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Evaluation of the "Most Probable Number" (MPN) and Wet-Sieving Methods

for Determining Soil-Borne Populations of Endogonaceous Mycorrhizal Fungi

Article  in  Mycologia · September 1990

DOI: 10.2307/3760048

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Mycological Society of America

Evaluation of the "Most Probable Number" (MPN) and Wet-Sieving Methods for Determining
Soil-Borne Populations of Endogonaceous Mycorrhizal Fungi
Author(s): Z.-Q. An, J. W. Hendrix, D. E. Hershman and G. T. Henson
Source: Mycologia, Vol. 82, No. 5 (Sep. - Oct., 1990), pp. 576-581
Published by: Mycological Society of America
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Mycologia, 82(5), 1990, pp. 576-581.
? 1990, by The New York Botanical Garden, Bronx,NY 10458-5126

EVALUATION OF THE "MOST PROBABLE NUMBER" (MPN)

AND WET-SIEVING METHODS FOR DETERMINING
SOIL-BORNE POPULATIONS OF ENDOGONACEOUS
MYCORRHIZAL FUNGI

Z.-Q. An, J. W. Hendrix

DepartmentofPlant Pathology,University
of Kentucky,Lexington,Kentucky40546

D. E. Hershman

of KentuckyResearch and Education Center,

DepartmentofPlant Pathology,University
Princeton,Kentucky42445

AND

G. T. Henson

McLean CountyExtension Office,Calhoun, Kentucky42327

ABSTRACT

The communityof endogonaceous mycorrhizalfungipresentin plots with two different cropping

historiesin a westernKentuckysoybean fieldwas analyzed by wet-sievingof spores fromfieldsoil
samples and determiningviabilityof spores of certainspecies witha vital stain,and by conductinga
"Most Probable Number" (MPN) bioassay procedure.The MPN proceduredetected 17 species, while
wet-sievingof fieldsoils detectedonly 10 species. Population densitiesof viable spores of individual
specieswereusuallylowerthanthoseoftotalspores,althoughthedifferences werenotalwaysstatistically
significant.InformationfromMPN bioassays on population densities of individual species is more
useful than informationon population densities of total propagules determinedonly by analysis of
colonization of roots because mycorrhizaleffectson plants probablyare due to effectsof individual
species. Wet-sievingof spores fromfieldsoils and MPN bioassay of propagulesboth yielddifferent and
usefulinformation,and both may oftenbe effectively employed.
Key Words: vesicular-arbuscularmycorrhizalfungi,mycorrhizae,Endogonaceae, Glomus, Gigaspora,
soil microbiology,soil ecology

Endogonaceous mycorrhizal fungi are ubiq-
uitous in soils on which their hosts grow, and
most plant species are hosts. These fungi have
been shown to affect crop productivity, either
positively or negatively; and they are suspected
of being essential for the survival of many plants
in natural competitive situations. Careful studies
have found the endogonaceous community
(herein defined as the total number of species
present in a soil) of a given soil to be rather
complex. The community is composed of a num?
ber of species, only some apparently active on a
given host (Schenck and Kinloch, 1980; McGraw
and Hendrix, 1984, 1986; Modjo et al, 1987;
Gould, 1988). Mycorrhizal effectson productiv?
ityof hosts logically will be affectedby the species
present in the community and their relative pop-
576
ulations ("population" herein used in a quanti-
tative sense).
Two methods have been used to measure the
mycorrhizal community: 1) isolation of spores
by wet-sieving of soil and identification and
counting using a microscope (Daniels and Skip-
per, 1982); and 2) bioassay of propagules by
planting an assay host on dilutions of the soil
followed usually by qualitative evaluation foren?
dogonaceous colonization and quantitative anal?
ysis by the "Most Probable Number" (MPN)
procedure (Alexander, 1965; McGraw and Hen-
drix, 1986; Porter, 1979; Wilson and Trinick,
1982). Both approaches have strengths and
weaknesses. Spores of some endogonaceous my?
corrhizal fungi are too small to be extracted by
sieving [e.g., Glomus tenuis(Greenall) Hall, 1977],

