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Applied and Environmental Microbiology, September 1998, p. 3473-3479, Vol. 64, No. 9
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Cellular Events Involved in Survival of Individual
Arbuscular Mycorrhizal Symbionts Growing in the Absence of
the Host
Cable
Logi,1
Cristiana
Sbrana,2 and
Manuela
Giovannetti1,*
Dipartimento di Chimica e Biotecnologie
Agrarie1
e Centro di Studio per la
Microbiologia del Suolo C.N.R.,2
Università degli Studi di Pisa, 56124 Pisa, Italy
Received 1 December 1997/Accepted 3 June 1998
 |
ABSTRACT |
A survival strategy operating in the absence of the host was shown
in obligately biotrophic arbuscular mycorrhizal
(AM) symbionts. When no host-derived signals from the surrounding
environment were perceived by germinating spores, fungal hyphae
underwent a programmed growth arrest and resource reallocation,
allowing long-term maintenance of viability and host infection
capability. The early stages of mycelial growth of AM
fungi were studied by a combination of time-lapse and
video-enhanced light microscopy, image analysis, and
immunodetection, with the aim of acquiring knowledge of cell events
leading to the arrest of mycelial growth. The time-course of growth
arrest was resolved by precisely timing the growth rate and magnitude
of the mycelium originating from individual spores of Glomus
caledonium. Extensive mycelial growth was observed during the
first 15 days; thereafter, fungal hyphae showed retraction of
protoplasm from the tips, with formation of retraction
septa separating viable from empty hyphal segments. This active process
involved migration of nuclei and cellular organelles and appeared to be
functional in the ability of the fungus to survive in the absence of a
host. Immunodetection of cytoskeletal proteins, metabolic
activity, and the retention of infectivity of germinated spores
confirmed the developmental data. The highest amounts of tubulins
were detected when hyphal growth had ceased but when retraction of
protoplasm was most active. This was consistent with the role of the
cytoskeleton during protoplasm retraction. Succinate dehydrogenase
activity in hyphae proximal to the mother spore was still detectable in
6-month-old mycelium, which remained viable and able to form
appressoria and produce symbiotic structures.
| |
INTRODUCTION |
Arbuscular mycorrhizal (AM) fungi
are obligately biotrophic organisms, widespread in soil,
which live symbiotically with most plant roots. It has been
calculated that they are able to colonize the roots of 80% of plant
species belonging to all phyla of land plants (29).
Fossil records and DNA sequence data have provided evidence that
arbuscular mycorrhizas were established as early as 410 million to 360 million years ago (24, 25, 27). Because of this long-lasting coevolution with their host plants, AM fungi have evolved unequivocal mechanisms of host recognition which play a fundamental role in their
life cycle. Only in the presence of signals released by host roots does
morphogenetic differentiation of infection structures occur, allowing a
functional symbiosis to be established (8, 11). Despite
this, the recognition process does not involve specific regulation
of the germination event by host-derived signals. AM fungal spores
are capable of germination and growth from a quiescent-like state in
response to different edaphic and environmental conditions,
irrespective of the presence of host plants (7, 12, 15, 19, 20,
30). This is contrary to findings described for other
biotrophs, such as some pathogenic fungi, whose resting structures
germinate only as a direct response to the presence of host plants
(4, 5). Such behavior by AM fungal spores is apparently
inconsistent, given that in the absence of the host these fungi are not
capable of extensive independent mycelial growth and consequently of
completion of the life cycle (8, 13, 19, 21). Thus,
many authors have observed that germlings of different AM
fungal species ceased growth within 15 days of germination
(2, 9, 13, 26).
The fate and behavior of individual AM fungal spores that germinate in
the absence of appropriate hosts may affect the survival of individuals
and populations. However, this fundamental event in the life cycle of
AM fungi has so far not been adequately investigated. In this
work we studied the early stages of mycelial growth of AM fungi,
adopting a combination of time-lapse and video-enhanced light
microscopy, image analysis, and immunodetection in order to acquire
knowledge of cell events leading to the arrest of mycelial growth in
the absence of host plants.
