FEMS Microbiology Letters 204 (2001) 101^104
www.fems-microbiology.org
E¡ects of long-term storage at di¡erent temperatures on conidia of
Botrytis cinerea Pers.: Fr.
Katia Gindro *, Roger Pezet
Swiss Federal Research Station for Plant Production of Changins, Secteur 1, Mycologie, Route de Duillier, CH-1260 Nyon, Switzerland
Received 3 July 2001; received in revised form 2 August 2001 ; accepted 6 August 2001
First published online 18 September 2001
Abstract
Survival of Botrytis cinerea conidia was studied after storage without pretreatments at different temperatures (380³C, 320³C, 4³C and
21³C). Germination tests performed during 3 years showed that viability at 21³C was completely lost after 1 month. Conidia stored for 30
months at 380³C, 320³C and 4³C were able to germinate, respectively, at 79%, 8% and 0.2%. Changes in adenylate level, energy charge and
respiration (O2 consumption) made on each set of conidia were correlated to the germination rate. The 30-month-old stored conidia showed
differences in pathogenicity tests on apples. While the pathogenic aggressiveness of conidia stored at 380³C was almost the same as for fresh
conidia, it decreased with increasing temperature of storage. An ultrastructural study made on conidia stored for 30 months at 380³C has
shown the emergence of a new wall layer in a retraction zone of the cytoplasm by comparison to fresh conidia. However, the integrity of the
cytoplasmic content was maintained. The effects of low temperature storage, maintenance of cell integrity and pathogenicity of conidia of
B. cinerea are discussed. ß 2001 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
Keywords : Botrytis cinerea; Conidium ; Storage ; Adenylate; Cell respiration
1. Introduction
Conidia of Botrytis cinerea are the most important
structures implicated in the infection process. Biochemical
and molecular studies linked to their metabolism during
dormancy, adhesion, germination and plant^host penetration are necessary for a better understanding of epidemiology and infection stages, but ¢rst there is a need for a
great quantity of conidia that are genetically stable. Thus,
it becomes necessary to store these conidia until use without loss of either their vital or pathogenicity characteristics.
Sclerotia of B. cinerea are considered the most e¤cient
structures for long-term survival of the fungus. However,
di¡erent studies have shown that conidia of B. cinerea can
survive cold periods [1] and desiccation [2]. However, few
experimenters have attempted to store conidia detached
from their conidiophores. The most current techniques
use cryoconservation in liquid nitrogen either in oil for-
* Corresponding author. Tel. : +41 (22) 363 43 53;
Fax: +41 (22) 362 13 25.
E-mail address : [email protected] (K. Gindro).
mulations [3], or on porous glass beads [4]. The germination rate is the usual criterion used to control spore viability. However, some other tests, including enzymatic
assays linked to the synthesis and concentration of ATP
in spores and growing hyphae, have also been developed
[5]. The aim of this work was to ¢nd an easy storage
method that would maintain large amounts of dormant
conidia of B. cinerea, detached from their conidiophores,
during long periods without loss of germination, vitality or
aggressiveness. These parameters were evaluated on conidia stored at di¡erent temperatures using germination
rate, di¡erent biochemical techniques (adenylate concentration and energy charge, cellular respiration and various
enzymatic assays), tests of aggressiveness on the host, and
ultrastructural analyses of the conidia.
2. Materials and methods
2.1. Organism and growth conditions
B. cinerea Pers.: Fr., isolate P69, was cultivated in Petri
dishes on oatmeal agar (Difco), and plated with 200 Wl of
a dense aqueous conidial suspension (7U103 conidia
0378-1097 / 01 / $20.00 ß 2001 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
PII: S 0 3 7 8 - 1 0 9 7 ( 0 1 ) 0 0 3 8 4 - 6
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K. Gindro, R. Pezet / FEMS Microbiology Letters 204 (2001) 101^104
ml31 ). The number of conidia was determined by turbidimetry with a spectrophotometer at 400 nm calibrated
with known concentrations of conidia. Cultures were then
placed at 21³C under alternating light and dark (12 h
each) for 1 week at a light intensity of 30 þ 5 Wmol photons s31 m2 . Conidia were harvested under vacuum according to the method of Pezet and Pont [6], sealed in
plastic tubes and stored dry at di¡erent temperatures
(380³C, 320³C, 4³C and 21³C) for 30 months.
2.2. Germination rate
Glass slides were covered with 1 ml potato dextrose
agar (Difco), plated with 100 Wl of a dense conidial suspension (7U103 conidia ml31 ) and incubated for 24 h at
21³C in moist chambers. The germination rate of each set
of conidia was evaluated regularly over 30 months by
counting 100 conidia. Each analysis was done in triplicate.
