Soil Biol. B&-hem. Vol. 23. No. 2, pp. 177-183. 1991 0038-0717,9153.00 + 0.

00
Printedin Great Bntatn. All rightsreserved CopynghcC 1991 BergantonPresspk

GERMINATION AND HYPHAL GROWTH OF VAM

FUNGI DURING AND AFTER STORAGE IN SOIL
AT FIVE MATRIC POTENTIALS

DAVID D. DOUDS JR* and N. C. SCHENCK

Plant Pathology Department, Fitield Hall. University of Florida, Gainesville, FL 3261 I, U.S.A.

(Accepted 20 June 1990)

Summary-Four vesicular-arbuscular mycorrhizal (VAM) fungi. Giguspora mclrgarito Reeker & Hall,
Glomru mosseae (Nicol. & Gerd.) Gerdemann & Trappe. Glomus intramdices Schenck & Smith. and
Acuulosporu longula Spain 8c Schenck. were studied to characterize the variety of responsesof VAM fungi
to storage. Spores were stored at WC for up to 4 months in soil at several matric potentials [$m]. then
removed and exposed for I month in soil at field capacity to induce germination. Each species studied
has a different response to storage duration and moisture availability, reflecting the complexity of the
problem of storage of VAM fungi in soil. Net germinability. the difference in percentage germination
between that which occurred in storage and germination after the subsequent month at field capacity. was
effectively zero for Gi. margarira. Hyphae continued to grow and additional germ tubes were produced
upon removal from storage. Gi. marguritu spores which produced one germ tube per spore during storage
in moist soil subsequently produced less root colonization of Puspalum notatum than spores stored in drier
soil. Net germinability of spores of G. inrrmadices increased with decreasing storage +m and was
independent of storage duration. Upon removal from storage. preexisting hyphae resumed growth. Net
germinability of spores of G. mosreae was inversely proportional to duration of storage and independent
of $m. Spores which germinated in storage did not resume growth upon removal from storage. Net
gcrminability of A. kongulaincreased with duration of storage and was independent of moisture availability
during storage, indicating a dormant period.

of VAM fungi to cxposurc to soil at room tcmpcra-

Vesicular-arbuscular mycorrhizal (VAM) fungi arc turc at several matric potentials. Wc report spore
most commonly mnintaincd in pot culture with a germination. germ tube production, and hyphal
plant. This method rcquircs much space and labor growth of four species of VAM fungi in storage and
and may not prcscrvc the genetic variability of the after a subsequent exposure of I month in moist soil.
original population collected from the field. Even so, The elTcct of storage upon the ability of one fungus
storage of VAM fungus spores and infected root to colonize plant roots also was examined.
pieces in pot culture soil, though bulky, may be the
most reliable and broadly applicable method of pres-
ervation available (Siqueira ef al.. 1985). VAM fungi MATERIALS AND METHODS
in excised roots can remain infective for ca 2 years
Experiment I
(Tommerup. 198 I). Spores of Glontus fasciculuwn
have remained viable after storage for 4 years in dry Spores of four species of VAM fungi were isolated
soil (Ferguson and Woodhead. 1982). Fine sand soil from pot cultures with Puspalum nototum Flugge and
containing spores of Glomus intrurodices remained Medicago surioum L. as plant hosts. Fungi used were:
infective after 175 days of storage at I % moisture and Glomus mosseue [International Culture Collection of
temperatures from I to 21°C (Nemec. 1987). Storage VA Mycorrhizal Fungi (INVAM) isolate number
of dried pot culture soil with spores at room tcmpera- 1561 (Nicol. and Gerd.) Gerdemann and Trappe,
turc has also been used for Glumus chum (Louis and G. inrrurudices (INVAM 208) Schenck and Smith,
Lim. 1988). Giguspora margarita [INVAM 185) Becker and Hall,
Knowlcdgc of the response of VAM fungus and Acaulosporo longula (INVAM 316) Spain and
spores to factors limiting viability in storage is Schcnck. Pot cultures ranged in age from 7 months
essential to the dcvclopmcnt of protocols for the (Gi. margarita) to 23 months (A. fongulu). Cultures
storage of pot-culture soil. Factors limiting survival with spore populations sufficient to supply spores
of spores in storage include tcmpcrature. moisture for the entire experiment were selected to ensure a
availability in the soil, and duration of storage homogeneous source of spores.
(Tommerup. 1983, 1987; Nemec, 1987). Our objective Spores were isolated from soil by wet sieving
was to characterize the variety of responses of spores (Gerdemann and Nicolson, 1963) and centrifugation
(Jenkins, 1964) and placed on membrane filters
(0.45 pm pore six. 2.5 cm dia). Three filters per
*To whom all correspondence should be addressed at: soil moisture per sample period per VAM fungus
USDA-ARS Eastern Regional Research Center, 600 E. species were prepared (5 x 5 x 4, respectively). The
Mermaid Lane, Philadelphia, PA I91 18. U.S.A. mean number of spores per filter (f SEM) was:

177
178 DAVID D. Oouos JR and N. C. SCHESCK
Gi. margarira, 23.0 + 1.0; G. mosseae. 32.2 f 1.4;
G. inrraradices. 33.2 f 1.4; and A. longula. 40.4 $
2.1. Filters were folded and placed in tissue spea-
men bags (Shandon Southern Instruments. Inc.,
Sewickley. Pa.). Spores have been shown to germi-
nate equally well when exposed between mem-
brane filters in soil or directly in soil (Tommerup,
1983).
The membrane filters in specimen bags were buried
in plastic bags filled with pasteurized (7oZC for 2 h)
soil at five matric potentials [$m]: -0.01, -0.05,
-0.50, - 1.30 and -2.20 MPa. Soil water contents
at various $ m were determined using a ceramic plate
extractor (No. 1500, Soil Moisture Equipment Co.,
Santa Barbara, Calif.). G. mosseae, A. longula and
Gi. margarira spores were originally cultured, stored
and germinated in Arredondo fine sand soil (loamy,
siliceous. hyperthermic Grossarenic Paleudult). G.
inrrarudices spores were originally cultured. stored
and germinated in a mixture of Arredondo fine sand
soil and calcined clay (“Emathlite”, Mid-Florida
Mining Co.. Lowell, Fla. U.S.A.; 1.4: I.0 v/v). Bags
were scaled and stored in the dark at 23°C. Samples
were removed at 2. 4, 8 and I6 weeks to assay sport
germination.
Spores wcrc germinated by placing the specimen
bags in a tray containing a layer of pasteurized soil
and then covering them with an additional I cm of
soil. The soil was watered with dcionizcd water and
maintained at field capacity and room tcmporaturc.
After I month, the filters wcrc rcmovcd and sports
and hyphac wcrc stnincd with 0.05% Trypan Blue.
Sports wcrc considcrcd gcrminntcd if germ tubes
longer than thick, darkly stained, residual attached
hyphac were prcscnt. Total lsngth of VAM fungus
hyphae on membrane filters removed at I6 weeks was
determined by the line-intersect method (Nswman,
1966). Hyphae intersecting parallel lines I mm apart
were counted and data expressed as Icngth of hyphae
per germinated spore.
A second sample of spores was removed from
storage at 4 months. These spores were stained
immediately with Trypan Blue to assay germination
which occurred during storage. Length of hyphae per
germinated spore was calculated as above.
Experiment II
A second experiment was conducted to charac-
terize the germination of spores of Gi. margarita and
to determine if germination during storage sub-
sequently afTccted the ability of the fungus to colo-
nize roots of P. no~arum. Spores first were isolated
from moist, fresh pot-culture soil (Arredondo fine
sand) and placed in the germination assay outlined
above. The remainder of the soil was allowed to air
dry. Water contents were dctermincd gravimctrically
on subsamplcs removed at each of four stages
of drying ($m = -0.0044. -0.0052, -0.0075 and
-0.675 MPa; 14.5. 12.0. 7.9 and 3.8% water [w/w]
respcctivcly). The remainder of soil rcmovcd at each
stage was placed in a plastic bag for cxposurc at 23°C.
This cxpcrimcnt diffcrcd from experiment I because
spores wcrc exposed in situ rather than on membrane
filters.