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 An et al. : Mycorrhizal
and some endogonaceous fungi may produce
spores rarely or not at all, although propagules
are present (Baylis, 1969). Spore large enough to
be extracted may not be completely recovered
(Clarke and Mosse, 1978). Spores extracted from
field soils often are coated with soil materials
which make identification difficult.Spores may
be embedded in or on root fragments or other
soil particles, and propagules may be hyphae or
some form other than spores. Spores which ap?
pear normal may be dead (An and Hendrix, 1988;
McGraw and Hendrix, 1986).
The MPN bioassay detects all propagules ca-
pable of infecting the assay host and thus may
detect spores embedded in debris or propagules
which are not spores. It does not enumerate non-
living spores. However, there are numerous
problems with MPN bioassays, such as effectsof
duration and temperature of the growth period
ofthe bioassay host (Wilson and Trinick, 1982),
specificity of individual fungi for the bioassay
host (Adelman and Morton, 1986) and edaphic
variables associated with the bioassay (Adelman
and Morton, 1985). Dormancy occurs in some
species (Tommerup, 1983), and the MPN bio?
assay may fail to detect dormant spores. To date,
the MPN bioassay has been used only to deter?
mine total propagules based on colonization of
roots of the assay host. The MPN bioassay is
also labor-intensive.
We recently studied the effectsof crop rotation
on the endogonaceous community in a soybean
field. For the firstsampling date ofthe 1988 sea?
son, before the 1988 crop was planted, we mea?
sured endogonaceous populations by both these
methods. Results for two field treatments, con?
tinuous soybeans for 3 years (1985-1987) and
soybeans in 1985 and fescue during 1986 and
1987, are given here.
MATERIALSAND METHODS
In 1986 and 1987, a 2-ha area with a prior
history of soybean cultivation in McLean Co.,
western Kentucky, was strip-planted to either
soybean [Glycine max (L.) Merr.] or tall fescue
(Festuca arundinacea Schreb). The soil type was
Melvin silt loam, a river bottom soil highly pro-
ductive for soybean, with a pH of 6.6. Plots were
9 m wide (12 rows for soybean) x 61 m long
and were replicated four times. The experiment
was laid out as a randomized complete block
design. Soil samples from the upper 15 cm of
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Fungi Methodology 577
each plot were taken March 1, 1988. The soil of
all plots had been disced earlier in late winter in
preparation for planting soybeans on all plots
later in the spring. For the analysis of populations
of spores of endogonaceous fungi in soil by siev-
ing, four subsamples (100 g) from each plot were
independently suspended in 1 liter of water with
a magnetic stirrerfor 2 minutes and allowed to
settle for 10 see. The suspension was decanted,
and soil fractions between 425 and 38 fim were
collected. The sediment was resuspended in 1
liter of water, and the procedure was repeated.
The sievings were composited and centrifuged
firstin tap water for 5 minutes at 1270 x g, then
in 2 M sucrose for 1 minute. After final centrif?
ugation, spores were rinsed in tap water and
counted in Petri dishes. Spores were identified
according to the "INVAM Species Guide" of
Schenck and Perez (1987,1988) and keys of oth?
ers (Gerdemann and Trappe, 1974; Hall and Fish,
1979). Nomenclature follows that of Schenck and
Perez (1988).
Viability of spores was determined with a vital
stain (MTT) (An and Hendrix, 1988). Because
stained spores of some species (e.g., Glomus
macrocarpum and G. fecundisporum) could not
be distinguished, spores of certain species were
collected with a Pasteur pipette before staining.
Equal volumes of a stock solution containing 0.5
mg MTT/ml deionized water and an aqueous
suspension of spores (2 ml each) were mixed in
6 cm diam Petri dishes and incubated at room
temperature for 50 h.
For the analysis of propagule populations and
species of endogonaceous mycorrhizal fungi by
MPN bioassay, soil of each sample was serially
diluted with steamed sand containing 1.1 g/L of
an 18N-2.6P-9.9K slow release fertilizer (18-6-
12 Osmocote, Sierra Chemical Co., Milpitas,
California) and 0.11 g/L of frittedtrace elements.
The dilution series was one part sand and one
part of the previous dilution. Seventy g of soil/
sand dilution mixture was placed in a Leach "Pine
cell" plant growth tube (Ray Leach, Canby, Or?
egon 97013). Each tube was seeded with one soy?
bean seed previously inoculated with commer?
cial Rhizobium inoculum. Five replicate tubes
were prepared for each dilution. The plants were
watered daily and grown 9 weeks in a greenhouse.
Spores were isolated by the same sieving and
centrifugingmethods described above and iden?
tified. MPN analyses were conducted for each
individual species, and population densities of
578
