Sclerotia Formation of Phlebopus Portentosus in Wild and Artificial Cultivation


 Phlebopus portentosus is a favorite wild edible mushroom in the Xishuangbanna region, Yunnan, China and in northern Thailand. This bolete has a unique biotrophy. It can be saprobic but also form a tripartite association with root mealy bugs and plants. P. portentosus is the only edible fungus of Boletales in the world that can be cultivated artificially and anniversary produced at present. Sclerotium is often found at its natural environment and cultivated media, but the regularity and growth characteristics of the sclerotium are unknown. In this study the whole process of birth, growth, death and rebirth of the sclerotium of P. portentosus at the national and lab conditions was reported for the first time. The sclerotium formation in the nature is related to adversity, such as reduced rainfall and low temperature. The more rainfall, the less sclerotia. It seems that the lower temperature increased the sclerotium formation, however the relationship of the sclerotium formation to temperature was not obvious as the rainfall. Under artificial conditions the sclerotium formation of P. portentosus is related to the fungus physiological maturation, and the sclerotium occurrence always accompanied by appearance of the water drops on the colony. The result will set up a platform for research on importance of the sclerotium in life circle of P. portentosus.


of the sclerotium in life circle of P. portentosus.
Sclerotium is a firm, frequently rounded resting body of fungal hyphae, which differentiated into a rind and a medulla 1 . It can give rise to a fruiting body, a stroma or mycelia 2 . The sclerotium can play a significant role in the fungal life cycle, such as overcoming adverse conditions and rapidly colonizing nearby substrates when favorable conditions return [3][4][5] . Normally the sclerotium is small, However, some edible or medicinal fungi, such as Poria ccos, Cordyceps sinensis and Grifola umbellata can develop big tuber-like sclerotia, which are harvested as food or medicine [6][7][8][9] . Recent success of morel cultivation showed that the morel sclerotia played a key role during production of its fruiting bodies. That is the sclerotia development is an essential stage for the producing morel fruit body [10][11][12][13][14] .
Phlebopus portentosus (Berk. and Broome) Boedijn is a delicacy in the tropical regions of China and Thailand. It is placed within the Boletinellaceae 2,15 . This mushroom is extremely popular and sold at ¥60-100/kg (US$ [9][10][11][12][13][14] in the Xishuangbanna region of Yunnan 16,17 . Harvesting and trading the mushroom is an important means of livelihood for the local people. In recent years the production of the mushroom has declined due to uncontrolled commercial harvesting. Research on cultivation of P. portentosus has been carried out at the Yunnan Institute of Tropical Crops of Yunnan, China since 2003.
Technologies for the cultivation of P. portentosus in mushroom houses and by field inoculation have been developed 18,19 . During our research, the unique biotrophy of P.
portentosus has been gradually unveiled. This mushroom can live a saprophytic lifestyle [20][21][22] . However, it usually has a symbiotic association with soil mealy bugs, forming a special insect gall on plant roots [23][24][25][26] . In addition, sclerotium has often been discovered abundantly at its filed soils and artificial media. It is understandable that P. portentosus will produce the sclerotium under low-temperature or in dry seasons in the natural environment, which is a stress response to the harsh environmental conditions. When the temperature and moisture were well controlled within the suitable ranges in the process of artificial cultivation of the mushroom 16,27,28 , however, a large number of sclerotia was still developed. This phenomena inspired us to study the interesting issue.
In this paper, the microscopic change process of Phlebopus portentosus sclerotia formation under field and artificial culture conditions was analyzed and established. The microscopic changes of Phlebopus portentosus from mycelium growth, mycelium kink, sclerotia formation, maturation, germination and senescence were clarified, which provides a platform to ascertain the importantce of sclerotium formation in the cultivation of P. portentosus.