Soil was removed from the bags after 3, 7.5, I2
and 19 weeks. Spores were isolated by wet sieving
and centrifugation, a process which broke hyphae
and left stubs of germ tubes and hyphal attach-
ments. Spores were placed on three membrane filters
and germinated as above. Healthy or unhealthy
appearing spores were not discriminated for or
against at the initiation of the assay so samples
reflected changes in the population of spores pres-
ent in the soil at the beginning of the experiment.
At the conclusion of the germination assay, spores
were scored for germination. hyphal length per
germinated spore and intact and broken germ tubes
per spore.
The last sample of soil + spores of Gi. margarita
was removed at 25 weeks and used in a colonization
test. A 5 g (adjusted dry wt) sample of soil of each + m
was mixed into I75 g (dry wt) of pasteurized Arre-
dondo fine sand soil and placed in a conical plastic
pot (“conetainer”, Ray Leach Cone-Tainer Nursery.
Canby, Ore.). Three replications per original incu-
bation $rn were prepared. Two P. notalum seedlings
were transplanted into each pot and grown under
controlled conditions: 14-10 h day-night; 28-21°C;
and 600-800 p mol m-* s-’ photosynthetic photon
flux density for 2 weeks. Entire root systems were
cleared, stained (Phillips and Hayman. 1970) and
assayed for percentage root length colonized and
number of penetration points.
&la unalysis
Net gcrminntion was dcfincd as the dificrcncc in
pcrccntagc gcrrnination bctwccn that which occurrod
in storage and after the subscqucnt month at field
capacity. Data were analyzed using analysis of vari-
ance or linear rcgrcssion where applicable. Pcrccntagc
germination and colonization wcrc analyzed using
arcsin transformed data.
RFSULTS
Experimenr I
Thirty-seven percent of the Gi. margaritu spores
isolated from pot culture germinated when exposed
to soil at field capacity (time = 0. Fig. I). Spores
showed a strong tendency to germinate in storage at
all $m studied (time = S. Fig. I). Approximately
85% of the spores germinated when stored on mem-
brane filters in soil of J/m = -0.01 MPa and 45% in
*m= -2.2 MPa. Germinated spores produced more
hyphae in storage at -0.01 MPa than at -2.2 MPa
(Table 1). Spores which germinated in storage
approximately doubled their length of hyphae upon
exposure for I month in the germination assay. This
was possible due to both continued growth from
previously-formed hyphae and the production of
new germ tubes. These responses were greater at
-2.2 MPa than at other moisture contents tested.
Due to the germination of Gi. margarita spores in
storage and their ability to rcsumc growth upon
removal from storage, survival was independent
of both storage duration and soil $m and net
germination was effectively zero.
Forty-eight percent of the G. inrrarudices spores
isolated from pot culture germinated when exposed
to soil at field capacity (time = 0. Fig. 2). More
than 70% germinated while in storage on mem-
brane filters at -0.01 and -0.05 MPa, but only 1%
 Germination and hyphal growth of VAM fungi 179