Our work was aimed at investigating (i) the time course of fungal
growth in the absence of the host, from spore germination to growth
arrest; (ii) cell events involved in developmental arrest; (iii)
metabolic activity of the mycelium after growth arrest and its
ability to restore growth and to differentiate infection structures; and (iv) differential expression of cytoskeletal proteins during the
various growth phases.
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MATERIALS AND METHODS |
Fungal material.
Spores of Glomus caledonium
(Nicolson et Gerdemann) Trappe et Gerdemann (Rothamsted isolate) and
spores or sporocarps of Glomus mosseae (Nicolson et
Gerdemann) Gerdemann et Trappe (Kent isolate) were obtained from the
collection of the Dipartimento di Chimica e Biotecnologie Agrarie,
University of Pisa, Italy. Voucher specimens of these isolates were
deposited in the Herbarium of the Department of Botanical Sciences,
University of Pisa, Herbarium Horti Botanici Pisani (PI), respectively,
as PI-HMZ 8 and PI-HMZ 4.
Time course of hyphal growth in the absence of the host
plant.
Spores of G. caledonium were surface
sterilized with 2% chloramine T supplemented with streptomycin (400 µg ml
1) for 20 min, rinsed five times in sterile water,
put on 20- by 20-mm cellophane membranes (Hoefer, San Francisco,
Calif.), and placed on 1% water agar (WA) in 9-cm-diameter petri
dishes. After 4 days' incubation in the dark at 28°C, spores were
checked for germination. Cellophane membranes bearing germinated spores
were transferred to 5.5-cm-diameter petri dishes on a thin layer of 1%
WA for growth measurements, which were carried out under a Reichert-Jung Polyvar light microscope. Hyphal length was assessed by
using Quantimet 500 image analysis software (Leica, Milan, Italy).
The lengths of both protoplasm-containing and empty mycelium were
measured daily on a group of 12 spores, up to the time of growth
arrest. The mean growth rate of the mycelium during the early phase was
calculated on eight spores by dividing the mycelial length by the
elapsed time (about 6 days). The length of total mycelium of a group of
12 spores was measured monthly, up to 6 months.
Time-lapse video microscopy.
Surface-sterilized spores of
G. caledonium and G. mosseae were
germinated as previously described. After 1 week of incubation, individual spores were mounted on microscope slides in sterile distilled water (SDW). The coverslips were sealed with 1% WA, which
was periodically wetted with SDW. With this method, individual spores
could be held for more than 6 h, allowing the entire process of
septum formation to be monitored. Spores were observed under a Polyvar
light microscope equipped with differential interference contrast
optics and with a 3 CCD color video camera connected to a
videocassette recorder (Hi 8, EV-C2000E; Sony Co., Tokyo, Japan)
and to a videographic printer (UP-890 CE; Sony Co.).
To observe vacuoles and vacuolar strands in hyphal tips at different
stages of protoplasm retraction, 1-month-old germinated
spores were
stained with the fluorescent dye MDY-64 (yeast vacuole
membrane marker;
Molecular Probes, Inc., Eugene, Oreg.) (10 µM)
in dimethyl
sulfoxide-10 mM HEPES buffer, pH 7.4. Other spores
were stained with
the cyanine dye 3,3'-dihexyloxacarbocyanine
iodide (Sigma Chemical Co.,
St. Louis, Mo.) (0.87 µM) in dimethyl
sulfoxide-water. All spores
were observed under epifluorescence
by using a Polyvar microscope
equipped with an HBO 200 mercury
lamp, with filter combination B1 (BP
450-495, LP 520, DS 510).
To visualize the occurrence and location of nuclei and mitochondria in
hyphae during the phase of protoplasm retraction, 1-month-old
germinated spores were mounted on microscope slides in
diamidinophenylindole
(DAPI) (5 µg/ml) in a 1:1 water-glycerol
solution and observed
under epifluorescence, with filter combination U1
(BP 330-380,
LP 418, DS 420).