2.3. Adenylate levels and energy charge
Conidia (10 mg) of B. cinerea were suspended in 300 Wl
NRB1 (Nucleotide Releasing Reagent for Microbial
ATP, Celsis), incubated for 90 s at room temperature
and ¢ltered through 0.45-Wm disk ¢lters. The nucleotides
were separated on a RP-HPLC column according to the
manufacturer (Supelcosil LC-18-T, 15 cmU4.6 mm ID
(3 Wm particles), Supelco). Each analysis was done in triplicate. The surface of the peaks corresponding to the adenylates of interest (ATP, ADP and AMP) was converted
to micrograms using standards of known concentration.
Energy charge was calculated according to Atkinson and
Walton [7].
2.4. Measurement of cell respiration
Conidial respiration was measured polarographically at
20 þ 0.01³C using a Clark-type oxygen electrode (PO2 Analyzer, Bachofer) in 1 ml of KD solution (glucose 0.04%
(w/v), L-serine 0.02% (w/v): in acetate bu¡er 0.02 M pH
5.2) containing 10 mg of each set of conidia according to a
modi¢ed method of Kosuge and Dutra [8]. Each measure
was done in triplicate.
2.5. Pathogenicity tests
Aggressiveness of each set of conidia of B. cinerea was
estimated by pathogenicity tests on apples (Golden Delicious) according to the method of Schu«epp and Ku«ng [9],
except that wounded cuticles were inoculated with 50 Wl of
a dense conidial suspension (7U103 conidia ml31 ). The
experiments were done in triplicate.
2.6. Transmission electron microscopy
An ultrastructural study was made on fresh and stored
conidia (380³C and 21³C for 30 months, and at 380³C
for 24 h), ¢xed with osmium tetroxide and glutaraldehyde,
and further stained with uranyl acetate and lead citrate
according to the method of Reynolds [10].
3. Results
Our results have shown that the viability of conidia was
completely lost after 1 month when stored at 21³C, while
79%, 8% and 0.2% conidia, respectively, still germinated
after 30 months when stored at 380³C, 320³C and 4³C
(Table 1). Interestingly, the germination rate of conidia
stored at 380³C remained at 99% for the ¢rst 6 months,
which is the germination rate of fresh conidia (result not
shown). In addition to the germination rate, various biochemical tests were performed on vital parameters such as
adenylate level, energy charge and cellular respiration (Table 1). In order to evaluate the viability of conidia after a
long storage period at di¡erent temperatures, the data in
Table 1 were used to compare measurements on fresh
conidia with conidia stored for 30 months. A general decrease of the energy level, O2 consumption and aggressiveness was observed as the storage temperature increased. A
shift from ATP to ADP and AMP was also observed, and
the shift was larger in conidia stored at higher temperatures. Conidia stored at 380³C for 30 months still had
50% of their original energy charge, 70% respiration
rate, 80% germination rate, and about the same aggressiveness (90%) as fresh spores.
We also analyzed the ultrastructure of conidia to assess
Table 1
E¡ect of storage temperature on germination, energy charge, and aggressiveness of conidia of B. cinerea conidia stored for 30 months
Storage temperature
(³C)
Germination
(%)a
ATP (Wg)a
ADP (Wg)a
AMP (Wg)a
ECa;b
O2 consumed
(nmol min31 )
Aggressivenessa
(diameter of necrosis in cm)
Fresh conidia
380
320
4
21
99.0 þ 0.1
79.0 þ 1.8
8.0 þ 1.4
0.2+0.1
0.0 þ 0.0
0.46 þ 0.08
0.14 þ 0.01
0.02 þ 0.01
0.00 þ 0.00
0.00 þ 0.00
0.30 þ 0.04
0.36 þ 0.09
0.08 þ 0.01
0.04 þ 0.01
0.00 þ 0.00
0.14 þ 0.02
0.54 þ 0.13
0.45 þ 0.12
0.49+0.04
0.10 þ 0.04
0.67 þ 0.04
0.33 þ 0.04
0.10 þ 0.01
0.01 þ 0.01
0.00 þ 0.00
32.0
22.0
8.0
0.7
0.0
3.1 þ 0.3
2.8 þ 0.2
0.8 þ 0.1
0.2 þ 0.1
0.0 þ 0.0
a
b
Mean of three replicates.
Energy charge: ATP+0.5 ADP/ATP+ADP+AMP.
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103
Fig. 1. Ultrastructure of B. cinerea conidia. A and B: Fresh harvested conidia. C and D: Conidia stored dry for 30 months at 380³C. E and F: conidia stored dry during 30 months at 21³C. as = abscission site, ew = external wall layer, f = ¢laments, i = inclusion, iw = internal wall layer, iwn = new internal wall layer, m = mitochondria, n = nucleus, nu = nucleolus, pm = plasma membrane, pmd = disrupted plasma membrane, r = ribosome, rc = retraction
zone of the cytoplasm, v = vacuole. Scale bars indicate 1 Wm.
changes due to long-term storage. Electron micrographs of
conidia stored for 30 months at either 380³C or 21³C
were compared to fresh conidia as shown in Fig. 1. An
additional ultrastructural analysis of conidia stored at
380³C for 24 h ruled out any freezing e¡ects as the structure of the conidia looked precisely like fresh conidia (data
not shown). The cell wall of fresh conidia has two layers,
the outer layer, denser to the electrons and covered with
micro¢brils, and the inner layer, wider and less dense to
the electrons (Fig. 1A,B). The fresh conidia have intact
mitochondria, vacuoles with or without dark inclusions,
dense bodies sometimes linked to the membranes, one to
several nuclei with nucleoli and ribosomes throughout the
cytoplasm surrounded by a plasma membrane (Fig. 1B).