100 c/posprro margarita

-0.01 MPO -a50 MPO -13oMpo -2.20 MPo
90

80

TO

s 60

s
._
Y
50 50

E
&
(3 40

30

20

10

0
if 0248

Storage duration (wk)

Fig. I. Germination of spores of Gi. morgarira in storage at 23°C in Arredondo fine sand with various
matric potentials [Jim]. Time 0 = germination upon isolation from original pot culture: germination at
2. 4. R and 16 weeks = perccmuge of sports removed from storage at these times and assayed as having
germinated after I monrh’s exposure in soil at field capacity: S = germination of spores while in storage.
assayed immediately after 4-months storage and bclicved to have occurred during the first weeks of
storage. Means of three umplcs of sports f SEM.

gcrminalcd in storigc on mcmbranc fillers at total gcrminntion minus 30% gcrrninalion in storage)
-2.2 Ml% (time = S, Fig. 2). Sports which germi- and 73% net germination after 4 weeks storage at
natcd in storage produced an additional 82-106% - 1.30 Ml% (75% total germination minus 2%
hyphal length (-0.05 and -0.01 MPa rcspcctivcly) gcrminalion in storage).
upon cxposurc for I month in the germination assay Fifty-two percent of the G. mosreue spores isolated
(Table 2). Production of a second germ tube was not from pot culture germinated when incubated in soil
evident. so the additional hyphnl growth resulted at field capacity (time = 0. Fig. 3). Germination of
from regrowth from original germ tube hyphae. Net spores in storage declined from 30% at -0.01 MPa
germinability of spores of C. inrrmdices stored at to 3-4% at -0.5 to -2.2 MPa (time = S, Fig. 3).
room temperature was inversely related to soil Jim Germinated spores produced significantly more
and was independent of time under the conditions hyphae while in storage at -0.01 MPa than at Jim of
of this experiment (net percentage germination = -0.5 to -2.2 MPa (Table 2). These spores did not
-27.70(MPa) + 13.48; r = -0.88). For example, produce additional hyphae when removed and
G. inrrurodices spores exhibited a net germination exposed at field capacity in the germination assay.
of 46% after 4 weeks storage at -0.050 MPa (76% This, and their vacuolated. discolored appearance

Tublc I. Hyphal length and number of germ wbcs produced by spores of Gi.
murguriru slorcd for 4 months in soil al various m&k potentials [*ml and upon
I month of further erposurc in soil at field capacity (germination assay).
cxperimcnl I’

Hyphiie
(mm germinated spore“) Germ tubes spore -’

$m (MPa) Storage Germ. uruy Storage Germ. assay

-0.01 39R UA I.IA I.2A

-0.05 ?ZA %A I .OA I.IA
-0.50 2SA 43A I .OA I.IA
- I.10 24R S2A I .OA I.lA
-2.20 208 91A I .OB I .3A
.. ..
Rcgrcssionb NS NS

‘Each number represents the mean of three observations. Numbers for a

comparison between storage and germination assay within a &m followed
by the same letter arc not significantly difkrcnt (z - 0.05. Duncan’s multiple
range ICSI).
%ignificancc of linear regression of each dependen: variable (hyphal length or
germ tubes) with em (MPa) as chc independent variable. NS. not significant;
l*. significant (P < 0.05).
180 DAVID D. DOLT JR and N. C. !SCHEKK

100
Glomus intrcraakes

90

60
F -0.01MPa -005 MPa -0 50 MPa -130 MPa -2.20 MPO

TO

7
;‘ 60

3
._
;; 50
.s
j 40

30

20

10

0
02 s Oi 16

Storoge durotion (wk)

Fig. 2. Germination of spores of G. inrruradicesafter storage at 23°C in Arrcdondo fine sand-calcined

clay soil mix with various Jim. See legend of Fig. I.

suggcstcd they wcrc dead. Germination of sports only 4% net germination after I6 weeks at that +m
of G. n~o~seue upon removal from storage and (8% total germination minus 4% germination in
cxposurc at ticld capacity was indcpcndcnt of storage storage).
t,bm and invcrscly proportional to duration at those Twenty-nine pcrccnt of the A. ~WI~I&Jsports iso-
tJm in which sports did not gcrminatc significantly latcd from pot culture gcrminatcd when cxposcd to
in storage [net percentage germination = - 1.87 soil at field capacity (time = 0, Fig. 4). Gcrmina-
(weeks) f 2g.88; r = -0.791. For example. G. tion of spores in storage declined from 19% at
nros.secrr spores exhibited a net germination of 23% J/m= -0.01 MPa to S-7% at $m= -0.5 to
after 2 weeks storage at -0.50 MPa (27% total -2.2 MPa (time = S, Fig. 4) and spores produced
germination minus 4% germination in storage) and few hyphae (Table 2). Otherwise, the etTect of

1oc I.
GLomus mosseoo

90

-00s MPO -0.50 MPo -1.30MPa -220hlPll

70

30

20

10

L
0
0246165 0246 I 16 S 024616s 02 46165 0 24 El65

Storage durotion twk)

Fig. 3. Germination of spores of G. mosseoeafter storage at 23’C in Arredondo tine sand soil with various
$ m. See legend of Fig. I.
 Germination and hyphal growth of VAM fungi 181