total propagules are the sums of the population
densities of the individual species. Roots were
cleared in KOH and stained (Kormanik and Mc-
Graw, 1982) forobservation forcolonization and
attached or intraradical spores.
Statistical analysis was complicated by the fact
that variances among means were unequal as in
previous studies (McGraw and Hendrix, 1984,
1986). Consequently, standard errors ofthe mean
are given with the population data, and proba-
bility values for comparisons of individual pairs
of means were computed by a least significant
differenceprocedure (Helwig and Council, 1979),
using log10transformation. Probability values for
contiguous means were compared and letters as?
signed.
RESULTS AND DISCUSSION
The MPN bioassay detected more species than
soil sieving (Table I). Two of these, Glomus in-
traradices and G. ambisporum, are unlikely to be
found by sieving. Glomus intraradices produces
spores inside roots, and few apparently are freed
and isolated by our sieving methods. G. ambi?
sporum produces spores in sporocarps, which
would also be removed by the coarse sieve used
to remove debris. Sporocarps of G. ambisporum
probably often become detached from roots re?
moved from field soils because extensive wash?
ing is usually required. Both of these species are
conspicuous when roots of the MPN bioassay
plants are stained and observed because the sand
used as a plant growth medium is removed from
roots with minimal washing. Caution must be
employed when identifying spores present in
stained roots. The solvents used in staining pro?
cedures change sizes and other spore character?
istics (Morton, 1986), and staining prevents de?
termination of color. Both are important criterion
for identification (Schenck and Perez, 1988). If
ambiguity is encountered, roots must be ob?
served before as well as after staining.
Five other species were also detected by MPN
bioassay that were not found by soil sieving.
Therefore, if an objective is to define the endo?
gonaceous community, the MPN bioassay, or
some kind of bioassay, is essential. Planting an
appropriate host in a soil may elevate a minor
member ofthe community to prominence (e.g.,
the growth of sorghum-sudangrass hybrid in?
creased the population of spores of G. caledon-
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Mycologia
ium ca 10-fold; McGraw and Hendrix, 1984). In
this study, growing fescue resulted in detectable
populations of spores of G. canadense, G. macu-
losum, and G. aggregatum, only one of which
was detected in the continuous soybean plots by
the MPN bioassay (Table I). Rotating with fes?
cue increased both number of species and pop?
ulations of many species, and the MPN bioassay
was more sensitive than sieving of spores in de-
tecting these changes.
Because of host specificity,the MPN bioassay
must be conducted with each host of interest. In
the present experiment, our objective was to de?
termine the effectof crop rotation on population
densities of fungi colonizing soybean; therefore,
only soybean was used as an assay host. Adding
hosts does not increase appreciably the labor re?
quired for setting up the MPN assay, but the
labor for the subsequent work and the green?
house space requirements are increased propor-
tionately. Therefore, treatments must be chosen
carefully.
Dead spores apparently often degrade slowly.
For the species for which vitality of spores was
determined, the proportion of vital spores was
often about half that of total spores (Table I),
although differences were not always significant
statistically because of the variability of these
data. For G. macrocarpum, populations of living
spores approximated populations of propagules.
It is reasonable that the propagules present might
be predominantly spores because a host had not
grown on the soils for about five months, in the
case of soybean, or less in the case of fescue. With
other species, the two procedures gave divergent
results, however. Populations of propagules of
Gigaspora margarita appeared to be higher than
populations of sieved spores, although the dif?
ferences were not statistically different.Popula?
tions of propagules of G. gigantea were much
higher than populations of spores in the contin?
uous soybean plots; perhaps this species pro?
duces propagules on soybean other than spores.
Conversely, populations of propagules of Glo?
mus microcarpum were much lower than pop?
ulations of spores. Throughout our study of this
rotation experiment, G. microcarpum did not be-
have in a manner consistent with the other major
species in the mycorrhizal community. In other
studies, Fang et al. (1983) were unable to obtain
single-spore cultures of G. microcarpum. These
observations suggest that the ecology of this spe-
 Table I
Population densities (number per kg soil) of propagules, total spores, and living spores in a soil planted to soyb
two years

