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\begin{document}

\title{Microstructure characterization of different types of chlamydospores in
Duddingtonia flagrans}



\author[1]{Bo-Bo Wang}%
\author[1]{Fenghui Wang}%
\author[1]{Youlei Li}%
\author[2]{Kui-Zheng Cai}%
\affil[1]{Affiliation not available}%
\affil[2]{Northwest University for Nationalities}%


\vspace{-1em}



  
  \date{May 21, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
The morphological and structural differences of different types of
chlamydospore of Duddingtonia flagrans, a nematophagous fungus, were
studied under light microscope and electron microscope to provide
reference for the biological control of parasitic nematodiasis. In this
study, D. flagrans isolate F088 dormant chlamydospore and non-dormant
chlamydospore were selected as the research objects. The structural
differences of these spores were observed by optical microscopy through
lactol cotton blue, Trypan blue and MTT staining. FUN-1, DAPI and CFW
staining were used to observe the metabolic activity, cell wall and
nucleus differences of the two types of spores under fluorescence
microscope. Ultrastructure of the two kinds of spores was observed using
scanning electron microscope (SEM) and transmission electron microscope
(TEM). Since lacto phenol cotton blue, trypan blue staining cannot
distinguish dormant spores from dead spores, MTT assay was performed.
Fluorescence microscopy observation showed that the cytoplasmic
metabolic activity of non-dormant spores was stronger than that of
dormant spores. The nucleus of dormant spores was bright blue, and their
fluorescence was stronger than that of non-dormant spores. The cell wall
of non-dormant spores produced stronger yellow-green fluorescence than
that of dormant spores. Ultrastructural observation showed that there
were globular protuberances on the surface of the two types of spores,
but with no significant difference between them. The inner wall of
dormant spore possesses a thick zona pellucida with high electron
density which was significantly thicker than that of non-dormant spores,
and their cytoplasm is also changed. In this study, the microstructure
characteristics of dormant and non-dormant chlamydospores of D. flagrans
fungi were preliminarily clarified, suggesting that the state of cell
wall and intracellular materials were changed after spores entered to
dormancy.%
\end{abstract}%



\sloppy


\textbf{Microstructure characterization of different types
ofchlamydospores in\emph{Duddingtonia flagrans}}

\textbf{Bobo Wang, Fenghui Wang, Youlei Li} *\textbf{, Kuizheng Cai} *

(Medical College of Yan' an University, Yan'an 716000, China)

*These authors contributed equally to this study.

*Correspondence: Kuizheng Cai, Medical College of Yan'an University,
Yan'an 716000, China.

E-mail: ckz000@126.com

Phone: 86-0911-2650158

Fax: +86-0911-2650158

*Correspondence: Li You Lei, Medical College of Yan'an University,
Yan'an 716000, China.

E-mail: liyoulei@yau.edu.cn

Phone: 86-0911-2650158

Fax: +86-0911-2650158

\textbf{Abstract:} The morphological and structural differences of
different types of chlamydospore of \emph{Duddingtonia flagrans} , a
nematophagous fungus, were studied under light microscope and electron
microscope to provide reference for the biological control of parasitic
nematodiasis. In this study, \emph{D. flagrans} isolate F088 dormant
chlamydospore and non-dormant chlamydospore were selected as the
research objects. The structural differences of these spores were
observed by optical microscopy through lactol cotton blue, Trypan blue
and MTT staining. FUN-1, DAPI and CFW staining were used to observe the
metabolic activity, cell wall and nucleus differences of the two types
of spores under fluorescence microscope. Ultrastructure of the two kinds
of spores was observed using scanning electron microscope (SEM) and
transmission electron microscope (TEM). Since lacto phenol cotton blue,
trypan blue staining cannot distinguish dormant spores from dead spores,
MTT assay was performed. Fluorescence microscopy observation showed that
the cytoplasmic metabolic activity of non-dormant spores was stronger
than that of dormant spores. The nucleus of dormant spores was bright
blue, and their fluorescence was stronger than that of non-dormant
spores. The cell wall of non-dormant spores produced stronger
yellow-green fluorescence than that of dormant spores. Ultrastructural
observation showed that there were globular protuberances on the surface
of the two types of spores, but with no significant difference between
them. The inner wall of dormant spore possesses a thick zona pellucida
with high electron density which was significantly thicker than that of
non-dormant spores, and their cytoplasm is also changed. In this study,
the microstructure characteristics of dormant and non-dormant
chlamydospores of \emph{D. flagrans} fungi were preliminarily clarified,
suggesting that the state of cell wall and intracellular materials were
changed after spores entered to dormancy.