Conidia that were stored for 30 months at 380³C showed
some di¡erences. The cellular wall had a new electrondense layer within the inner cell wall. The cytoplasm was
retracted, and ¢laments seemed to join the new inner cell
wall layer to the intact plasma membrane (Fig. 1C,D).
Both vacuoles and their dense inclusions were enlarged.
Conidia that had been stored at 21³C for 30 months
showed signi¢cant retraction of the cytoplasm (Fig. 1E).
Recognizable cellular structures were altered. The plasma
membrane was disrupted at many places, and the cell wall
showed only the external dense layer (Fig. 1E,F).
4. Discussion
The present work has shown that conidia of B. cinerea,
detached from their conidiophores, can be stored for 6
months without any loss of their germination rate. Like
most conidial fungi, ungerminated conidia of B. cinerea
contain stored nutrients such as glycogen, lipids and phospholipids available for ATP synthesis and maintenance of
vital functions [11]. Metabolism of lipids results in a
marked vacuolization of the fungal cell [12]. It is di¤cult
to consider that, at temperatures below 320³C, active metabolism occurs, resulting in hydrolysis of stored nutrients ; however, such vacuolization was observed in conidia stored for 30 months at 380³C. As other
ultrastructural modi¢cations, such as denser mitochondria, granulated and slightly retracted cytoplasm, were
also observed, we suspect that these structural modi¢cations were due to a desiccation process that occurred during long-term storage even at such a low temperature. The
FEMSLE 10132 17-10-01
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loss of water is even more important at higher storage
temperatures and depends, at atmospheric pressure, on
air^water saturation capacity. Alteration of cellular structures in conidia stored for 30 months was enhanced with
increasing storage temperature. Freezing of B. cinerea conidia at 380³C for 24 h did not modify the cell ultrastructure. The low temperature itself had no apparent destructive e¡ects.
Water content in cells plays a crucial role and is considered to be a biological solvent [13]. Hydrogen bonds are
responsible for the most important weak interactions between biological molecules such as proteins, carbohydrates, nucleic acids and hydration layers. It stabilizes vital
structures, ensures mobility and cohesion between the
macromolecules, and forms a single vital system with
them [14]. Loss of water will ¢nally result in a complete
disorganization of this equilibrium, leading to an unstructured accumulation of non-aqueous compounds such as
membrane lipids and macromolecules [15]. In air-borne
conidia, such as those of B. cinerea, the water content is
very low [16]. In such a biological system, vitri¢cation of
water can occur even at ambient temperature, and is maintained under this physical stage at freezing temperatures.
This particular state of aqueous solution, the glassy state,
has been suggested as a mechanism for membrane protection during dehydration and freezing [17]. Once water exists in a glassy state, dehydration is very limited, and
membranes remain in a £uid lamellar phase during further
hydration [18]. During long-term storage at low temperature, however, sublimation of water cannot be avoided,
even at normal atmospheric pressure (personal communication of Dr. A. Steiner, Swiss Federal O¤ce of Metrology and Accreditation, Bern-Wabern, Switzerland). This
slow but progressive loss of water is most likely responsible for the ultrastructural modi¢cations observed in conidia after 30 months at 380³C. It is expected that longer
storage periods will lead to signi¢cant structural alterations.
ATP within the cell is a thermodynamically unstable
molecule [12]. For dormant conidia of B. cinerea, the
best preservation regimen is storage at the lowest temperature possible. At temperatures of 320³C or 380³C,
where all oxidative metabolism is frozen, the size of the
ATP pool is only linked to the physico-chemical degradation rate. After 30 months at 380³C, 30% of the ATP pool
in conidia was converted to ADP or AMP when compared
to fresh ones. At a storage temperature of 320³C, ATP
was barely detectable after 30 months, and almost half of
it was degraded beyond the AMP stage. At temperatures
above freezing, detached conidia do not store well. The
loss of germination was related to degradation of cellular
organelles, and the decline in respiration and energy level.
At an energy charge less than 0.1, vital functions dropped
drastically. We conclude that the protocols for conservation described in this paper provide suitable conditions for
the storage of conidia of B. cinerea over several years.
Acknowledgements
The authors thank Dr. H. Richter for critically reading
the manuscript and Mrs. I. de Groote for very helpful
technical assistance.
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