Phylogenetic analysis of dematiaceous fungi isolated from the soil of Guangdong , China

1 Department of Dermatology, The Fifth Affiliated Hospital, Sun Yat-Sen University,Guangdong, Zhuhai 519000, China. 2 Department of Dermatology, The Second Affiliated Hospital, Sun Yat-Sen University,Guangdong, Guangzhou 510120, China. 3 Department of Endocrinology, The Fifth Affiliated Hospital, Sun Yat-sen University, Guangdong, Zhuhai 519000, China. 4 Zhuhai Entry-Exit Inspection and Quarantine Bureau of PRC, Guangdong, Zhuhai 519015, China.

Dematiaceous or darkly pigmented fungi are uncommon causes of human disease but can be responsible for life-threatening infections in both immunocompromised and immunocompetent individuals (Revankar and Sutton, 2010;Schell, 1995).They are a heterogeneous group.The distinguishing characteristic common to all these various species is the presence of melanin in their cell walls, which imparts dark color to their conidia or spores and hyphae.The colonies are typically brown to black in color as well.Dematiaceous fungi are commonly found in the soil and generally distributed worldwide (Montenegro et al., 1996;Dixon et al., 1980;Lopez et al., 2004).They are the etiologic agents of phaeohyphomycosis, chromoblastomycosis and mycetima.Over 100 species and 60 genera of dematiaceous fungi have been implicated in human disease (Matsumoto et al., 1994).
As these diseases usually occur by the penetration of the causative agent through skin wounds, it is significant to search for the agents in nature, clarify their habitat and the environmental circumstances in which they may infect man.Agents (Dixon et al., 1980;Yegres et al., 1991) have been isolated such as Phialophora spp., Cladosporium spp., Exophiala spp., Sporothrix sp., Wangiella dermatitdis, Bispora betulina, and Scytalidium lignicola, which demonstrated the presence of pathogenic dematiaceous fungi in nature, although the identity of most of these strains has not been verified by molecular data.Nishimura (Nishimura, 1994) and Nishimura et al. (Nishimura et al.1989) investigated the ecology of pathogenic fungi in natural and living environments in Colombia, Venezuela, Brazil, China and Japan and succeeded in isolating various species of pathogenic dematiaceous fungi including Fonsecaea pedrosoi, Phialophora verrucosa and Exophiala spinifera.They did not find the fungus Cladophialophora spp., which are the mainly causative agents of chromoblastomycosis in China.
The taxonomy and identification of dematiaceous fungi are difficult due to a lack of phenetic characters and high degree of morphological plasticity.In the present study, we isolated 60 dematiaceous fungal strains out of 367 soil samples; these were further identified by molecular biological method.If the phylogenetic relationship and the geographical distribution of dematiaceous fungi from soil of Quangdong, PR china, are revealed, it will be useful for future study.

Sample collection
There are three climatic zones in Guangdong: the central subtropical (Nanxiong, Lianshan, Lianxian and Shaoguan), the sourthern subtropical (Yingde, Meixian, Shantou, Guangzhou, Yangjiang), and the northern tropical (Zhanjiang, Xuwen).365 samples were collected from the three climatic zones where four to ten collecting sites were set up randomly (Table 1).The work was done during autumn and winter (October 2006 to January 2007), the dry season in Guangdong.Samples were collected from the surface soil upward of 15 cm.Utilizing a spoon which was rinsed with sterile water after each use, approximately 25 g sample were placed in 100 ml plastic bottles containing a small crystal of paradichlorobenzene (for arthropod control), and returned to the laboratory for processing on the same day.