\textbf{Keywords:} \emph{Duddingtonia flagrans} ; nematophagous fungi;
dormant chlamydospore; non-dormant chlamydospore; metabolic activity;
ultrastructure

\textbf{Introduction}

Nematophagous fungus \emph{Duddingtonia flagrans} is a promising
candidate for biological control of animal helminthic diseases. The
chlamydospore of this fungus was produced very early in the growth
process. Compared with other nematopgagous fungi that did not produce
chlamydospore, this fungus could produce chlamydospore in three days and
produced few conidia {[}1,2{]}. This fungus is the only species of
chlamydospore producing among the nematopgagous fungi of the Orbiliaceae
family {[}3{]}. In addition, the advantage of this fungus in the
biological control of animal parasitic nematodes is that the
double-walled chlamydospore added to the feed can resist the adverse
environment of the digestive tract {[}4-7{]}. The spores then develop
along with the worm eggs in the feces of the animal, the spores of the
fungus begin to germinate, and the larvae that hatch further stimulate
the mycelium of the fungus to specialize into feeding organs to feed on
the larvae as nourishment for the fungus itself {[}6{]}. Due to the
strong resistance of chlamydospore to harsh external environment, such
as freezing and drying, chlamydospore as an effective component of feed
additives, such as various formulation, lick blocks and powders, has
good commercial value {[}8-10{]}. Both powder and pellet formulation can
effectively reduce infectious larvae in pasture, and have obvious
control effect on gastrointestinal nematodes in grazing horses, cattle,
sheep, goats and other livestock {[}11-13{]}.

The dormancy of spores is one of the most mysterious life forms because
of their resistance to adverse conditions and their ability to remain in
a state of hypometabolism (a sort of half-dead) for long periods of
time, which might be the best form for the source of all life on Earth
{[}14{]}. The study of fungal spore dormancy is much later than that of
bacterial spore. There are still many questions to explain the
phenomenon of fungal spore dormancy. \emph{D. flagrans} is a species of
fungi belonging to the Deuteromycotina. At present, no sexual stage has
been found, and the chlamydospore is asexual.

These spores are often found dormant in nature or in biocontrol products
{[}15{]}. And this dormancy is a deep endogenous dormancy, which is
different from the exogenous dormancy of some fungi such
as\emph{Aspergillus} conidium {[}16{]}. Endogenous dormancy spore cannot
germinate even in the presence of sufficient nutrients such as water,
carbon and nitrogen sources. At present, researchers pay more attention
to the dormancy of some species of \emph{Aspergillus}
and\emph{Trichophyton rubrum} conidium {[}17-20{]}, and these fungi are
mainly opportunistic pathogens and cause skin diseases in humans.
However, there are relatively few literatures on the observation of the
ultrastructure of fungal spores. Anjo et al. observed the structural
characteristics of conidia of \emph{A. fumigatus} at different culture
stages by transmission electron microscopy {[}21{]}. Some studies
observed the ultrastructure of the resting and germinating conidia
of\emph{A. ochraceus} and the resting conidia of \emph{Botrytis} cinerea
{[}22,23{]}. The structure of dormant chlamydospore of fungi was less
observed. Among these studies, Citiculo et al. observed the metabolic
activity of chlamydospore at different days of age by fluorescence
microscopy against the conditioned human pathogens \emph{Candida
albicans} and \emph{Candida Dublin} {[}24{]}. Yan et al. observed the
ultrastructure of dormant and non-dormant chlamydospore of \emph{M.
oryzae} {[}25{]}. In some reports, the interaction of \emph{D.
flagrans}with L3 of \emph{C. contortus} was observed by SEM {[}26,27{]}.
However, the morphological data of dormant and non-dormant of \emph{D.
flagrans}chlamydospore are still lacking. In the practical application
of biotechnology, producers encounter the problems of chlamydospore seed
material inspection and final product quality inspection. Due to the
existence of structural dormancy of chlamydospore in the product, it
could not germinate on the basis of normal culture, so the conventional
methods of spore incubation and colony counting could not reflect the
actual number of live spores. Therefore, to fix this problem, it is
necessary to find an effective method to distinguish dormant spores from
dead spores by morphology. Meanwhile, it is also of great scientific
significance to study the morphological and structural differences
between dormant spores and non-dormant spores.