Immunoblotting of cytoskeletal proteins.
To yield comparable
amounts of total proteins from mycelium of different ages and from
ungerminated spores, different numbers of fungal propagules were used:
300 sporocarps of G. mosseae and 1,000 spores of
G. caledonium were incubated for 30 days; 350 sporocarps of G. mosseae and 2,000 spores of
G. caledonium were incubated for 15 days; and 400 sporocarps of G. mosseae and 2,000 spores of
G. caledonium were utilized as nongerminated controls.
One-dimensional sodium dodecyl sulfate-polyacrylamide gel
electrophoresis was carried out with a discontinuous buffer system
(
18). Extracts (4 µg of protein) were loaded onto linear
12%
polyacrylamide gels (10 by 8 by 0.75 mm) and run in a Bio-Rad
(Richmond, Calif.) Mini Protean II slab cell system.
Immunodetection of cytoskeletal proteins was carried out as previously
described (
1). After electrophoresis, the proteins
were
transferred to nitrocellulose membranes by using a Trans-Blot
apparatus
(Bio-Rad). Electrotransfer was performed first overnight
at 100 mA, 20 V, and then for 1 h at 350 mA, 100 V, at 0°C. Nitrocellulose
sheets were then blocked for 1 h in 2% bovine serum albumin in
phosphate-buffered saline (PBS) (pH 7.3) and incubated for 1 h
at
room temperature with mouse monoclonal anti-
- and anti-
-tubulin
and antiactin antibodies, which were diluted 1:500 in PBS containing
0.1% bovine serum albumin and 0.1% Triton X-100. Bound antibodies
were visualized with rabbit anti-mouse immunoglobulin G antibody
conjugated to peroxidase and diluted 1:3,000 in PBS containing
0.1%
Triton X-100. The peroxidase reaction was carried out with
0.3 mg of
3,3'-diaminobenzidine per ml in 1:1 (vol/vol) Tris-HCl
(100 mM) (pH
7.6)-water, supplemented with 25 µl of H
2O
2
immediately
prior to use. Color development was stopped by rinsing the
membranes
with SDW.
Metabolic activity and infectivity of 1- to 6-month-old
mycelium.
Succinate dehydrogenase (SDH) activity was assessed, as
described by Smith and Gianinazzi-Pearson (28), on 1- to
6-month-old germinated spores of G. caledonium.
Surface-sterilized spores were prepared as described previously, and at
least five germinated spores were utilized for each
assay. The
lengths of hyphae showing formazan salt depositions
on 6-month-old
mycelium and the numbers of stained and unstained
hyphal tips on
1- to 6-month-old mycelium were recorded by using
a Polyvar light
microscope equipped with Quantimet 500 image analysis
software.
To test infectivity by the mycelium, surface-sterilized spores were
germinated as previously described and individually used
to inoculate
the roots of six 2-week-old
Ocimum basilicum L. (basil)
plants placed on a 47-mm-diameter Millipore membrane
(0.45-µm-diameter
pores). Another Millipore membrane was used to make
up a sandwich
system. Ten replicates were set up for each trial, which
consisted
of 1-, 2-, 3-, 4-, 5-, and 6-month-old germinated spores. One
month after inoculation, sandwiches were carefully opened and
roots
were stained to reveal fungal structures (
23). The number
of
entry points formed by each germinated spore was recorded for
each
plant root system and for each sandwich.
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RESULTS |
Time course of hyphal growth in the absence of the host plant.
Observations carried out for 15 to 20 days on spores of G. caledonium growing in the absence of the host showed that within a
few days the elongating germ tubes gave rise to a mycelial network whose extension was highly variable between individuals. After a period
ranging between 5 and 10 days postgermination, total hyphal length
reached a plateau and the mycelium entered a state of developmental
arrest (Fig. 1). The mean growth rate of
the mycelium during this early phase was 1.97 ± 0.39 µm/min
(mean ± standard error). An interesting feature of the mycelial
net was the occurrence of many hyphae completely devoid of protoplasm. Time course observations showed that during the plateau growth phase,
corresponding to arrest of hyphal elongation, fundamental modifications
occurred in the mycelium: protoplasm was retracted from most hyphae,
leading to the formation of cross walls delimiting an empty mycelial
network. This phenomenon was further studied by daily measurements of
the extension of protoplasm-containing hyphae and empty hyphae.
