Mycelium from C. Three biological replicates were employed for each setup. Beta-tubulin was used as an endogenous control for normalization. Negative control was employed for each primer pair to eliminate false positive results. While the control group developed into mature fruiting bodies, 1. These results showed that LiCl of higher concentrations had stronger inhibitory effect on C. Mature fruiting bodies were produced in the control group, while the 1. No initiation was observed in the groups treated with higher concentrations of LiCl, in the following 30 days.
The plates treated with 1 mL saturated cisplatin solution had mature fruiting bodies and began autolysis, while the control group only developed into stage-2 primordia. The three delivery methods of LiCl, either mixed in the agar, on the surface of agar, or under the agar, showed no differences and all efficiently inhibited the fruiting body development. LiCl is not sensitive to heat treatment and can be autoclaved to sterilize the solution.
Any of these delivery methods can be chosen in large-scale manufacture of the materials. These results showed an stronger inhibitory effect on C. To demonstrate that GSK-3 inhibitor is also important in fruiting body formation in other mushrooms, the effect of LiCl on P. LiCl was added to PDA medium before autoclave. These results showed an inhibitory effect on P. With the aforementioned positive results that GSK-3 inhibitors can inhibit fruiting body development, we hypothesized that GSK-3 activity is associated with the fruiting body development.
A GSK-3 activator, cisplatin, was then tested for its effect on C. The cisplatin treated group showed an accelerated development since the formation of hyphal knot, and the mature fruiting bodies appeared two days earlier than the control group, which only developed into young fruiting bodies.
These results showed a promoting effect of cisplatin on C. These data support the observation that among fungal species of the order Agaricales, GSK-3 inhibitors inhibit fruiting body formation, whereas GSK-3 activator activates fruiting body formation. Mycelial growth area of the biological triplicates was recorded daily.
Fig 2A shows the average mycelial growth area of each group with error bars showing the maximum and minimum values. As the inoculum usually needs time to adapt to a new environment and absorb nutrients, the mycelium grew only slowly in the first two days. On day 5 and day 6, the mycelium from 1. The results showed that proper concentrations of LiCl would accelerate the mycelium growth. The average mycelial growth area of inoculums on medium mixed with 1.
The error bars encompass the lowest and highest values. This is an ideal property of GSK-3 inhibitor for large-scale production of mycelium-based material. The modified recipe with addition of a proper concentration of LiCl, can not only inhibit fruiting body formation but also speed up mycelium growth, and hence shorten the manufacture cycle and lower the cost.
To investigate the LiCl effect on gene expression in the mycelium stage, the expression levels of GSK-3 and its target genes were tested. The bottleneck to produce living mycelium-based material is to avoid the formation of fruiting bodies.
Initials then develop into stage-1 and -2 primordia, young and eventually mature fruiting bodies. We explored all possible chances in existing production lines to introduce a GSK-3 inhibitor, specifically, LiCl, which is cheaper than the other GSK-3 inhibitors. The previous sections demonstrated that LiCl could be added from pre-inoculation to mycelium extension.
Hyphal knot is a short stage that is difficult to define by naked eyes. So only stages after hyphal knot were tested. As shown in Fig 3 , the addition of LiCl at stages of initial and stage-1 primordium led to the arrest of development. However, stage-2 primordium and young fruiting bodies continued to develop into mature fruiting bodies after LiCl treatment. So, the stages of mycelium, initial and stage-1 primordium are sensitive windows to LiCl. Addition of LiCl at stages of initial and stage-1 primordium inhibited further development.
Addition of LiCl at stages of stage-2 primordium and young fruiting body did not inhibit the fruiting body development. In many instances, one would prefer for a living fungal mycelium to refrain from developing into fruiting bodies so that the mycelium could be easily maintained without concerns of loss in its shape, form, or consistency.
Compared to the current method of heat-killing fungal mycelium to prevent fruiting body formation, a living version of mycelium that simply does not form fruiting bodies is far more desirable, considering its living nature and thus self-healing potential.
Therefore, we suggest the inclusion of LiCl or CHIR trihydrochloride in recipes to manufacture living fungal mycelium-based materials that exhibit controlled fruiting body development. In other cases, promoting fruiting body development may be of interest. For instance, when the intended goal is to produce as many fruiting bodies e. Although cisplatin could accelerate fruiting body formation, more studies about its safety and influence on human health are needed.
LiCl affected the expression levels of GSK-3 and three substrates. In a previous study of C. GSK-3 has important role in cell-fate specification, leading to cell differentiation or apoptosis or development through a number of signaling pathways[ 32 — 35 ]. We proposed that GSK-3 could be the links between environmental stimuli and developmental responses, as a master-switch of fruiting body formation Fig 4.
While GSK-3 is constitutively active under favorable conditions, any unfavorable stimuli could inactivate it and turn off the fruiting body development. The activity of GSK-3 may directly or indirectly determine fruiting body development. GSK-3 could be the links between environmental stimuli and developmental responses, as a master-switch of fruiting body formation.
While GSK-3 is constitutively active under favorable conditions, any unfavorable stimuli could inactivate it and turn off fruiting body development. Hence, the aim of this study is to understand relationship between fruiting body formation and colony pigmentation and vegetative compatibility in C.