In this study, we aim to elucidate the morphological differences of
different types of chlamydospore under light microscopy and electron
microscopy. Firstly, the structural differences of dormant, non-dormant
and dead spores were observed by optical microscopy with different
staining methods. Secondly, the differences of metabolic activity, cell
wall and nucleus between dormant and non-dormant spores were observed by
fluorescence microscopy. Finally, the ultrastructure of the two types of
spores was compared by scanning electron microscope and transmission
electron microscope. The data obtained in this study provide a
scientific basis for the application of \emph{D. flagrans} in the
biological control of animal parasitic nematodes and for understanding
the dormancy mechanism of the fungal chlamydospore.

\textbf{1 Materials and methods}

\textbf{1.1 Test strains}

The \emph{D. flagrans} isolate used in this study was F088 (CGMCC No.
11209), the gene database entry number is KU881774.1, which was isolated
and purified from sheep feces collected in Heilongjiang Province, China
in 2014 by an improved spreading separation technique. The isolate was
stored in refrigerator at 4  on test tube containing 2\% cornmeal agar.
Mycelium and spores were taken from the preserved inclined plane and
inoculated on Potato Dextrose Agar (PDA, Beijing Landbridge Technology
Co., LTD.) plate containing chloramphenicol and incubated at 28  for 7
days.

\textbf{1.2 Strain culture and chlamydospore preparation}

\textbf{1.2.1 Preparation of non-dormant chlamydospore}

The agar containing mycelium grown on PDA was separated into
6\textasciitilde{}8 even small rounds with a sterilized hole punch
(aperture 5\selectlanguage{ngerman}×5 mm) and inoculated in the above triangular flask
containing Sandburg's liquid culture medium. The flask was shocked at 28
\selectlanguage{english} at 200 r/min for 72 h and the culture was stopped. 10 mL of the
culture containing mycelium was inoculated into a triangle flask
containing 1000 mL of 200 g grains {[}28{]}. Then the culture was placed
in an incubator at 28 \selectlanguage{english} for 21 days. When more mycelium grew on the
grain and yellow powder culture was observed by naked eye, it was proved
that more chlamydospore had been produced, and the culture was
terminated. An appropriate amount of 0.05\% sterilized Tweene-80
solution was added to the triangular flask, and the solution was swirled
for 1-2 min, then mycelium and spores were washed off the grains, and
the crude spore suspension was collected and placed in ultrasonic cell
pulverization instrument (SCO-2500, Shanghai Shengyan Ultrasonic
Instrument Co., LTD.), and subjected to ultrasonic treatment for 5 min
at 150W and 20 KHZ. At an interval of 30 s, the spore suspension was
filtered through a 300-mesh copper screen, the filtrate was centrifuged
at 1000 r/min for 5 min, and then washed with sterilized distilled water
for 3 times. The sediment (i.e. chlamydospore) was suspended with
sterilized distilled water and counted with a blood cell count plate.
Finally, the concentration of the spore suspension was adjusted with
distilled water to about 1x10\textsuperscript{4} spores /mL for further
use.

\textbf{1.2.2 Preparation of dormant chlamydospore}

With the same batch of culture incubated at 28 \selectlanguage{english} and relative humidity
of 30-40\% to 50 d, and dried at 35 \selectlanguage{english} for 10 d, the spore germination
rate was determined to be less than 5\% {[}28{]}.

