The Identification And Phylogenetic Relationship Of Rare Pathogenic Fungi Based On The Mitochondrial Cytochrome B Gene

In recent years, the tendency of the morbility and mortality in systematic fungal infections has been raised with the increase of immunologic deficiency people which causes by the abusage of medication; the development of organ transplantation and treatment technic; and fundamental diseases. Simultaneously, the style of fungal infection and the species of pathogenic fungi has also been changed. Results from the rapid progress of fungal infections, difficult diagnosis, lack of safe and higher efficiency antifungal drugs, the treatment of fungal infection becomes extraordinary difficult and the mortality and fatality are getting increased. Hence, the research of pathogenic fungi has become one of the attractive aspects in the medical fields.Recently, some opportunistic infections caused by Geotrichum sp. (Ge) and Graphium sp. (Gr) have been occasionally reported. Ge. candidum, Ge. clavatum and Ge. capitatum are more common than other Geotrichum species. They can cause geotrichosis of skin, tunica mucosa and internal organs. The most common known species of the genus Graphium are Gr. eumorphum, Gr. fructicola and Gr. putredinis. They may cause infections of subcutaneouly and deep tissue. The morphological character of Geotrichum is similar to Trichosporon, Malbranchea, Coccidioide, Moniliella, Scytalidium, Oidiodendron and Arthrographis. And Graphium is similar toPseudallescheria,Petriella,Ceratocystis,Pesotum,Phialographium and Ophiostoma. This makes it difficult to identify the species of Geotrichum and Graphium. That takes trouble to the related research. Thus, gene analysis is necessary to make the correct identification and classification of them.At present, some researchers overseas had used methods to study the interspecies and intraspecific gene polymorphism of Geotrichum sp. and Graphium sp., such as microsatellites PCR, RAPD-PCR, isozyme analysis, karyotype analysis, and so on. This research makes mitochondrial cytochrome b (cyt b) gene as target gene because it is a functional gene which is discribed as molecular clock. The analysis of mitochondrial cytochrome b gene has conquered the shortage of that many targets gene such as ITS, 18S or 28S rDNA which can not be used for classification, identification and phylogenic evolution at the same time. As the new target, cyt b gene can show more genetic information.This research will study the classification, identification of Geotrichum sp., Graphium sp. and related species bases on cyt b gene and DNA sequence analysis and make sure the phylogenetic relationship of them. The related reports of this study had not been seen.First we cultured the tested strains on PDA medium at 25℃, and observed the characteristics of the colony and morphology. Geotrichum strains grow rapidly and produce white, cottony or powdery, a little dry or cream, slimy, smooth and wet colony. The hyphae have abundant septa and branches. The fragmentation of undifferentiated hyphae produces arthrospores which are unicellular, in chains, rectangular or doliform in sharp and with the characteristic of tight connection between two arthrospores. Graphium coloniesgrow moderately rapidly and pitchy, brown woolly to cottony, a little dry or wet. The hyphae are septate and conidiophores are simple, long, mostly straighten at the alongside. And we also can see synemmata which are bundles, tightly arranged of erect hyphae. Rhizoid-like structures may be observed at the base of the synnema. On the top of each synnema clusters one-celled, oval or claviform conidia.Second, we extracted the DNA from the hypae, then, PCR using the universal primer of fungi with heat start. Geotrichum sp. and Graphium sp. both showed one band (almost 420bp). Based on the related sequence got from GenBank, we compared the homology and got the information that Geotrichum sp. was 399bp and Graphium sp. was 402bp. According to the mitochondrial genetic code of yeast fungi, we gained the corresponding amino acid sequence that Geotrichum sp. was 132aa and Graphium sp. was 133aa. We constructed the phylogenetic trees separately based on the DNA and amino acid sequence using UPGMA and found the same species can be classfied to different group, while the different species are in the same group. If the sequences of strains are the same, we concluded that they are the same species. It is shown that some different species has the same sequence, while some same species has the different sequence. Though the sequence of DNA is different, the amino-acid sequence may be the same.The shorter the branches of the phylogenetic tree are, the closer is the relationship, on the other hand, The longer the branches of it are, the further is the relationship.The DNA and amino-acid sequence of Geotrichum is 399bp and 132aa, Graphium is 402bp and 133aa. The sequence analysis of Geotrichum contains 6 DNA types and 4 amino-acid types in 20 strains of 5 species. Ge. candidum andGe. pseudocandidum are in high similarity and their genetic relationships are close. The relationship between Ga. citri-aurantii and other Geotrichum sp. is farther. The phylogenetic tree of Graphium contains 20 DNA types and 14 amino-acid types in 29 strains of 13 species. It is different with 8 strains of Gr. penicillioides, and the genetic relationship is fairly closed among Gr. eumorphum, Gr. cuneiferum, Gr. fructicola, Gr. putredinis and Gr. tectonae.The morphological classification and identification is very confused, and different from molecular biology methods. The morphology can not act as the only standard of identification. We need gene analysis to make the morphological classification and identification correct and consummate, and make further research of the strains difficult to identify. We made sequence alignment with Geotrichum sp., Graphium sp., related species and the common species in clinical infections, and had gained the partial sequences of genus-specific nucleotides. With this sequence, we can design the specific primer of probe using in rapid identification and diagnosis.In summary, the analysis of cytb gene sequences can make the morphological classification and identification correct and consummate. Sequence alignment can show the genus-specific or interspecific difference, which will be good at the rapid identification of pathogenic fungi, diagnosis and therapy of fungal infections and research of mechanisms.