Tabk 2. Length of byphaa produced by spores of VAM fungi while Experiment II

in storage for 4 months in soils at various matric potentials (Sm)
and upon I month of further exposure in soil al field capacity Nearly 90% of the spores of Gi. margarita sur-
(gcrmimtion assay), capcrimcnt I’ vived to produce new germ tubes after 19 weeks of
Hyphac (mm germinaled sport ’) storage in siru at room temperature at all IL m studied
#m (Table 3). With each moisture tension, germination
SDccin uufw StoraKc Gwminarion assay appeared to be lower after 37.5 weeks exposure
G. intraradicrs -0.01 5.28 10.8A than after 19 weeks. Germination after 7.5 weeks
-0.05 5.3A 9.6A exposure was significantly lower than after 19 weeks.
-0.50 IO.OA IS.SA
-1.30 3.48 12.7A Hence. a quadratic equation was used to analyze the
- 2.20 I .60 10.7A data. Germination after 7.5 weeks exposure was
Regression . NS significantly lower than after 19 weeks for all +rn
G. mossc4c -0.01 19.5A IO.IA
studied.
-0.05 l2.7A 2.9A
-0.50 3.5A 5.OA The amount of hyphae produced by each germi-
- 1.30 1.8A 0.5A nated Gi. margarita spore during the germination
-2.20 O.JA O..cA assay was affected little by time (r = -0.210,
Regression .. ..
P > 0.10) or moisture tension during incubation
A. longula -0.01 0.98 3.7A
-0.05 0.3A ?.6A (r = 0.354, P > 0.05) (Table 3). The amount of
-0.50 0.7A 3.?A hyphae produced after an exposure of 19 weeks was
- 1.30 O.lB 3.4A not statistically different from that produced by
-2.20 0.58 3.8A
spores not subjected to air drying and storage.
Regression NS NS
The number of new germ tubes produced by Gi.
‘Numben arc the means of three obscrvalions. Compare numbers
margcvira during the germination assay tended to
bc~wecn columns for a particular species x $m combinalion
only. Slnrirlics as in Table I. P c 0. IO.
l
increase with duration of storage and moisture
tension during storage (Table 3). New germ tube
production was affected by $rn at the I2 and
I9 week sample periods and by storage duration at
+rn upon survival and germinability was minor -0.0044 and -0.0075 MPa. Total germ tubes, new
relative to the efTcct of storage duration [net pcrccnt- plus those produced in storage and broken upon
age germination =I 2.84 (weeks) + 13.77; r = 0.871. isolation from the soil. incrcascd with duration of
For example. A. longul~ sports exhibited net cxposurc (Table 3).
germination of 19% after 4 weeks storage at lnoculum of Gi. nrcrrguritc: stored at room tcmpcra-
-2.2 MPa (26% total germination minus 7% turc for 25 weeks was able to colonize P. noturum
germination in storage) and 73% net germination seedlings within 2 weeks of inoculation (Table 4).
aficr 16 weeks at that $rn (80% total gcrmina- Inoculum stored at - 0.0044 M Pa produced fcwcr
tion minus 7% germination in storage). Length of pcnotration points per length of root than other
storage enhanced germination of A. lort~~rlu sports inoculn. indicating lower infectivity even though
uoon removal from storage at all soil moisture sport numbers and pcrccntagc germination were the
tensions tested. same for all inocula after I9 weeks (Table 3).

100
Acou~~spOra lon9t.h

90
-0.01 MPa -0.05 MPO -0!30MPa -130 MPa -2 20 MPa
80

70

8 16 0248165 024816s 02 4 916s 024816s

Storogo duration 1wk I

Fig. 4. Germination of spores of A. longula after storage at 23°C in Arredondo fine sand soil with various
+m. See legend of Fig. I.
 Dlrvl~ D. Douos JR and N. C. SCHEF~CK

Table 3. Percentage gertnioation and hyphal growth and germ tube production per germinated spore for
Gi. margarita stored in soil aL various mat& p0tmtids and rubsequm~ly exposedI month in moist soil.
expefimml II’