\textbf{1.2.3 Preparation of artificially lethal chlamydospore}

The purified fresh chlamydospore was placed at 121 \selectlanguage{english} under high pressure
for 15 min.

\textbf{1.3 General optical microscopy and staining observation}

Suspended drops of dormant, non-dormant and dead chlamydospore were
prepared and added to the slide. The slide was covered and photographed
under an optical microscope. The purified spore suspension was placed in
a 2 mL centrifuge tube at 1500 r/min for 5 min, 2/3 of the supernatant
was removed, and the milk phenol cotton blue dye solution (Guangzhou
Exon Biotechnology Co., LTD.) was added for 5 min at room temperature,
and the spore suspension was absorbed, observed and photographed on a
slide. 0.9 mL of the spore suspension was added to 0.1 mL of 0.4\%
Trypan blue dye solution, mixed evenly for 3 min, and observed under a
microscope. Take a 5 mL centrifuge tube and firstly add 1 mL MTT dye
solution, 1 mL PBS buffer solution, at the same time add 2.5 \selectlanguage{greek}μ\selectlanguage{english}L cobalt
chloride solution with a concentration of 2.5 mM, take 100 \selectlanguage{greek}μ\selectlanguage{english}L spore
suspension to be dyed and purified, swirl and mix for 1 min, and then
incubate at 27  for 24-36 h. The spore fluid was absorbed and observed
under an optical microscope.

\textbf{1.4 Metabolic activity of dormant and non-dormant chlamydospore}

To determine the level of metabolic activity of dormant and non-dormant
chlamydospore, FUN-1 fluorescent probe (Shanghai Yubo Biotechnology Co.,
LTD.) was used for staining. The chlamydospore suspension 20 \selectlanguage{greek}μ\selectlanguage{english}L was
placed on a clean slide, and the prepared 10 \selectlanguage{greek}μ\selectlanguage{english}mol/L FUN-1 (containing
2\% glucose) was added to the slide and stained for 30 min at room
temperature, away from light. Firstly, the fluorescence intensity was
examined under a fluorescence microscope (PWS-Scan-F, Shanghai Powerscin
Biotechnology Co., LTD.), then the fluorescence intensity was excited
under a confocal laser scanning microscope (Leica, Beijing Derica
Biotechnology Co., LTD.) with a wavelength of 488 nm laser, and then
with a wavelength of 488 nm and 633 nm laser at the same time. The
fluorescence changes of the two types of spores were observed and
photographed after the images at the two wavelengths were superimposed.

\textbf{1.5 DAPI and CFW staining of dormant and non-dormant spores}

The spore suspension to be tested was centrifuged 200 \selectlanguage{greek}μ\selectlanguage{english}L at 1500 rpm for
5 min, and the supernatant was discarded, and then centrifuged with PBS
(pH7.2) twice, and the supernatant was discarded. The washed spores were
fixed with 5\% paraformaldehyde at 4  for 24 h, and then gently washed
with PBS for 3 min. The spores were suspended in 10 mL DAPI dye working
solution (1 \selectlanguage{greek}μ\selectlanguage{english}g/mL, Shanghai Jizhi Biochemical Technology Co., LTD) for
20 min. The stained samples were placed on slides, and the fluorescence
was analyzed under confocal laser scanning microscope with maximum
excitation wavelength of 340 nm and maximum emission wavelength of 488
nm. According to the instructions recommended by the reagent
manufacturer (Wuhan Purity Biotechnology Co., LTD.), the CFW dye drops
were added to the chlamydospore fixed with 5\% paraformaldehyde, the
fluorescence intensity was adjusted, and the staining of the cell wall
was observed and photographed under a fluorescence microscope.

\textbf{1.6 Electron microscope}

According to the published method {[}29{]}, the above dormant and
non-dormant chlamydospores were coated on cellulose film respectively,
and incubated at 28  for 12 h, then fixed overnight with 2.5\%
glutaraldehyde solution (special for electron microscopy, Beijing
Mycorrhizal Biological Company), and then treated with PBS (0.01 M,
pH7.4) briefly rinsed, alcohol gradient dehydration, tert-butanol vacuum
drying. Samples on the charged rubber were gold-plated fixed, and then
were observed and photographed by using scanning electron microscope
(SEM) (JEOLS-3400IV, Peabody, Massachusetts) under 15 KV voltage.