These showed that when total hyphal growth reached the plateau, the
length of protoplasm-containing mycelium remained constant (Fig.
2a and c) or tended to decrease (Fig. 2b
and d). Long-term observations, up to 80 days, showed that this
decrease was paralleled by rising values of the length of empty hyphae
(Fig. 3).
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FIG. 2.
Time courses of mycelial growth of four individual
spores of G. caledonium. Lengths of total ( ),
protoplasm-containing ( ), and empty ( ) mycelium are shown.
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FIG. 3.
Long-term time course of mycelium developing from
an individual spore of G. caledonium. Lengths of total
(), protoplasm-containing (), and empty () mycelium are
shown.
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In some spores, a renewal of mycelial elongation following growth
arrest was observed. An example is shown in Fig.
4: the
spore, 23 days old, resumed growth
after 16 days of stasis. Accordingly,
assessment of spore growth
was carried out monthly, for a period
ranging from 1 to 6 months
after germination. The results showed
that total mycelial length
increased from the 1st to the 3rd month
(from 45.13 ± 2.94 to
68.19 ± 6.05 mm, respectively), remaining
constant up to the 6th
month (70.83 ± 7.08 mm).
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FIG. 4.
Long-term time course of mycelium developing from
an individual spore of G. caledonium. Lengths of total
(), protoplasm-containing (), and empty () mycelium are
shown.
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Formation of retraction septa.
Time-lapse video microscopy
provided evidence of the phenomenon of withdrawal of protoplasm from
the hyphal tip and the formation of retraction septa. The tip, usually
rich in dense granular protoplasm, initiated vacuolization from the
apical zone. Vacuoles, usually small when observed in protoplasm of
growing hyphae, became progressively enlarged in protoplasm-retracting
hyphae, leading to the formation of empty areas (Fig.
5). In the distal zone of the apex, a
steady dense area of protoplasm became evident where a cross wall was eventually formed. Protoplasm retraction followed by the formation of
further septa occurred in the same way, and the completion of each
empty portion (delimited by septa) was carried out in about 2 to 3 h (Fig. 6).
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FIG. 5.
Time-lapse video microscopy showing protoplasm
retraction and septum formation in apical segments of two G. caledonium hyphae. Note the vacuole extension process (a to d),
leading to the formation of empty tips (e to h). Bar, 6.25 µm.
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FIG. 6.
Time-lapse video microscopy showing the vacuole
extension process accompanying protoplasm retraction (a to c) and
leading to the formation of an empty hyphal segment (d) in
G. caledonium. Bar, 6.25 µm.
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DAPI staining and epifluorescence microscopy showed that the active
process of protoplasm retraction also involved the migration
of nuclei
and cellular organelles (Fig.
7).
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FIG. 7.
Epifluorescence microscopy of nuclei (visualized by
staining with DAPI) of G. mosseae hyphae. (A) Nuclei
(arrowheads) located below the first retraction septum. Note the
hyphal tip completely devoid of protoplasm. (B) Empty intercalary
hyphal segment (large arrow), separated by a septum from the contiguous
tract containing nuclei (arrowhead) and mitochondria (small arrow). (C)
Elongated nuclei occurring in hyphae showing protoplasm retraction.
Bars, 7.1 µm (A and B) and 12.5 µm (C).
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Cytoskeletal proteins.
Proteins extracted from ungerminated
spores or spore mycelium of the AM fungi G. mosseae and
G. caledonium, separated by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis and immunostained after
Western blotting for - and -tubulins and actin, revealed one band
for each cytoskeletal protein in mycelial samples, whereas signals from
ungerminated spores were very weak or absent (Fig. 8). A weaker signal was more regularly
detected for -tubulin than for -tubulin in both fungal species,
but their mobilities were similar, with one band of
Mr 55.5 × 103 to 56.5 × 103. The actin antibody revealed one band of
Mr 41.5 × 103 to 43 × 103, typical of actin. The signals for - and -tubulin
and actin of mycelium incubated for 30 days appeared stronger than
those of mycelium incubated for 15 days in both fungal species (Fig. 8).