Soybean
Sieved spores Sieved
Species Total Living Propagules Total
Total 1438?282ac 587 ? 177 d 8582 ? 986 a
Gigaspora spp.
G. margaritaBecker & Hall 37 ? 17 a 30 ? 13 a 66 ? 22 a 26 ? 5a
G. gigantea (Nicol. & Gerd.) Gerd. & Trappe 5? 4b 2 ? 1b 85 ? 22 a 11 ? 5b
Glomus spp.
G macrocarpumTul. & Tul. 168 ? 30 c 96 ? 32 c 110 ? 43 c 1610 ? 366 a
G. microcarpumTul. & Tul. 250 ? 89 b 133 ? 60 b 21 ? 7c 3653 ? 922 a
G. intraradicesSchenck & Smith 0c 49 ? 49 b 0b
G. ambisporumSmith & Schenck 0c 13 ? 8b 0c
G. canadense (Thaxter) Trappe & Gerd. 0c 0c 3080 ? 241 a
G. fecundisporumSchenck & Smith 8c NDb 44 ? 20 b 108 ? 62 b
Unidentifiedbrown spp.c 965 ? 229 a ND 115 ? 78 b 57 ? 19b
G. maculosum Miller & Walker 0b 21 ? 21b 6 ? 6b
G. leptotichumSchenck & Smith 13 ? 13 c ND 36 ? 19 b 0c
G. aggregatumSchenck & Smith emend Koske 0b 0b 31 ? 24 a
G. caledonium (Nicol. & Gerd.) Trappe & Gerd. 0b 14 ? 8ab 0b
G. claroideumSchenck & Smith 0a 7?7a 0a
G. manihotisHoweler, et al. 0b 0b 0b
G. monosporumGerdemann & Trappe 0b 0b 0b
G. mosseae (Nicol. & Gerd.) Gerd. & Trappe 0b 7 ? 7b 0b
a Mean of 4 replications? standard deviation. Means followedby same letterin a row do not differat P = 0.05 (LSD).
bND = not determined.
c UnidentifiedGlomus chlamydospore(Schenck and Perez, 1988), probably a single species, diameter 95-125 Mm,wall th