These dormant and non-dormant chlamydospores were fixed in 2.5\%
glutaraldehyde, then washed three times with PBS buffer (pH7.4), placed
in 1\% osmic acid for 2 h, and washed three times with PBS buffer. After
gradient dehydration with 30\%\textasciitilde{}90\% acetone at room
temperature, the ultrathin sections were prepared by penetration
embedding and polymerization at 60  for 36 h according to the
conventional process of electron microscopy. The sections were treated
with lead citrate staining solution for 10 min, and then stained with
uranium acetate for 30 min. After washing and drying, JEM-1230
transmission electron microscope (TEM) was used to observe and
photograph.

\textbf{2 results}

\textbf{2.1 Observation and staining of different types of
chlamydospores under general light microscope}

Under an optical microscope, the dormant or non-dormant chlamydospore
appears round or oval and yellowish brown in the absence of staining
(Figure 1A \& 1B), while the staining of artificially killed spores were
dark (Figure 1C). The normal mature chlamydospore was double walled,
spherical, 11.3\textasciitilde{}35 \selectlanguage{greek}μ\selectlanguage{english}m in diameter, and oval in size
16.3\textasciitilde{}27.5\selectlanguage{ngerman}×27.5\textasciitilde{}67.5 \selectlanguage{greek}μ\selectlanguage{english}m. The average
spore wall thickness under oil microscope was 2\selectlanguage{ngerman}±0.1 \selectlanguage{greek}μ\selectlanguage{english}m. The immature
chlamydospore or the spore in liquid medium has a smooth surface and has
globular protuberances on the surface when mature. The above three types
of spores can be stained blue by lactol cotton orchid staining (Figure
1D, 1E \& 1F). The artificially killed spores were pale and dark in
color. The three types of spores stained with trypan blue were all black
(Figure 1G, 1H \& 1I), is difficult to distinguish. In MTT assay,
dormant and non-dormant spores were stained black or blue, and no matter
the spores died artificially or naturally, they were not stained (Figure
1 J, 1K, 1L \& 1M). Their differences were significant, suggesting that
this staining method could distinguish dead and dormant spores.

\textbf{2.2 Comparison of metabolic activity and cell structure of
different types of chlamydospores}

FUN-1 staining showed that the dormant spores could only see green
fluorescence after the simultaneous excitation of 488 nm and 633 nm
laser (Figure 2A), while non-dormant spores showed orange fluorescence.
Non-dormant spores can be superimposed under both fluorescence
conditions, while dormant spores cannot be superimposed, suggesting that
non-dormant spores have stronger metabolic activity than dormant spores
(Figure 2B). DAPI staining results showed that the nuclei of resting
spores were regular, round and dense, showing bright blue fluorescence
(Figure 2C \& 2D). Non-dormant spore nucleus materials emit weak
fluorescence, showing blue circles. CFW staining showed that non-dormant
spores could produce strong yellow-green fluorescence, while dormant
showed weak yellow-green fluorescence (Figure 2E \& 2F), indicating that
there was a significant difference between the binding strength of cell
walls and CFW dye.

\textbf{2.3 Comparison of the ultrastructure of dormant and non-dormant
chlamydospore}

Under scanning electron microscope, the dormant spores did not germinate
after 12 h of culture (Figure 3A), but the shape of spores were round
and full. The non-dormant spores had germinated (Figure 3B), the
germination tubes were extended, and the surface was locally deflated.
There were nodules on the surface of both types of spores but no obvious
differences. The transmission electron microscope (TEM) image shows the
internal structure of the spore. The spore wall is composed of outer
shell, outer membrane and cortex from the outside in (Figure 3C, 3D, 3E
\& 3F). The white transparent part of the spore wall, with certain
electron transmittance, is the inner wall of the spore, or the cortex.
Due to the high electron permeability, the inner wall of the dormant
spore forms a thick white zona pellucida, which is significantly thicker
than that of the inner wall of the non-dormant spore, and several times
the difference can be observed. The cytoplasm of non-resting spores is
full of many round, regular and small liposome contents, and there are
also a few glycogen particles with low electron density. As the resting
spores aging process, many cytoplasmic vesicles gradually fuse to form
several large nearly spherical masses (possibly liposome spheres). The
nuclei are crowded to the near edge, and the nuclei are lobulated. There
are still some glycogen-like particles with low electron density in the
edge of the cytoplasm.

