Morphological Characteristics of Hyphal Interaction between Grifola umbellata and its Companion Fungus

Morphological characteristics of hyphal interaction between Grifola umbellata (Pers. Ex Fr.) Pilat and its companion fungus which related to sclerotia formation from hyphae were investigated by external observations, light microscopy and transmission electron microscopy (TEM). External observations showed that a dense antagonism line was formed by both G. umbellata and companion fungus after their hyphae contacted each other in dual culture. Many hyphal strands emerged on the colony of G. umbellata and differentiated to sclerotia from where hyphal strands crossed. Light microscope observations revealed the process of antagonism line formation. Mature antagonism with structural differentiation, was composed of three main layers: the rind, the rind underlayer and the hypha layer. TEM observations showed that after colonies hyphal contact, a series of reactions always occurred in both G. umbellata and companion fungus. Cells in the center of antagonism line were dead. Cells of G. umbellata adjacent to the antagonism line were usually large and hollow, with unilateral thickened wall, whereas those of companion fungus were empty, with thin or thick wall. Both hyphal interaction at the antagonism line may be one of the main reasons for sclerotia of G. umbellata differentiation from hypha.

Morphological Characteristics of Pseudosclerotia of Grifola umbellata in In Vitro

The present study was carried out to investigate morphological characteristics of pseudosclerotia of Grifola umbellata formed by artificial cultures. Isolate G. umbellata DUM GUS-01 was obtained from sclerotium cultivated in field. The fungal isolate was cultured on PDYM broth, PDYMA(potato dextrose yeast malt agar) and oak sawdust media at 20degrees C under the dark condition. G. umbellata DUM GUS-01 showed a volumetric increment of fungal lumps rather than mycelial growth. Particularly, G. umbellata DUM GUS-01 produced a large amount of melanin pigments in all culture treatments. The color of the fungal mass has been changed into grey gradually, and then formed melanized rind-like structure on its superficial part. The fungal structures which were covered with melanized rind-like layer were named as pseudosclerotia of G. umbellata. The pseudosclerotia of G. umbellata DUM GUS-01 formed a new white mycelial mass, which was swollen out of the melanized rind structure for its volumetric increment. When the pseudosclerotia were sectioned, their structure was discriminated from two structures such as a melanized rind-like structure layer formed by aggregation of aged mycelia and a white mycelial mass with high density. As results of scanning electron microscopic examination, the pseudosclerotia of G. umbellata DUM GUS-01 which were formed in in vitro conditions were similar to the sclerotia of G. umbellata cultivated in natural conditions except for the crystals formed in medula layer of natural sclerotia. Although size, solidity of rind structure and mycelial compactness of pseudosclerotia were more poor than those of natural sclerotia, the morphological structure and growth pattern of pseudosclerotia were very similar to those of natural sclerotia. Therefore, it is probable to induce pseudosclerotia to sclerotia of G. umbellata in in vitro conditions. Consequently, it seems that the induced pseudosclerotia can be used as inoculum sources to substitute natural sclerotia in field cultivation.

A New Method for Cultivation of Sclerotium of Grifola umbellata

Sclerotia of Grifola umbellata were cultivated by two methods such as burying and root inoculation methods. The sclerotia of G. umbellata produced by the burying method were 6.0~6.8 x 3.4~4.6 x 1.8~1.9 cm (Width x Length x Thickness) in size and 17.3~19.6 g in weight, respectively. Their increase rate was 1.10~1.12 times. On the other hand, the sclerotia cultivated by the root inoculation method were 18.3~31.5 x 12.5~26.4 x 3.1~3.7 cm (Wx L x T) in size and 219.1~576.6 g in weight, respectively. Their growth increment was 11.18~39.77 times. The rhizomorphs of Armillaria mellea were developed with a high density under fallen leaves layer covering cultivation site, and distributed mainly between soil surface and soil depth of about 10 cm as well as colonized prominently on the inoculated wood logs. Fungal interaction between G. umbellata and A. mellea were observed mainly in the stage of white sclerotium of G. umbellata. The sclerotia of G. umbellata which were developed newly and harvested in the root inoculation method were twined with root hairs of host tree and rhizomorphs of A. mellea. The sclerotia of G. umbellata decomposing root hairs of host tree were confirmed through SEM examination. Physiochemical characteristics of soil in all cultivation sites had no significant differences. Soil pH were in the range of pH 3.98~4.40. Organic matters were the range of 17.97~23.86% and moisture contents of soil were 12.00~18.20%. Soil temperatures showed 12.9~13.8degrees C in November and 22.0~23.9degrees C in August, respectively. In conclusion, the root inoculation method seems to be a practical method for cultivating sclerotia of G. umbellata due to its many advantages such as simplicity of inoculation process, shortening of cultivation periods and facility of harvest.

Sclerotial Development of Grifola umbellata

Sclerotial development of Grifola umbellata (Pers. : Fr.) Donk was investigated through microscopic examinations. The sclerotium of G. umbellata was bumpy and rugged, multi-branched, and dark-brown to black in color. The sclerotial development of G. umbellata was categorized into three stages such as sclerotial initial, development and maturation. Sclerotium development was initiated as the white fungal mass. The superficial part of white sclerotium changed into gray, light brown and then black as its development proceeded further. As a distinctive characteristic of this fungus, a large number of crystals were observed in the medulla layer of sclerotium during its maturation. For development of new sclerotium, G. umbellata formed a white sclerotial primordium on the matured sclerotium. Development of sclerotium in G. umbellata was intimately associated with rhizomorphs of Armillariella mellea and the developing sclerotia were often penetrated by rhizomorphs of A. mellea into medulla layer.

THE PHYLOGENETIC RELATIONSHIPS OF GRIFOLA UMBELLATA AND ITS COMPANION FUNGUS: EVIDENCE FROM ITS SEQUENCE ANALYSIS / 微生物学通报

The sequences of 5.8S rDNA and the flanking internal transcribed spacers (ITS1 and ITS2) were sequenced from hypha, fruit body and sclerotia of Grifola umbellata and its companion fungus. Their ITS sequences similarity was 99.36%. The results suggested that G. umbellata was closely related to its companion fungus.

PRODUCTION OF ACTIVE OXYGEN SPECIES AND CHANGES OF ENZYMES IN GRIFOLA UMBELLATA INDUCED BYARMILLARIA MELLEA ELICITOR / 微生物学通报

The changes of active oxygen species and some enzymes in Grifola umbellata induced by Armillaria mellea elicitor were studied.The results showed that active oxygen species appeared in both mycelia and sclerotia of G.umbellata after treated with A.mellea.There were two phases of active oxygen production upon addition of A.mellea elicitor.Phase I occured at 10 minute after addition of A.mellea elicitor.Phase Ⅱ occurred about 90 minute.The changes of some enzyme activity were also studied in this paper.Compared with control,the A.mellea elicitor could reduce the activity of superoxide dismutase and peroxidase.The catalase activity changed only little.The phenylanine ammonia lyase activity declined in the early stages and then increased in the late stages.