Isolation of dematiaceous
3 g of soil were transferred to a sterile 15 ml glass tube; 10 ml of sterile saline were then added, mixed by agitation for 1 min and set for 20 min, after which it was diluted to the concentration of the proportion of 1:100, when 0.2 ml was collected from the middle part of the soil suspension and placed on two plates.Then, the solution was poured on two media; potato dextrose agar (PDA) Rose Bengal respectively, both containing antibiotics (50 mg of chloramphenicol, 10 6 units of penicillin, 200 mg of streptomycin and 200 mg of cyclohexamide per liter).Pulled a medium to 3 plates, sealed the plates with plastic film, left a hole of 3 to 5 mm.The plates were incubated at 26°C for 2 to 3 weeks.The suspected colonies were subculture on Sabouraud dextrose agar (SDA) at 25°C and cheeked grossly and microscopically.All isolates were further evaluated by molecular biology methods.
Ultrapure water was added to increase the volume to 50 μl.Each reaction mixture was heated to 95°C for 4 min, followed by 30 cycles of 94°C for 30 s, 55°C for 30 s, and 72°C for 60 s., followed by incubation for 10 min at 72°C.

Direct sequencing and phylogenetic analysis
Direct sequencing of PCR products was done with an ABI PRISM 3100 sequencer (ABI, America) after labeling with BigDye Terminator Cycle Sequencing Ready Reaction (Applied Biosystems, Foster City, California).The ITS sequences of reference sequence from GenBank collection (Table 2) and isolated dematiaceous fungal (Table 3) in this study were aligned by using Clustal W software.Phylogenetic tree was then constructed by the neighborjoining (NJ) method in the Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0 (Tamura, 2007).Bootstrap analysis with the MEGA program was performed by taking 500 random samples from the multiple alignments (values > 50 are shown with the branches).The evolutionary distance between organisms is indicated by the horizontal branch length, which reflects the number of nucleotide substitutions per site along that branch from node to the endpoint.
The rDNA ITS regions (including part of 18S, ITSⅠ, 5S, ITSⅡ, part of 26S) were successfully amplified from all the dematicacious fungi by universal primers.The size of acquired PCR products ranged from 500 bp to 650 bp.Each strain of dematicacious fungi tested was shown to have unique ITS base sequences, although some of them were very similar.

