (ppm)
Mg (ppm)
pH(H 2 O)

of contaminants, three lOg of soil inoculum of

each VAM species were respectively placed
5.0cm below the surface layer of 500g Carey soil
in black polythene bags. A single rooting A Ilium
ascalonicum bulb was then planted 2.0cm above
the inoculum layer (i.e. one bulb per planting
bag giving a total of 24 bags).
The plants were allowed to grow for 3
months in the glasshouse (with a temperature
range of between 30 to 38C). At the end of this
period, the tops of the plants were cut off and
removed. One hundred gram soil samples, in
three replicates per species were wet-sieved and
the number of spores isolated were counted and
recorded following the method of Gerdemann
and Nicolson (1963). The onion roots present in
the remaining soil samples were cut up into
1.0cm segments and thoroughly mixed with the

TABLE 1
Species and origin of the "imported" VA mycorrhizal fungi
Fungus

Origin

Acaulospora laevis

Gerd. & Trappe

Te Anau, New Zealand

Gigaspora calospora

(Nicol & Gerd.)

Gerd. & Trappe

Perth, Western Australia

Glotnus mosseae
Glomus caledonium

Gerd. & Trappe

(Nicol & Gerd.)
Gerd. & Trappe

Dunedin, New Zealand

Ink Ten, Western Australia

Glotnus
Glomus
Glomus
Glomus

Gerd. & Trappe

Porter & Hall
Porter & Hall
(Thaxter sensu Gerd.
Gerd. & Trappe

Perth, Western Australia

Meridin, Western Australia
Meridin, Western Australia

272

monosporum
merredus
spp.
fasciculatum

Rothamsted, Harpenden, U.K.

PERTANIKA VOL. 10 NO. 3, 1987

PROPAGATION AND MAINTENANCE OF VAM CULTURES

soil. This treatment was repeated for all the 8

VAM species tested. These soil inocula were subsequently sealed in plastic bags and kept in the
refrigerator (4C) until required.
Experiment II: Establishment of Pot
Cultures. Stock cultures of the eight VAM
fungi were further propagated in the greenhouse
in large clay pots of 15.0cm diameter, filled with
10kg of black organic soil with lOOg of soil
inoculum (comprising colonized onion root
segments, spores and hyphae) spread in a thin
layer 8.0cm below the surface of the soil. A
pasture grass, Setaria anceps var. splendida was
used as the host plant. Three well-washed stem
cuttings of this grass 10.0cm long and with at
least one node were planted per pot, with at least
6.0cm of the stem in the soil.
Basal dressings of urea (O.lg), triplesuperphosphate (TSP 0.2g) and muriate of potash
(MOP 0.2g) were applied to each pot (quivalent
to 30kg N/ha, 60kg P/ha and 60kg K/ha). Subsequently, prewashed granite pebbles were then
spread over the soil surface to reduce evaporation. Ten replicates were used per species of
fungus.
Twenty grams of soil were sampled from
each of the 4 randomly selected pots per species
at fortnightly intervals for a period of 6 months.

The number of spores isolated was only recorded

at the end of the sixth month. Spore counts were
repeated 12 and 24 months later. Setaria roots
were also examined under 40 magnification for
presence of mycorrhizal hyphae, arbuscules or
vesicles.
RESULTS
Experiment I
Spore counts of the various mycorrhizal species
propagated in the Carey soil with A. ascalonicum as the host plant were greatest for: Gigaspora calospora, Glomus merredus and Glomus
spp. (Table 3).
Experiment II
Table 3 also shows the spore count of the individual VAM species in the organic soil with the
grass Setaria as the host plant. Successful clonization of the Setaria roots was detected as early
as 4 weeks after planting. Species which are
rapid colonizers include: Acaulospora laeins,
Glomus merredus,
Glomus
fusciculatum,
Glomus monosporum and Glomus caledonium.
Species such as Gigaspora calospora and Glomus
mosseae showed positive root colonization only 8
weeks after planting. The fungus Glomus spp.