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580 Mycologia

cies is differentfrom that of other common en?
dogonaceous fungi. Populations of G. microcar?
pum usually paralleled those of G. macrocarpum.
This study is the firstuse ofthe MPN bioassay
which measured populations of individual spe?
cies. We consider the value of the information
obtained to be well worth the extra work re?
quired. The value of determinations of total
propagules, obtained by assessment of coloni?
zation only, is dubious, because mycorrhizal ef?
fects on hosts probably are due more to the ac?
tivity of individual species than of the total
mycorrhizal community.
For studies of the ecology of endogonaceous
fungi, the use of both methods is advisable. Both
procedures measure some parameters which are
differentas well as some which are the same.
Populations of spores may be more an indicator
of past events than of inoculum potential. Col?
onization of roots and subsequent sporulation
appears to occur near maturity of an annual crop
(Hetrick et al, 1983). In this study, populations
of spores may be indicative of events ofthe pre?
vious fall. If sampling had been done during the
growing season, populations of spores might be
low relative to those of propagules due to ger? Helwig, J. T., and K. A. Council (Eds). 1979. SAS
mination of spores in the presence of host roots
and to extramatrical hyphae acting as propa?
gules. In some studies, it may be important to Hetrick,
determine mortality of spores. Therefore, both
procedures offer different and useful informa?
tion.
acknowledgments
We thankJanetFinley,Chris Hoskins, and Yiying
Huang forassistance and the KentuckySoybean As? McGraw, A.-C,
sociation forfinancialsupport.Paper No. 89-11-187,
KentuckyAgriculturalExperimentStation.
LITERATURECITED
Adelman,M. J., and J. B. Morton. 1985. Variation
in MPN's withinteractionsbetweennative VAM
fungi,hosts,and soils. p. 305. In: Proceedingsoj Modjo, H. S., J. W. Hendrix, and W. C. Nesmith.
the 6thNorthAmericanConferenceon Mycorrhi?
zae. June25-29, 1984. Bend, Oregon,U.S.A.
-, and-. 1986. Infectivityof vesicular-
arbuscular mycorrhizalfungi:influenceof host- Morton,J. B. 1986. Effectsof mountantsand fixa-
soil diluentcombinationson MPN estimatesand
percentagecolonization. Soil Biol. Biochem. 18:
77-83.
Alexander,M. 1965. Most-Probable-Numbermeth? Porter,W. M. 1979. The "Most Probable Number"
od formicrobialpopulations.Pp. 1467-1472. In:
Methods of Soil Analysis,Part 2, Chemical and
MicrobiologicalProperties.Ed., C. A. Black. Amer.
Soc. Agronomy,Madison, Wisconsin.
An, Z.-Q., and J. W. Hendrix. 1988. Determining
viabilityofendogonaceoussporeswitha vitalstain.
Mycologia SO: 259-261.
Baylis, G. T. S. 1969. Host treatmentand spore pro?
ductionbyEndogone. New Zealand J.Bot. 7:173-
174.
Clarke, C. A., and B. Mosse. 1978. RecoveryofVA
mycorrhizalsporesaftergermination.Pp. 239. In:
RothamstedExp. Stn. Rep. for 1977, Part 1.
Daniels, B. A., and H. D. Skipper. 1982. Methods
for the recoveryand quantitative estimation of
propagulesfromsoil. Pp. 29-35. In: Methodsand
Principles of MycorrhizalResearch. Ed., N. C.
Schenck. Amer. Phytopath.Soc, St. Paul, Min?
nesota.
Fang, Y.-C, A.-C. McGraw, H. Modjo, and J. W. Hen?
drix. 1983. A procedurefor isolation of single
spore culturesof certainendomycorrhizalfungi.
New Phytol 93: 107-114.
Gerdemann,J., and J. M. Trappe. 1974. The En?
dogonaceae in the Pacific Northwest.Mycologia
Memoir No. 5. 76 p.
Gould, A. B. 1988. Mycorrhizalfungiin relationto
reclamation of surface mined land in Western
Kentucky.Ph.D. Dissertation,Univ. ofKentucky,
Lexington.119 p.
Hall, I. R. 1977. Species and mycorrhizalinfections
of New Zealand Endogonaceae. Trans. Brit.My?
col. Soc. 68:341-356.
-, and B. J. Fish. 1979. A keyto theEndogona?
ceae. Trans. Brit.Mycol. Soc. 73: 261-270.
UsersGuide. StatisticalAnalysisSystemInstitute
Inc. Raleigh,North Carolina. 495 p.
B. A. D., W. W. Bockus,and J. Bloom. 1983.
The role ofvesicular-arbuscular mycorrhizalfungi
in the growthof Kansas winterwheat. Canad. J.
Bot. 62: 735-740.
Kormanik,P. P., and A.-C. McGraw. 1982. Quan-
tificationof vesicular-arbuscularmycorrhizaein
plantroots.Pp. 37-45. In: Methodsand Principles
ofMycorrhizalResearch.Ed., N. C. Schenck.Amer.
Phytopath.Soc, St. Paul, Minnesota.
and J. W. Hendrix. 1984. Host and
soil fumigationeffectson spore population den?
sities of species of endogonaceous mycorrhizal
fungi.Mycologia 76: 122-131.
-, and-. 1986. Influenceof soil fumiga?
tionand sourceofstrawberry plantson population
densitiesof sporesand infectivepropagulesof en?
dogonaceous mycorrhizalfungi.Plant andSoil94:
425-434.
1987. Mycorrhizalfungiin relationto controlof
tobacco stuntdisease withsoil fumigants.Soil Biol.
Biochem. 19: 289-295.
tives on wall structureand Melzer's reaction in
spores of two Acaulospora species (Endogona?
ceae). Mycologia 78: 787-794.
method for enumeratinginfectivepropagules of
vesicular arbuscular mycorrhizalfungi in soil.
Austral.J. Soil Res. 17: 515-519.
Schenck, N. C, and R. A. Kinloch. 1980. Incidence

This content downloaded from 139.52.204.26 on Tue, 3 Feb 2015 18:20:37 PM

All use subject to JSTOR Terms and Conditions
 An et al. : Mycorrhizal Fungi Methodology 581

of mycorrhizalfungion six fieldcrops in mono- arbuscularmycorrhizalfungi.Trans. Brit.Mycol.

cultureon a newlycleared woodland site.Mycolo?
gia 72: 445-456.
-, and Y. Perez. 1987. Manual for the Identi?
fication of VA MycorrhizalFungi. 1st Ed. Uni?
versityof Florida, Gainesville, Florida. 245 pp.
and-. 1988. Manual for the Identifi?
cation of VAMycorrhizalFungi. 2nd Ed. Univer?
sityof Florida, Gainesville, Florida. 241 pp.
Tommerup,I. C 1983. Spore dormancyin versicular-
Soc. 81: 37-45.
Wilson, J. M., and M. J. Trinick. 1982. Factors af-
fectingthe estimation of numbers of infective
propagules of Vesicular Arbuscular Mycorrhizal
fungibytheMost Probable NumberMethod.Aus-
tral.J. Soil Res. 21: 73-81.
Accepted forpublicationJanuary25, 1990

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