\textbf{3 Discussion}

\emph{D. flagrans} is a new species first reported by microbiologist
Duddington in 1948 (Duddington, 1948), which was then recorded as a
spherical structure with a diameter of 24-30 \selectlanguage{greek}μ\selectlanguage{english}m {[}1{]}. There are also
elliptic ones with a size of 15\textasciitilde{}29\selectlanguage{ngerman}×28\textasciitilde{}59
\selectlanguage{greek}μ\selectlanguage{english}m, which is consistent with the observed results in our study. Because
of its thick wall, chlamydospores are thought to be more resistant to
adverse environmental conditions than zoospores and ascospores. In some
species of \emph{Phytophthora} , lipid vacuoles and dense inclusion
bodies similar those found in other resistant spores are present in aged
chlamydospores {[}30{]}. It has been reported that 3 to 10 chlamyspores
of \emph{Phytophthora cinnamomi} were formed at the apex or interstream
of mycelium during in vitro culture. They are formed by the expansion of
the mycelium wall and its protoplasm into the expanded structure
{[}31{]}. Candida albicans and Candida Dublin chlamydospores generally
forms four to five beaded structures. In this study, the formation of
\emph{D. flagrans} chlamydospore is similar that of other fungi
mentioned above {[}24{]}.

The chlamydospore of fungi is considered to be an important survival
structure and a major source of inoculation. \emph{D.
flagrans}chlamydospore is not only important in the survival cycle, but
also an active component of biocontrol preparations and a component of
strain preservation. Due to the widespread structural dormancy of the
chlamydospore, the chlamydospore will become dormant during the
preparation process. This situation precludes the use of the method of
counting yield and quality by viable tests during product testing in the
preparation of this bacterium, as spores cannot germinate on their
normal medium within a short time after structural dormancy is formed.
Therefore, in order to find a simple and effective method for detecting
the product quality, various tests were carried out to prove the
structural differences between resting chlamydospore and dead spore
under the optical microscope. The results showed that the observation
without staining under the optical microscope could not distinguish the
resting spore from the dead spore. The appearance of artificially killed
spores is rather dull, but it is difficult to distinguish it with
natural dead spores in practice by this phenomenon alone, because the
spores have thick walls and normal internal structure cannot be observed
by optical microscopy. Unexpectedly, the cell wall of chlamydospore
remained intact after high temperature and high pressure, and the spore
remained undisturbed and could not be distinguished from live spores.
Although the artificial lethal spores showed light and dark color by the
cotton orchid staining of the fungus staining solution, it was proved
that the dormant, non-dormant and dead spores could not be distinguished
in practice. Trypan blue dye is often used to stain living cells. This
method is used to detect the integrity of cell membranes and determine
whether the cells are alive or not. Because the living cell membrane is
complete, the dye solution cannot enter the cell, while the dead cell
membrane is incomplete and the dye solution can enter the cell. However,
in our study, no matter dormant spores, non-dormant spores or dead
spores could be stained black by trypan blue, which made it impossible
to distinguish between dead and alive spores, possibly because the
permeability of fungal cell wall was different from that of animal cell
membrane. MTT dye can act on mitochondria of succinic dehydrogenase
reduction into blue-purple crystals deposited in cells, dead cells
because of the absence of this enzyme, cannot combine with MTT dye into
blue-purple crystals. In our study, MTT staining was used to incubate
spores at 27\textasciitilde{}30 for 24 h, which could distinguish live
spores (including dormant spores) from natural dead spores, and
artificial death was not stained. Our method is simple and reliable, and
can be used to detect and count the live chlamydospore.