Ten single ascospore isolates were derived from in vitro stromata produced from crossing two isolates of C. Among the isolates, isolate numbers. The meeting lines between the isolates on SDAY agar plates were observed. The isolates were also inoculated in brown rice medium for fruiting body formation, as previously mentioned. Single ascospore isolates of C. In wild, the color of C. White colonies have also been reported, though much less in numbers, in C.
Here, it can be noted that colony pigmentation does not correlate with the fertile fruiting body production. In this experiment, none of the crossings involving white and yellow isolates produced perithecial fruiting bodies, while crossings involving only white isolates or yellow isolates produced fertile fruiting bodies. However, synnemata production was very frequent.
For synnemata production, crossing was not necessary; rather it was even detrimental. Isolates 1 and 6, and 5 and 8 produced synnemata while inoculated in single, but their crossings did not produce any synnemata at all Figs. Similarly, isolate 7 in single produced synnemata, but crossing with isolate 2 did not enhance synnemata production Fig. None of the crossings produced perithecial fruiting bodies.
In this study, vegetative compatibility was assessed by observing the growth patterns at the meeting line between two isolates inoculated on opposite sides of agar plates. The isolates overlapped each other and induced thicker mycelial growth at the meeting line, showing vegetative compatibility between these isolates Fig.
Colony pigmentation did not show any correlation with vegetative compatibility. White isolates showed both vegetative compatibility and incompatibility between them, similar to yellow isolates Fig. A, A distinct line could be observed between two isolates; B, No distinct line was observed between the isolates. Further, mycelial growth was enhanced at the meeting line.
Thus, it could be said that vegetative incompatibility can induce fertile fruiting body production, while vegetative compatibility does not guarantee it.
Further studies are necessary to understand the nature of vegetative compatibility in C. A, Fruiting body formation from vegetative incompatible isolates; B, Fruiting body formation from vegetative compatible isolates. Although C. Also, synnemata production is much more common than perithecial stromata production in C. For example, powdery mildew forms white, powdery spores called Oidium imperfect stage.
It also forms a resting stage that is a perfect stage of the fungus, such as Erisyphe. You can see this as the black, pinhead-sized structures on a leaf with white, powdery growth of powdery mildew. How do we identify fruiting bodies? It is not necessary to know the fungal names or even whether the stage is imperfect or perfect. The first step to disease and fruiting body identification is to use reference books that describe the disease on your host plant. When the fruiting body is listed as an example as a pycnidium or an acervulus, pathologists know what to look for on the plant.
The most significant structures in fungal ID are spores, fruiting bodies, and sometimes mycelium. The poisonous Amanita muscaria fly agaric is recognizable by its bright red cap with white patches.
Pigments in fungi are associated with the cell wall. They play a protective role against ultraviolet radiation and can be toxic. The poisonous Amanita muscaria : The poisonous Amanita muscaria is native to temperate and boreal regions of North America. The rigid layers of fungal cell walls contain complex polysaccharides called chitin and glucans.
Chitin, also found in the exoskeleton of insects, gives structural strength to the cell walls of fungi. The wall protects the cell from desiccation and predators. Fungi have plasma membranes similar to other eukaryotes, except that the structure is stabilized by ergosterol: a steroid molecule that replaces the cholesterol found in animal cell membranes. Most members of the kingdom Fungi are nonmotile. The vegetative body of a fungus is a unicellular or multicellular thallus.
Dimorphic fungi can change from the unicellular to multicellular state depending on environmental conditions. Unicellular fungi are generally referred to as yeasts.
Example of a unicellular fungus : Candida albicans is a yeast cell and the agent of candidiasis and thrush. This organism has a similar morphology to coccus bacteria; however, yeast is a eukaryotic organism note the nucleus. Most fungi are multicellular organisms. They display two distinct morphological stages: the vegetative and reproductive. The vegetative stage consists of a tangle of slender thread-like structures called hyphae singular, hypha , whereas the reproductive stage can be more conspicuous.
The mass of hyphae is a mycelium. It can grow on a surface, in soil or decaying material, in a liquid, or even on living tissue. Example of a mycelium of a fungus : The mycelium of the fungus Neotestudina rosati can be pathogenic to humans. The fungus enters through a cut or scrape and develops a mycetoma, a chronic subcutaneous infection.
Most fungal hyphae are divided into separate cells by endwalls called septa singular, septum a, c. In most phyla of fungi, tiny holes in the septa allow for the rapid flow of nutrients and small molecules from cell to cell along the hypha. They are described as perforated septa. The hyphae in bread molds which belong to the Phylum Zygomycota are not separated by septa. Instead, they are formed by large cells containing many nuclei, an arrangement described as coenocytic hyphae b.
Fungi thrive in environments that are moist and slightly acidic; they can grow with or without light. A bright field light micrograph of c Phialophora richardsiae shows septa that divide the hyphae.
Like animals, fungi are heterotrophs: they use complex organic compounds as a source of carbon, rather than fix carbon dioxide from the atmosphere as do some bacteria and most plants. In addition, fungi do not fix nitrogen from the atmosphere.
Like animals, they must obtain it from their diet. However, unlike most animals, which ingest food and then digest it internally in specialized organs, fungi perform these steps in the reverse order: digestion precedes ingestion. First, exoenzymes are transported out of the hyphae, where they process nutrients in the environment. Then, the smaller molecules produced by this external digestion are absorbed through the large surface area of the mycelium.
As with animal cells, the polysaccharide of storage is glycogen rather than the starch found in plants.
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