TABLE 3
Number of VAM spores isolated from soil with A, ascalonium (Expt. 1) and Setaria (Expt. 2)
as the respective host plants
Fungus

Spore no. per g soil

Setaria (Expt. 2)

Spore no.
per lOOg soil
A. ascalonium
(Expt. 1)

6 months

18 months

24 months

355 13.20

3.000.17

5.75 0.60

240.03 1.33

1939 9.50

1.530.31

1.93 0.25

84.33 19.28

Glomus mosseae

311 9.50

2.60 0.35

4.730.40

122.60 19.74

G. fasciculatum

184 6.00

5.80 0.45

12.330.16

N.D.

G. spp.

485 12.00

4.23 0.60

6.03 2.08

N.D.

G. caledonium

148 11.00

3.80 0.10

5.17 0.40

412.63 35.81

G. monosporum

233 12.50

3.47 0.51

5.131.46

168.23 3.25

G. merredus

518 15.80

3.77 0.42

6.80 0.46

N.D.

Acaulospora laevis
Gigaspora calospora

N.D. not determined

PERTANIKA VOL. 10 NO. 3, 1987

273

H. AZIZAH CHULAN

showed very poor root colonization even up to

the tenth week of observation.
Hyphae, hyphal coils, vesicles and arbuscules which were observed in the mycorrhizal
roots between weeks 2 to 10 showed stages of
infection. Soil borne auxiliary cells were seen in
roots infected by Gigaspora species only after "he
eighth week.
At the first two counts the low spore number
recorded is however compensated by a high percentage of root colonization (60%) and also
presence of abundant fine extramatrical hyphae
in the soil. At the second spore count made 12
months after sowing, the number of VAM spores
recorded per g soil was found to be almost
double that at 6 months. This is true for species
such as A. laevis and Glomus fasciculatum. The
number of spores isolated per g soil for the eight
VAM species at the third count (24 months after
the planting date) more than doubled again.
DISCUSSION
Results of the present study indicate that the two
soil types used are suitable media for growth and
multiplication of these mycorrhizal fungi tested.
The fine sandy loam property of the Carey series
soil allows multiplication of these fungi without
addition of any basal fertilizer, but the organic
soil while having a low fertility level was preferred because it was readily available.
Allium ascalonicum appears to be a good
host plant for rapid propagation of these fungi.
However, these plants were not quite suitable for
long term pot cultures because they are annuals
and the bulbs were prone to parasitization by
fruit fly maggots (Drosophila species) and hence
rotting. Similar observations were made by
Nadarajah (1982) in her studies on A. cepa.
Earlier studies on several plant species (including
at least 4 species of grasses) also failed to give
satisfactory propagation of selected VAM species
(Noraini & Maziah, pers, comm.). Within the
limitations of the present experiment, it was
found that the grass Setaria could be used to propagate the VAM spores because it not only supported rapid multiplication of the inoculum but
also enabled the long term maintenance of pot
cultures. While Setaria infections tended to
support few spore populations initially, soil plus
hyphae and infected root segments can be used
274

as source of inoculum for future inoculation

studies. Indeed Hall (1976) had reported that
the use of root and spore inoculum usually
results in more rapid formation of mycorrhiza
than inoculation with spores alone. Mycelium
and infected roots have in fact been shown to be
important in the survival and activity of nonsporulating endophytes, and they may also
represent a considerable proportion of the
inoculum potential of the spore formers
(Hayman, 1982).
The pasture grass Setaria offers the potential of
being a good host plant because:
i) it can be grown very easily and rapidly from
stem cuttings with just one node.
ii) it takes only four days for the stem to produce new leaf blades and about a week to
produce the fibrous roots from the single
node.
iii) it is easily maintained in the greenhouse i.e.
the plants need to be watered once on every
alternate day and the tops cut once every
eight months.
iv) fungal colonization occurs even on the very
fine feeder roots which are produced very
close to the soil surface, thus enabling easy
sampling of the soil inoculum.
v) soil inoculum can be harvested after every
three months by digging out a portion of the
soil together with the roots.
Fresh soil is then added to fill up the holes.
(A similar procedure is being practised at
Rothamsted Experimental Station, England
using clover as the host plant (Tinker, pers.
comm.).
ACKNOWLEDGEMENT
This project was supported by the International
Foundation for Science (IFS) Grant No: 684.
Technical help from Ms Fouziah is greatly
appreciated.

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PERTANIKA VOL. 10 NO. 3, 1987

PROPAGATION AND MAINTENANCE OF VAM CULTURES

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PERTANIKA VOL. 10 NO. 3, 1987

(Received 24 October, 1986)

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