3 Discussion
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 μm [1]. There are also
elliptic ones with a size of 15~29×28~59
μm, 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 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 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 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. D. flagranschlamydospore 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 27~30℃ 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 D. flagrans spores. If 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 D. flagranschlamydospore in our study has not been reported so far. Some studies on
the ultrastructure of the spores of two species of M. oryzae by
scanning electron microscopy showed that the diameter of the
chlamydospore of M. oryzae was 3~5 μm, and the
length of the spore was 200~500 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 458~1130 nm, the latter is
288~721 nm, there is a significant difference between
the two. In our study, 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 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 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 D. flagranschlamydospore 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 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.