Use of molecular methods for identifying culture of soil fungi from tropical forests of vietnam

 Những vấn đề chung 
Tạp chí Khoa học và Công nghệ nhiệt đới, Số 11, 12 - 2016 15
USE OF MOLECULAR METHODS FOR IDENTIFYING CULTURE OF 
SOIL FUNGI FROM TROPICAL FORESTS OF VIETNAM 
KALASHNIKOVA K. A. (1*), KONOVALOVA O. P. (1), ALEXANDROVA A. V. (1, 2) 
1. INTRODUCTION 
Soil microscopic fungi are always present in all biological communities and 
they are also one of the key elements in detrital food web, providing a circulation of 
nutrients. They grow on all possible substrates and play important role in the decay of 
organic matter. They are actively involved in the processes of soil formation and the 
formation of plant communities and also have an influence on the abundance and 
species composition of other groups of organisms in the soil [1]. The fungi provide 
rapid mineralization of organic residues, immobilization of macro- and micronutrients 
and plant nutrition, so it explains their great role in tropical forests [2]. 
Tropical regions are characterized by an exceptional diversity of biotic 
organisms, including fungi [3, 4]. Their species composition in the soil and on plant 
residues is a relatively little-studied component of ecosystems [5]. In this case, the 
main attention is usually focused on particular groups, which have practical 
importance. There are pathogens for plants or animals and humans [6] and producers 
of biologically active substances [7]. 
Estimates of species diversity of fungi vary widely, but experts are consentient 
that it has been insufficiently studied. There are described more than 1,200 species 
of fungi annually, a lot of them are associated with the soil [8]. Poorly studied areas 
of tropical forests have a huge potential of undescribed species inhabiting different 
substrates. There are a lot of features that make difficulties in studies of species 
diversity of microscopic fungi in the soil, like the impossibility of their observation 
directly in the nature conditions, complexity and difficulty of isolation of different 
groups of micromycetes on nutrient media, data comparability, obtained by different 
methods, and problems associated with species identification. 
Identification is possible only by morphological characteristics associated with 
sporification, however, not all species of the microscopic fungi can form it in the 
culture and accordingly they can not be determined by the standard methods. 
Also, there are many cryptic species or sibling species among microscopic fungi, 
which are morphologically similar, but have differences at the genetic level [8, 11]. 
Use of molecular genetic techniques helps in solving such problems. These 
techniques provide additional information in some cases and allow to identify or 
specify the species of fungi. However, this problem can not be solved completely by 
this method because of the incompleteness of the information in the database. 
Database of GenBank contains information of no more than about 20% of the 
presently described fungal species [8]. 
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Tạp chí Khoa học và Công nghệ nhiệt đới, Số 11, 12 - 2016 16
The objective of this research is a complete study of the species diversity of 
soil fungi of tropical forests in Vietnam with using morphological and molecular 
genetic methods for identification of the obtained cultures. 
2. RESEARCH METHODS 
Systematic research of the soil mycobiota of rainforest was organized in 2009 
and based on the resources of Joint Russian-Vietnamese Tropical Research and 
Technology Center [10, 11, 12]. The research material (upper horizon soil samples 
and plant litter) is collected between 2009 and 2014 in the monsoon rainforests of 
the six especially protected areas in Vietnam: in national parks Cat Tien, Bi Doup - 
Nui Ba, Chu Yang Sin, Bu Gia Map, in national reserves Dong Nai and Loc Bac 
forestry. The samples of the upper soil horizon and leaf litter were taken by the 
standard method [13] in sterile packages and were quickly dried. 
Laboratory work was carried out at the Department of mycology and algology 
of Biological faculty of Moscow State University. Isolation of micromycetes was 
performed by inoculation of serial dilutions of Z. Waxman on the solid nutrient 
media in the modification of D.G. Zvyagintsev [15]. There were two culture media 
for use in the research, that allow to reveal a wide range of micromycetes and 
provide easily differentiate the morphological types of colonies in samples: Czapek 
agar with 0.3% of sucrose and malt extract agar. Pure cultures of fungi were 
inoculated for identification on recommended for specific groups culture media [14]. 
Identification with using morphological features was performed according to 
generally accepted determinants and articles containing the research of individual 
genera and species description [17]. The names of species and systematic position were 
given in databases: The MycoBank Fungal databases ( and 
CABI Bioscience Databases ( 
The molecular-genetic method was used to identify sterile cultures and 
cultures that can not form sporification and clarify the definition of species which 
morphological criteria required verification. 
The sequences of the ribosomal gene cluster ITS1-5.8S-ITS2, including both 
variable regions of intergenic spacer sequences ITS 1 and ITS 2 and the conserved 
region of the ribosomal subunits 5.8S were determined by this method. For this sector 
in the GenBank were collected more information than for the other ones and it is 
offered to use it in the barcoding program unit [8, 9]. These sectors are recommended 
to a sequence for primary molecular identification, and only after that to continue 
work with sectors that give more precise results for specific groups of fungi [14]. 
Pure cultures of microscopic fungi were grown in Petri dishes on malt extract 
agar for 7÷10 days. Inoculating was produced by three injections into the culture 
medium. Colonies were aseptically removed from the surface of the medium and 
were placed into microtubes. DNA was isolated from the mycelium using Wizard® 
kit SV Genomic DNA Purification System A2361 (Promega, USA) according to the 
manufacturer's protocol. 