FUN-1 is a specific fluorescent dye for fungal metabolic activity. In
order to detect the metabolic activity and vitality of chlamydospore,
the resting and non-resting spores were used as experimental materials
in this study. The molecular probe FUN-1 was used to detect the
metabolic activity of these spores. The results showed that the
non-resting spores had high metabolic activity, and the resting spores
had no orange fluorescence, indicating that the metabolic activity was
very weak. Studies on Candida albicans and Candida Dublin showed that
chlamydospore of 5 days of culture had high metabolic activity detected
by this probe. After 5 days of culture, the metabolic activity would be
weakened under the condition of nutrient deficiency, and the metabolic
activity could not be detected completely after 30 days of incubation.
Without adding a new medium, the spores could not recover activity after
30 days, indicating that the spores had died {[}24{]}. In our study, the
non-dormant spores were cultured at the age of 25 days, which still
showed strong metabolic activity compared with the spores of Candida
albicans, while the dormant spores showed very weak metabolic activity
but did not die, which was inconsistent with the results of the above
study. The reason may be that Candida albicans is a parasitic fungus in
human body rather than in nature. Its spores have a shorter life span
than \emph{D. flagrans} spores. If \emph{D. flagrans} dormant spore is
subjected to a period of cold or moderate temperature and humidity, or
accompanied by a special phase of external stimulation that induces
increased metabolic activity, it will re-germinate and begin a new life
cycle {[}15{]}.

DAPI is a fluorescent dye that binds strongly to DNA and can adhere to
the sulci region of the DNA double helix. The excitation and emission
wavelengths of the DAPi-DNA complex are 360 nm and 460 nm, respectively,
and because DAPI can pass through intact cell membranes, it can be used
for staining both living and stationary cells. DAPI has a high
photo-bleach tolerance level and can be used to detect chloroplast DNA,
yeast mitochondrial DNA, viral DNA and chromosome DNA. In this study,
DAPI staining results showed that resting spores had stronger blue
fluorescence than non-resting spores, which might be related to the DNA
density of chromatin in the nuclei of resting spores. CFW can bind to
cellulose and chitin of fungal cell wall, and light green fluorescence
can be observed under fluorescence microscope. The results of our study
showed that the non-dormant spores were stained with light green and had
stronger fluorescence, while the dormant spores had weaker fluorescence,
which might be because the non-dormant spores had more contents of
chitin and cellulose than the dormant spores. It was proved that the
types and proportion of cell wall components and their binding forms
were different between the two types of spores, leading to their
different permeability {[}14{]}.

The ultrastructure of different types of \emph{D. flagrans}chlamydospore
in our study has not been reported so far. Some studies on the
ultrastructure of the spores of two species of \emph{M. oryzae} by
scanning electron microscopy showed that the diameter of the
chlamydospore of \emph{M. oryzae} was 3\textasciitilde{}5 \selectlanguage{greek}μ\selectlanguage{english}m, and the
length of the spore was 200\textasciitilde{}500 nm {[}25{]}. The surface
of the dormant spore (black spore) had protuberances. The surface of
non-dormant spore (yellow spore) has protuberances, the height of the
former is 458\textasciitilde{}1130 nm, the latter is
288\textasciitilde{}721 nm, there is a significant difference between
the two. In our study, \emph{D. flagrans} chlamydospore also had
protuberances on its surface, and there was no significant difference
between dormant and non-dormant spores. In addition, the spores with
protuberances are generally found on solid medium, and with the aging of
the verruca has a tendency to increase, in liquid medium culture has not
been found to have protuberances. Under transmission electron
microscopy, Yan \emph{et al.} found that the yellow chlamydospore had a
thin wall, while the black chlamydospore had a thick wall, especially
the inner wall, with a wide white band with high electron transmittance
{[}25{]}, and this finding is consistent with our results on \emph{D.
flagrans} chlamydospore. Under transmission electron microscope, the
yellow chlamydospore (non-resting spore) had complete nucleus, distinct
nucleoli and nuclear membrane, and some starch granules with different
shapes, while the black chlamydospore had no complete cell structure,
and the cytoplasm was filled with a large lipid sphere. However, no
obvious nucleolus or nuclear membrane was seen in \emph{D.
flagrans}chlamydospore under TEM. Perhaps the nondormant chlamydospore
we collected was so old that the liposomes filled the entire cytoplasm
and covered other cellular structures, and this needs further study.
Interestingly, we found that the cytoplasm of the non-dormant
chlamydospore was filled with small circular liposome contents, and the
liposome particles in the dormant chlamydospore further fused and became
larger and less in number, which was similar to the phenomenon observed
in the black chlamydospore.