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FIG. 8.
Immunoblots of -tubulin, -tubulin, and actin
from 30- (lanes 1) and 15 (lanes 2)-day-incubated mycelium and from
ungerminated spores (lanes 3) of G. caledonium (a) and
G. mosseae (b). Molecular weight markers were stained
with amido black.
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Metabolic activity and infectivity of 1- to 6-month-old
mycelium.
Viability of the mycelium was determined in 6-month-old
germinated spores by evaluating SDH activity. The mean percentage of
SDH-positive hyphae was 41.98 ± 14.19 (after angular
transformation of data). Metabolic activity declined steeply with
increasing distance from the spore, with little SDH activity in distal
hyphal branches. These results were confirmed by the number of active hyphal tips recorded on 1- to 6-month-old germinated spores. Fractions of SDH-positive hyphal tips were inversely correlated with the age of
the mycelium (Fig. 9).
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FIG. 9.
Relationship between the fraction of SDH-positive
hyphal tips and time after spore germination in G. caledonium mycelium. The solid line indicates the result of linear
regression analysis.
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Germinated spores retained the ability to infect plants for several
months, but infectivity decreased greatly after the 3rd
month. The
total number of entry points formed by 1-, 2-, and
3-month-old mycelium
(13.10 ± 3.83) was significantly different
from that formed by
4-, 5-, and 6-month-old mycelium (1.97 ± 0.50).
After 6 months of
growth, a low number of protoplasm-containing
hyphal tips (Fig.
10A and B) which were still capable of
infecting
host roots (Fig.
10C) was observed.
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FIG. 10.
Light micrographs of hyphae and mycorrhizal
infection by G. caledonium of O. basilicum.
Viable (A) and empty (B) hyphal tips of 6-month-old mycelium are shown.
(C) Viable tip producing appressoria (arrow) capable of root
colonization and arbuscule formation (arrowhead). Bar, 25, 25, and 27.8 µm (for panels A, B, and C, respectively).
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| |
DISCUSSION |
The results of our work led to the identification of a survival
strategy in obligately biotrophic AM symbionts operating in the
absence of the host. When no host-derived signal from the surrounding
environment is perceived by germinating spores, fungal hyphae undergo a
programmed growth arrest and resource reallocation, allowing long-term
maintenance of viability and host infection capability.
In our study, we resolved the time course of growth arrest by precisely
timing the growth length of the mycelium originating from individual
spores. In the absence of the host, the AM fungus G. caledonium, maintained in axenic culture, produced extensive mycelial growth during the first 15 days but thereafter underwent developmental arrest, although it proved able to sustain further limited mycelial growth up to 3 months after spore germination. Previous short-term studies showed that in the absence of host roots,
hyphal growth of G. mosseae, Gigaspora
margarita, and Glomus etunicatum ceased within 15 days
of germination (2, 9, 13, 26). Interestingly, growth arrest
occurred regardless of the amount of mycelium produced, suggesting that
the growth period could be a critical factor involved in growth arrest.
The mycelium produced by all spores examined underwent the process of
protoplasm retraction with formation of retraction septa, separating
viable from empty hyphal segments. The occurrence of retraction septa
in AM fungi has been previously mentioned by a few authors (19,
22), but the cellular events involved in the phenomenon were not
described in detail. The dynamics of formation of retraction septa in
various other fungal species and genera were described precisely by
Ingold (14). In the present study, we documented the same
phenomenon occurring in AM fungi by using time-lapse video microscopy.