DISCUSSION
Studies of an infectious disease usually approach from pathogens, so isolations and identificatons of pathogens are the most important parts in the approach.The usual identification of fungi by the morphological method, combining with some biochemical approaches, processes the observation of colonial textures, shapes, and colors in culture mediums, and the inspection of conidiophores, morphologies and generations of conidia.However, it is difficult because many dematicacious fungi appear in multi-morphologies, which the isolate may generate more than one kind of conidium or may be generated by various conditions.Therefore, it is not easy to determine whether a conidium is yielded by the multi-morphological fungi or by a mixture of fungi.To obtain an isolate with high purity, it requires sub-cultures from the similar culture medium.Even with the required conditions of subculture, some colonies are complicated to isolate.Furthermore the members of mitosporic fungi are taxonomically closely related, morphological identification of mitosporic fungi becomes more difficult.Early studies have shown that the results from the molecular biological identification of fungi are in accordance with of the morphology identification (Pechere et al., 1999).In this study, a series of colony complexes are involved.We identified all the strains by the molecular biological method and processed the morphology method to classify some isolates.Results indicated that dematiaceous species were found widely in Guangdong soil.However, the distribution amounts were not in a pattern.Defined by climatic zones, the western species of the southern subtropical are with the highest abundance and density; the eastern species of the southern subtropical are with the lowest abundance and density.The most found genus was Scolecobasidium.There were 17 samples found and it 28% (17/60) of the positive results.There is no detailed report found on its pathogenicity.Ten strains of Cladophialophora were found from 10 samples, which was 17% (10/60) of the positive results.Among them, the isolates of Cladophialophora carrionii were from the garden of Yuanshan Qingyuan.It has been the first time that C. carrionii which is the common pathogen for chromoblastomycosis in China was isolated from samples of Guangdong environment.Seven strains of Exophiala were recognized, including E. dermatitidis, E. xenobiotica, E. oligosperma, E. pisciphila, E. mesophila, Exophiala sp., E. eucalyptorum.Except E. eucalyptorum, the rest strains were common pathogens.Phaeococcomyces is a member of black yeast, which is hard to identify because its confusion on the morphology.Five strains were found from the experiment, but the species was not determined.Two isolates of Phialophora parasitica were found.There are 25 members in Phialophora of which five species are human pathogens, including P. verrucosa, P. richardsiae, P. repens, P. parasitica, and P. cyanescens (Park et al., 2005).The common pathogen P. verrucosa of chromoblastomycosis was not found in this experiment which may be because it intends to distribute in the cold zone (Liu et al., 2004).Cladosporium, dispensing widely, is the saprophyte found usually in soil and on plants, and is also the pathogen for plants.Some of Cladosporium are relative to human infections (Chew et al., 2009;Gugnani et al., 2006).Seven strains were found in this experiment, including four strains of C. oxysporum, two strains of C. cldosporioides, and one strain of C. sphaerospermum.The three species can cause human phaeohyphomycosis.C. oxysporum presents in the warm condition (Mckemy and Morgan, 1991).It is compliant with the four strains that have been from farms of Zhuhai and Zhanjiang Leizhou.The locations are warmer than the Chinese lettuce field in Meizhou from which the mould of the C. sphaerospermum strain is Meizhou is in the further northern region.One strain of D. bryoniae, plant pathogen, was isolated.Some studies have shown the fungi of Didymella are related to asthma (Pulimood et al., 2007), because the amount of the fungi increases during the storm season, which may lead to asthma.
There is no human disease reported for the rest eleven findings, including two strains of S. suttonii, one strain of Capnoclium sp., one strain of Melanized limestone ascomycete, two strains of Leptosphaeriaceae sp., two strains of Ascomycete sp., one strain of Mycosphaerella Alistairii, one strain of C. lunatus, one strain of Microdiplodia hawaiiensis.The major pathogen Fonsecaea pedrosoi of chromoblastomycosis was not found in the experiment.Are the substances foci suitable for Fonsecaea pedrosoi growing not the soil but other material, such as plant, litter and wheat stalk?May the condition factors of the experiment, such as culture medium, temperature or other involved fungi also play the role to inhibit the species survive?Further studies will be needed to address the above questions.
Using rDNA ITS sequences from 60 newly isolated strains and 9 reference strains, we constructed phylogenetic tree of dematicacious fungi.The phylogenetic method based on the ITS rDNA sequence to identify fungi agreed with the morphological method, even if more quick and accurate.The NJ tree indicates the relationships between genetic distances and some biological habits of strains, for example, the strains that can cause diseases in human and animal often group together (Cladophialophora carrionii, Cladophialophora devriesii, Cladophialophora oxysporum, Cladophia-lophora cldosporioides, E. dermatitidis, E. xenobiotica, E. oligosperma, E. pisciphila, E. mesophila, D. bryoniae), on the other hand, the strains that can cause diseases in plants usually get into other crowd.There is no evidence showing that the sorting relates to the geographical location, while the same kind strains from different regions reveal similar genetic distances.Therefore, the strains within the crowd or with closer genetic distance would be also with potential pathogenicity.Development Phylogenetic tree constructed by neighbor-joining (NJ) method.The tree was constructed using 500 bootstrap replications (values > 50 are shown with the branches).The evolutionary distance between organisms is indicated by the horizontal branch length, which reflects the number of nucleotide substitutions per site along that branch from node to the endpoint.of NJ tree would not only effect on identifications of fungal strains but would also play a role on guiding the studies on some biological habits of the strains.
Figure1.Phylogenetic tree constructed by neighbor-joining (NJ) method.The tree was constructed using 500 bootstrap replications (values > 50 are shown with the branches).The evolutionary distance between organisms is indicated by the horizontal branch length, which reflects the number of nucleotide substitutions per site along that branch from node to the endpoint.

Table 1 .
Localities, sites and number of soil samples collected in Guangdong, PR china.: indicates the north region of Guangdong with the medio-subtropical region; †: the middle region of Guangdong with the south subtropical region; ‡: the south region of Guangdong with the north tropical region; §: the east of the middle region of Guangdong; ¶: the middle region of Guangdong; #: the west of the middle region of Guangdong.The west of the middle region of Guangdong with the south subtropical region is Zhaoqing Gaoyao West; the central of the middle region of Guangdong with the south subtropical region is Zhaoqing Gaoyao East to Heyuan; the east of the middle region of Guangdong with the south subtropical region is Heyuan East. *

Table 2 .
List of reference sequences.

Table 3 .
List of strains isolated and source.