Results
Formation of sclerotium in the field. When the environmental conditions were getting non-conducive (lower temperature or less rainfall, or both) to the fungal growth, the fungal mycelia and rhizomorphs in the soil began to converge and tangle (Fig. 1a), and then a pale yellow, globose or irregular soft hyphal ball formed (Fig. 1b). As the hyphal ball gradually grew bigger its tissue became tighter and darker, and dense air hyphae developed on the surface (Fig. 1c). At this stage the ball case was soft, rough and covered by rhizomorphs (Fig. 1d), and its contained dark brown honey-like juicy inside (Fig. 1e).
Finally, the hyphal ball solidified and separated from surrounding hyphae becoming a matured sclerotium. The matured sclerotium was solid, dark, glabrous, vein-like rhizomorphs scattered on the surface (Fig. 2a). Its peridium and internal tissues (pith) have differentiated. The peridium was made from dark hyphae, and the internal tissue was made from dense interwoven hyphae, wax-like, grayish to brownish with brown spots (Fig. 2b). The sclerotium normally produced singly (Fig. 2c), rarely beads-like (Fig. 2d), can be globose, subglobose or irregular, 1.42~14.98 mm in diam. Seasonal dynamics of sclerotium occurrence in the field. In the field, the sclerotium can be developed year around except July and August when they have good temperatures and adequate precipitations. From the December through the April of the following year when the temperature became lower and the rainfall was scarce, sclerotia were produced abundantly in the soil ( Table 1, Fig 3). From the December through the February of the following year both young and matured sclerotia were discovered simultaneously. From the March through the June only matured and aging sclerotia were observed. After the April the sclerotia started to geminate as the temperature and rainfall gradually increased, and then amount of sclerotia discovered was gradually declined. In the meantime a few mushrooms of P. Portentosus emerged. From the May through the June a few matured and aging sclerotia were still existing in the soil.  Table 1. Seasonal dynamics of sclerotium formation from 2017 to 2018. Aging and died young of sclerotium in the field. Sclerotia dried out becoming shriveled when the climate was extremely dry and the soil was short of water ( Fig. 4a).
Their surfaces obviously wrinkled or became unevenness. At this moment the hyphal growth and accumulation of nutrients in sclerotia were interrupted due to water loss. The consequence is that the sclerotium became hollow and its peridium dried out becoming fragile, and finally ruptured to death (Fig. 4b, c). remained only the withered peridium left from the dead sclerotium (Fig. 4d). Amount of the dead sclerotia increased in the soil, as the dry weather continued, especially in the upper soil layer (0-10 cm). However, some of the matured sclerotia could survive during the difficult period even although they almost dried out. When the dried matured sclerotia were put in a petri dish lined with moistened double filter paper for 24 hours they could win rebirth.

Germination of sclerotium in soil.
When the temperature and moisture of soil reached the optimum conditions the survived sclerotium began to germinate. New mycelia developed from a single or multiple points of the sclerotium (Fig. 5a). The mycelium extended into the soil and the entire sclerotium was gradually surrounded by dense mycelium (Fig. 5b). As more and more mycelia continued to grow out from the sclerotium its accumulated nutrients were consumed and run out, and the sclerotia eventually disappeared from the soil. And then a new colony of P. portentosus formed, which fruiting bodies produced from the colony when the environmental conditions became suitable. Culture of sclerotium in the lab. The 3 isolates of P. portentosus grew well on the agar medium and produced round colonies. When the colonies started developing cotton woollike tangled interwoven hyphae a few small transparent liquid drops would appear on their surfaces (Fig. 6a). More liquid drops developed as the fungal colonies grew further and they became bigger and darker. And then a mass of curly, fluffy tangled mycelia knots raised up from the surface of colony (Fig. 6b). They soon developed into dense mycelia balls and had a lot of liquid drops on their surfaces. They were baby sclerotia ( Fig. 6c)。At this stage the peridium differentiated from its juicy internal tissue (Fig. 6d).
As the sclerotium grew up, the interior tissue solidified due to nutrient accumulation and hyphal intensive growth (Fig. 6e). And then the sclerotium became harder and harder and the color of the liquid drops turned dark red brown (Fig. 6f). At this stage fluffy hyphae disappeared from the hardening peridium surface and a few dark pits appeared (Fig. 6g).
The interior tissue was composed of fresh hyphae with more nutrients accumulated, juicy, brown to dark brown (Fig. 6h). Most of the sclerotia were globose to subglobose when young and became irregular cluster shapes due to adjacent sclerotia fused together, which could grow up to 20 mm long or more (Fig. 6g).

Discussion
The whole process of the sclerotium formation of P. portentosus at both the national and lab conditions was reported for the first time. At the national conditions the sclerotim occurrence was closely related to climate seasonal dynamics, especially to the rainfall (Fig. 3). More sclerotia were discovered in the drier months, December through April of the following year, that is the dry season in the Xishuangbanna region. During the dry season the rainfall was lower than 50 mm/month. When the rainfall is over than 100 mm/month, the amount of sclerotia discorvered was lower than 15/month. The more rainfall, the less sclerotia. It seems that the lower temperature increased the sclerotium formation. However the relationship of the sclerotium formation to temperature was not obvious as the rainfall. As can be seen, in the nature the sclerotium formation is an important strategy of the fungus, P. portentosus for overcoming the unfavorable conditions to survive.
It is not easy to distinguish the different kinds (formation stages) of sclerotia, especially the died from shriveled but still alive ones. After the sclerotium germination test was done we recognized that the died sclerotiun could not germinate but the shriveled germinated well under the suitable conditions. No any residues were left from the germinated shriveled sclerotia. However, when the sclerotium died out it left the dried out peridium in the soil (Fig. 4).
On the agar medium all the 3 isolates produced abundant sclerotia under the optimum conditions. It means that the sclerotium formation under artificial cultivation is not related to the unfavorable temperature or water conditions. The sclerotium occurrence always accompanied by appearance of the water drops on the colony (Fig. 6). The fungal colony on the agar medium produced water drops known as "spitting water",which is a common phenomena when it reached the reproductive stage from vegetative stage 4,30 .
The results of recent research on Morchella cultivation indicated that occurrence of fruiting bodies followed the sclerotium formation, which accompanied by the water spitting 31,32 . Morchella spitting water was due to the changes of cytoplasmic movement