The morphological and structural observation of different types of
chlamydospore in this study showed that there were differences in the
structure and morphology between resting and non-resting spores, which
were not only reflected in the difference of staining under the optical
microscope, but also, more importantly, reflected in the difference of
ultrastructure. In this study, we found a staining method that can
distinguish dead and dormant spores, which provides a useful strategy
for determining spore survival in future quality detection and other
studies. As for the differences in the ultrastructure of the two types
of spores, some phenomena are still difficult to explain due to our
incomplete work and limited knowledge. From the microscopic level, it
may be reflected in the differences in gene expression and the chemical
composition of spores, which need to be further studied in the future.

The dormant spore in \emph{D. flagrans} is not a ''dead'' state, as was
previously thought, but a living structure in nature. What role do they
play in the history of life under natural conditions remains unclear.
The dormancy and germination of chlamydospore is a complex and
mysterious phenomenon, which probably requires some complex signaling
and regulatory pathways and still needs further study. In this study, it
was shown that after the structural dormancy of the spores, the material
state or cytoplasm of the cells changed, which led to the structural
changes. This study has certain scientific significance for the study of
dormancy mechanism, and the results also provide certain theoretical
support for the application of chlamydospore in the production and
application of biocontrol preparations.

\textbf{Funding}

This work was supported by the Shaanxi University Science and Technology
Association youth Lifting project (20220217), Doctoral Research Project
of Yan'an University (YAU202212288 and YDBK2021-07), Scientific Research
Program Funded by Education Department of Shaanxi Provincial Government
(Program No. 22JK0613), the Yan'an City Key Science and Technology
Research Development Planning Project (No.2020ZCSY-005), and the
Scientific Research Project of Shaanxi Provincial Department of
Education (No.20JG041).

\textbf{Compliance with ethical standards}

\textbf{Conflict of interest statement} The authors declare that they
have no financial or commercial conflicts of interest.

\textbf{Ethics statement} This article does not contain any studies with
human participants or animals performed by any of the authors.

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\textbf{Figure 1} \textbf{Light microscope observation of different
types of chlamydospores.} A. Dormant-spores; B. Non-dormant; C. Dead
spores; D. Dormant spores stained with lactophenol cotton blue; E.
Non-dormant stained with lactophenol cotton blue; F. Dead spores stained
with lactophenol cotton blue; G. Dormant spores stained with trypan
blue; H. Non-dormant stained with trypan blue; I. Dead spores stained
with trypan blue; J. Dormant spores stained with MTT; K. Non-dormant
stained with MTT; L. Naturally dead spores stained with MTT; M.
Artificially dead spores stained with MTT.

\textbf{Figure 2 Observation on metabolic activity, cell nucleus and
cell wall of chlamydospores with fluorescent dye.} A. Dormant spores
stained with FUN-1; B. Non-dormant spores stained with FUN-1; C. Dormant
spores stained with DAPI; D. Non-dormant spores stained with DAPI; E.
Dormant spores stained with CFW; F. Non-dormant spores stained with CFW.

\textbf{Figure 3 Electron microscopic images of dormant and non-dormant
chlamydospores.} A. Dormant spores under SEM; B. Non-dormant sporesunder
SEM; C. Non-dormant spores under TEM; D. Non-dormant spores under TEM
(showing spore walls and cytoplasmic vesicles); E. Dormant spores under
TEM; F. Dormant spores under TEM (showing the spore wall and a part in
the spore).

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