Germ tubes
+m Storage Gnnination Hyphae
(MPa) (weeks) peranti@ (mm prminatcd spore -I ) NW Old
-0.0044 0.0 76 f 4 95? II 1.01 f 0.01 I.01 + 0.01
3.0 67 2 4 I0927 I.14 ?O.Ol 1.14~0.01
7.5 42 f 4 a3 2 a I .O? f 0.02 I .oa + 0.02
12.0 6825 III +a I .07 fi 0.02 1.17*0.05
19.0 aa k 4 742 I2 I .32 * 0.08 2.03 + 0.36
Rcgrcssior? .. NS .. ..
-0.0052 0.0 76 k 4 95* II I.01 + 0.01 I.01 * 0.01
3.0 67 2 6 123+7 I. I I + 0.05 I. I I f 0.05
7.5 55 f a 97 * 4 l.l4+0.07 1.14~0.07
12.0 65 + 4 1145 I3 1.06 f 0.03 1.17~0.03
19.0 a7 + 2 al it 13 I .07 k 0.02 I.31 +0.11
Regressionb .. NS NS ..
-0.0075 0.0 76 5 4 95 * II I.01 _+0.01 I.01 f0.01
3.0 62 + 6 I26 f I2 I .04 * 0.02 I .04 * 0.02
7.5 46 i 3 I47 f 14 I .05 f 0.03 I .05 f 0.03
12.0 64*2 134kll I .03 * 0.02 l.lo*o.o2
19.0 a4 i 4 a6 f 3 I. I I f 0.04 I .24 f 0. I I
Regression’ .. .. .. ..
-0.6750 0.0 76 k 4 95 f II I.01 * 0.01 1.01 f 0.01
3.0 41 *5 I25 f I2 I .05 & 0.05 I .05 f 0.05
7.5 47 f I 113*25 1.09 f. 0.04 1.09*0.04
12.5 a9 * 2 II9 f. 3 I.01 f 0.01 I .O? f 0.02
19.0 90*3 962 I? I.11 +0.02 I.41 + 0.07
Regression” .. NS ..
NS

Regression’ 3.0 .. NS NS NS
7.5 NS NS NS NS
12.0 .. NS . ..
19.0 NS NS . NS
‘Each number represents Ihe mean of three observations f SEM.
“Significunccof liocur regression of each dcpcndcnl vuriublc with weeks + (weeks)’ of storngc as indcpcndcnl
vsriablcs. NS. not significant; l * signilicunl (P < 0.05).
‘Significuncc of linear regression of cuch dcpcndcn~ vuriublc using *m (MPa) as the indcpcndcnt vuriublc.
i.e. within a slump duration across #m. NS. not signiRcant; lP < 0.10. l*P < 0.05.

IHSCUSSION though previously shown to have no effect upon

the change of dormant sports to a quiescent state
Spores of Gi. murguriru germinated in storage
(Tommerup, 1983). also may have stimulated gcrmi-
at all moisture contents in experiment I. possibly
nation by leaching potential inhibitors of germina-
due to hydration of spores during wet sieving and
tion from the spore or spore wall of Gi. marguriru
placement on moist membrane filters prior to
and C. nrosseue. Washing G. culedonium spores has
storage. Rapid hydration of Clomus caferlonium
been shown to enhance germination (Tommerup.
spores has been reported (Tommerup, 1984b). After
1985). but washing Gi. murgurita spores with water
rehydration. spores of G. culehniw~~ and A. h&s
did not stimulate germination nearly as well as
would germinate equally well in soil with Jim as
surface sterilization with sodium hypochlorite
low as - I.58 MPa as in wetter soil (Tommerup,
(Sward, 1981). In experiment II where Gi. margorifo
1984b). Germination of rehydrated spores in dry
spores were dried in siru, and hence, no leaching
soil may have an adverse effect upon reserves of
occurred, there was no firm evidence of germination
VAM fungi in soil (Tommerup. 1984b; Thompson,
in storage until the l9-week harvest in which there
1987).
were significantly more old, broken germ tubes
Spore isolation and placement on membrane
presumed to have been produced during the
filters prior to incubation in dry soil in experiment I.
storage period. Even so. at Jim = -0.675 MPa in
experiment II. a maximum of 27% of spores germi-
Table 4. foul root lcng1h and root coloniurtion of Pqdum
nated in storage after I9 weeks (0.3 old germ tubes
nowurn seedlingsby GI. margurirrr after 2 weeks of growth. lnocula
wcrc stored 25 weeks in soil a~ four mrtric potentials (Sm) UI 23°C’ per spore x 89.7% germination) (Table 3) compared
to 83% germination in storage at -0.5 MPa in
Storage Tolit root VAM fungus Penetration
rLm (MPa) length (cm) colonizalionb poinls cm - ’ experiment I (Fig. I). The data show that hydration
or leaching of potential inhibitors of germination
-0.W4.8 282 f 30 a.3 f 2.2 0. I9 f 0.03
-0.0052 172 f: 20 15.6 2 2.2 0.52 r 0.06 were significant factors in the storage of Gi. margoriru
-0.0075 233 + la 21.5 + 2.0 0.55 * 0.08 in expcrimcnl 1.
-0.6750 278 f 62 13.8 f 1.0 0.4a f 0.02 The result of germination in storage, whether due
Regression’ NS . ..
to the soil $rn being conducive to germination or
‘Each number rcprexnls the mean of three observations f SEM. previous hydration or leaching of spores, differed
bPcrcentagc of root length.
‘Significance of linear regression of each dependent variable with
among the VAM fungi tested. Germination of
6rn (MPP) as the indcpcndcnl variable. NS. no1 significant. G. mosseae spores in storage yielded spores which
‘P < 0.10. and l*P < 0.05. were unable to grow when removed from storage.
 Germination and hyphal growth of VAM fungi 183