Our studies detected an interesting feature of this phenomenon:
in G. caledonium, retraction of protoplasm was
initiated from the hyphal tips, which eventually became isolated by the formation of retraction septa; this is in contrast to findings reported for other fungi, where the hyphal segments proximal to the
germinating propagule appeared to be empty and septate (14). In both cases, the early cue indicating the initiation of retraction of
protoplasm from hyphal tips was the formation and progressive extension
of vacuoles, followed by development of a septum.
Previous studies reported that prolonged incubation of spores in
the soil without host plants induced a progressive increase in
the proportion of empty hyphae (31). Decreasing amounts of free sterols and a parallel increase in bound sterols were observed in
G. caledonium germinated spores after 14-day growth in
axenic culture, suggesting the occurrence of a senescence phase in the mycelium of AM fungi, as described for other fungal species (3, 6).
Systematic observation of mycelial growth in axenic conditions
suggested that the formation of retraction septa might be associated not only with mycelial senescence and aging but also with a mechanism involving control over allocation of the limited energy resources of
spores. The active process of protoplasmic retraction, involving migration of nuclei and cellular organelles, appears to have a function
in enhancing the fungal ability to survive in the absence of a carbon
donor.
Data on immunodetection of cytoskeletal proteins and on metabolic
activity and retention of infectivity of germinated spores confirmed
this behavioral explanation. Immunoblotting of cytoskeletal proteins
showed that signal strengths for - and -tubulins and actin
increased with mycelial age, in contrast to the growth pattern of
germinated spores, which showed a growth arrest as early as 10 days
after germination (Fig. 1). It is noteworthy that the strongest signals
for tubulins were detected in 30-day-incubated mycelium, when hyphal
growth had ceased but protoplasmic retraction activity was at its
highest, as demonstrated by long-term observations and video
microscopy. These results suggest the occurrence of higher amounts of
cytoskeletal proteins in old mycelium, consistently with their role in
movements of nuclei and organelles and transport during protoplasm
retraction (Fig. 7C).
Moreover, SDH activity in hyphae proximal to the mother spore was still
detectable in 6-month-old mycelium. These data showed that after the
spore had carried out a prolonged exploration of the surrounding
environment and reached the phase of growth arrest, a variable length
of mycelium with metabolic activity was maintained. This confirmed our
previous results showing that 26-day-old mycelium was resting, still
viable, and capable of renewed growth in response to host roots
(10).
The long-term ability of AM mycelium to retain infectivity in the
absence of the host was tested in soil by Tommerup (31), who showed that after a 4-month incubation, germinated spores of G. caledonium and Acaulospora
laevis could develop infection structures and colonize
Trifolium roots. Experimental evidence presented here
indicates that the longevity of individual germinated spores can
be even greater, since G. caledonium mycelium
remained viable and able to form appressoria and produce symbiotic
structures for as long as 6 months after germination. The decreasing
ability of germinated spores to form infection structures on host roots could be due to the correlated decrease in the number of
protoplasm-containing hyphal tips. The progressive retraction of
protoplasm from hyphae and its reallocation proximally to the mother
spore caused a marked decrease in the length of viable mycelium and
reduced the chances of the fungus locating host roots.
In conclusion, our developmental and spatiotemporal studies provided
evidences of a survival strategy evolved by the obligately biotrophic AM fungi
an energy-saving mechanism controlling
lifespan and the growth arrest decision, allowing long-term infectivity of mycelium growing in the absence of the host.
This strategy may cooperate with other mechanisms, e.g., a wide host
range (29), the ability of multiple germination (16, 17, 19), and the regulation of infection structure
differentiation (8, 11), in the survival of individuals and
populations of AM fungi.
| |
ACKNOWLEDGMENT |
This work was supported by National Research Council Italy,
Special Project R.A.I.S.A., and by a University of Pisa grant (Fondo di
Ateneo).
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Dipartimento di
Chimica e Biotecnologie Agrarie, Via del Borghetto 80, 56124 Pisa, Italy. Phone: 39-50-571561. Fax: 39-50-571562. E-mail:
mgiova{at}agr.unipi.it.
| |
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Applied and Environmental Microbiology, September 1998, p. 3473-3479, Vol. 64, No. 9
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