Pregerminated spores of Gi. margarita. G. infra- REFEREYCES

radices and A. longula. however, produced additional
Fcrguson J. J. and Woodhead S. H. (1982) Production of
germ tubes or hyphal growth upon removal from endomycorrhizal inoculum. A. Increase and maintenance
storage and exposure to soil at field capacity. Their of vesicular-arbuscular mycorrhizal fungi. In Merhodr and
capacities to colonize roots, however, may have Principles of Mycorrhiz/ Research (N. C. Schenck. Ed.).
been affected. Spores of Gi. margarita, incubated pp. 47-54. American Phytopathological Society, St Paul.
25 weeks in soil at the highest $rn studied in Gemma J. N. and Koske R. E. (1988) Seasonal variation in
experiment II. showed decline in infectivity over spore abundance and dormancy of Gigaspora gigrmrea
spores incubated at the other Jim (Table 4). The and in mycorrhizal inoculum potential of a dune soil.
Mycologic 80. 21 I-216.
data of the l9-week sample showed the same per-
Gerdemann J. W. and Nicolson T. T. (1963) Spores of
centage germination and total hyphal lengths for
mycorrhizal Endogonr species extracted by wet sieving
spores. thus growth to potential host roots may and decanting. Transactions of the Brifirh Mycological
not have been the limiting factor. Tommerup Sociery 46, 235-24-t.
(1984,) found that G. caledonium and A. laecis Jenkins W. R. (1964) A rapid centrifugal-flotation tech-
were limited in their capacity to produce prepene- nique for separating nematodes from soil. Plunr D&use
tration and penetration structures when pregermi- Reporter 4. 692.
nated up to 16 weeks before exposed to plant Louis I. and Lim G. (1988) Effect of storage of inoculum on
roots. The infectivity of pregerminated VAM fun- spore germination of a tropical isolate of Glomur &rum.
Mycologic 80. 157-16 I.
gal spores has been shown to decrease to 50% of
Nemec S. (1987) Effect of storage temperature and moisture
maximal capacity 8 weeks after germination and
on G1omu.s species and their subsequent efTect on citrus
to 0% at I6 weeks (Tommerup. 1981). Therefore, rootstock seedling growth and mycorrhiza development.
the combination of pregermination in moist soil Transactions of rho British Mycological Society 89.
and storage duration is detrimental to the potency 205-2 12.
of pot-culture inocula. Newman E. I. (1966) A method of estimating the total
Spccics of VAM fungi undergo dormancy. A length of root in a sample. Journul of Applied Ecology 3.
dormancy period was found here for A. longula and 139-145.
has been shown also for /1. luwis (Tommerup, 1983). Phillips J. M. and Hayman D. S. (1970) Improved pro-
cedures for clearing roots and staining parasitic and
Gi. rtrur~uritu and G. in~rurudices cxhibitcd a dor-
vesicular-arbuscular mycorrhiral fungi for rapid asscss-
mancy period of C(I 2 weeks in this study. Storage of
ment of infection. Trunsucrions of the British Myrologicul
dried inoculum of G. cltrrrtrn at 25-30°C has been Society 55. IS8 - 160.
shown to cnhancc germination. suggesting a dormant Siqucira J. 0.. Sylvia D. M., Gibson J. and tlubbell
pcriotl for this spccics (Louis and Lim. I9Xg). A 2-9 D. II. (19115) Spores. germination. and germ tubes of
week dornumcy period was cstimatcd for ticld popu- vesicular-arbuscular mycorrhizal fungi. Cunucliun Juurnul
lations of (;i~~q~orl~ ~i~rmrc*~ (Gcmma and Koskc. of Micrubioiugy 31, 965-972.
1%X). Incubation in dry soil has been shown to Sward R. J. (1981) The structure of the sports of Gixusporu
rclcasc spores of VAM fungi from dormancy sooner murKorifrr. II. Changes accompanying germination.
New Phytologisr 88. 66 I-666.
than incub;ttion in moist soil (Tommcrup. 19g3). This
Thompson J. P. (1987) Decline of vesicular-arbu.scular
was not seen for spccics used here
mycorrhixae in long fallow disorder of flcld crops
Each VAM fungus in cxperimcnt I cxhibitcd a and its expression in phosphorus deficiency of sun-
dilfcrcnt rcsponsc to stomgc duration and moist- flower. Ausrruliun Journul of Agriculrurul Resrurch 38.
ure availability. Gi. nrurguriru displayed low net 847-867.
germinability throughout the experiment. indcpen- Tommerup I. C. (1981) Survival mechanisms of VA mycor-
dent of both storage duration and moisture Gcr- rhizal fungi. In Progrum und Absrrucrs of rhe Fifth North
nunability of G. HIOSSCU~W;IS inversely proportional Americun Conference on Mycorrhixe. p. 16. Quebec,
to duration. Survival of G. inrruradiccs wets dcpen- Canada.
Tommerup I. C. (1983) Spore dormancy in vesicular-
dent upon $m and independent of time Storage
arbuscular mycorrhizal fungi. Trunsucfions of rhe Brirish
at room temperature released A. long& from
Mycological Sociery 81. 37-45.
dormancy. Tommerup 1. C. (1984,) Persistence of infectivity by germi-
Stordgc of spores of G. inrrorudices [208] and nated spores of vesicular-arbuscular mycorrhizal fungi in
A. /OII~I& [216] in soil at 23’C and $rn less than soil. Transactions of the British Mycological Sociefy 82,
-0.05 MPa appears to bc an effective method 275-282.
to maintain and cvcn enhance the germinability Tommerup I. C. (1984b) Effect of soil water potential on
of thcsc fungi. G. mosseue [I561 may bc stored spore germination by vesicular-arbuscular mycorrhizal
at -0.5 to -2.2 MPa for 2 months only with signifi- fungi. Transuctions of the British Mycological Society 83,
193-202.
cant loss of germinability. Storage of Gi. margurira
Tommerup I. C. (1985) Inhibition of spore germination
[ 1851 spores in soil at 23-C at $rn less than
of vesicular-arbuscular mycorrhizal fungi in soil. Truns-
-0.01 Mfa appears to hc an incffcctivc method
ocfions of the British Mycological Sociery 85. 267-278.
to ensure infcctivily. Tommerup 1. C. (1987) Physiology and ecology of VAM-
spore germination and dormancy in soil. In Mycorrhizoe
rl~~no~~~k~cf~t.n~~,~~f--Supportcd by a National Science in rhe Next Decode. Procricol Applicorions ond Research
Foundation Grant. Florida Agricultural Experiment Prioriries (D. M. Sylvia, L. L. Hung and J. H. Graham,
Station Journal Series No. 9562. Eds). pp. 175-177. University of Florida, Gainesville.