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ITS-nuclear rDNA portions (ITS1 and ITS2) were amplified by PCR, which 
divide the two macromolecular rRNA genes - 18S (SSU) and 25-28S (LSU) - and 
include low molecular weight rRNA sequence 5.8S, using an appropriate pair of primers 
ITS- 1F [16] and ITS-4 [17]. Amplification program was set through many experiments 
and carried out in specific mode: denaturation at 95°C for 5 minutes, annealing of 
primers 35 cycles at 95°C - 15 seconds, 55°C - 20 seconds, 72°C - 30 seconds, the final 
elongation step - 7 min. PCR was performed by using a kit for the amplification of 
DNA polymerase Colored Taq (art. K0132) of Sileks Company. The reaction products 
were analyzed by electrophoresis using a 2% agarose gel with ethidium bromide. After 
30 minutes of the electrophoresis, the authors assessed the presence of DNA under UV 
light. The total yield of DNA was quite high. Sequencing was performed with the same 
primers in both directions on an automated sequencer ABI Prism 3100 Genetic 
Analyzer ("Applied Biosystems HITACHI", USA) using a kit of reagents BigDyev.1.1. 
The sequences were processed and analyzed by Codon Code Aligner programs 
(www.codoncode.com). Alignment is done manually at Codon Code Aligner program 
and the Clustal W 1.6 [20]. Comparison and identification of nucleotide sequences was 
carried out at Nucleotide Blast Search (www.ncbi.nlm.nih.gov/BLAST/) data. This 
results of the sequence have been deposited in the GenBank database (NCBI) at 
numbers KP074967 ÷ KR075007, KR747689 ÷ KR747712. 
3. RESULTS AND DISCUSSION 
The collection of pure cultures of micromycetes that isolated from samples of 
the soil and the leaf litter of tropical forests of specially protected areas of Vietnam 
was created as a result of this work. The collection includes 1149 isolates, and 252 
of them are not formed sporulation in culture. There were 349 species identified as a 
result of morphological research. 
ITS parts of rDNA were sequenced for 184 strains, only 65 strains (35%) were 
found in the GenBank Data Base as a closest match with existed strains, 42 were 
identified up to species level and 23 were identified up to genus level. As a result of 
this work, at a list of a collection of the soil micromycetes of Vietnam were added 
30 species with a total amount of 379 species from 117 genera. The rest of the 
strains with no sufficiently close conformity could be the part of non-information 
species in the GenBank database or, as a supposition, they are new for the science 
undescribed species. The research on them will be continued. 
Among the strains with match at the GenBank database, the vast majority was 
belonged to division Ascomycota (Table 1.) 31% of them owns to order Xylariales, to 
order Eurotiales - 15%, in order Botryosphaeriales and Pleosporales - 12%, 
Hypocreales - 11%, Diaporthales - 3%, and a group of uncertain taxonomic position 
(Incertae sedis) - 5%. Division Basidiomycota was presented by strains of orders 
Agaricales - 3%, Cantharellales - 3%, Hymenochaetales - 2% and Incertae sedis - 3%. 
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Тable 1. Taxonomic position of strains of soil microscopic fungi isolated 
from the soil of tropical forests in Vietnam, for which a close match 
was found in the GenBank database 
Division Class Order 
Amount 
of 
strains 
The 
proportion of 
the total 
number of 
strains 
obtained, % 
Ascomycota 
Dothideomycetes
Botryosphaeriales 8 12 
Pleosporales 8 12 
Eurotiomycetes Eurotiales 10 15 
Sordariomycetes Diaporthales 2 3 
 Hypocreales 7 11 
 Xylariales 20 31 
 Incertae sedis 3 5 
Basidiomycota Agaricomycetes 
Agaricales 2 3 
Cantharellales 2 3 
Hymenochaetales 1 2 
Incertae sedis 2 3 
Total 65 100 
Most of soil micromycetes cultures of tropical forests of Vietnam, that not 
forming sporulation in culture, belong to the order Xylariales (20 strains classified to 
12 species): Annulohypoxylon sp. - in the collection of strains F138 number (number 
in GenBank KP747712); Hypoxylon sp. - № FV-13193 (KP747705); Neopestalotiopsis 
sp. - FV-13275 (KP747700); Pestalotiopsis clavispora (G.F. Atk.) Steyaert - № 362 
(KR074999), 381 (KR075005); P. mangiferae (Henn.) Steyaert - № 297 (KR074988), 
128 (KR074973), 139 (KR074975); P. vismiae (. Petr) J. Xiang Zhang & T. Xu - № 
FV-13 300 (KR747694), FV-13299 (KP747698), FV-13 47 (KP747709); 
Pestalotiopsis sp. - № FV-13213 (KP747695); Pestalotiopsis sp.1 - № 358 
(KP074997); Pestalotiopsis sp.2 - № 359 (KP074998); Pestalotiopsis sp.3 - № 335 
(KP074992), 376 (KP075003); Pestalotiopsis sp.4 - № 378 (KR075004); Xylaria sp. - 
№ FV-13 120 (KP747692), FV-13 335 (KP747691), FV-13 89 (KP747707). 
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The genus Pestalotiopsis provides the largest abundance - 8 species, 5 of them 
possibly are newly identified. Species of this genus are widely distributed mainly in 
regions with tropical and subtropical climates. This genus in recent years attracts the 
close attention of mycologists. On the one side, these species are endophytes and 
phytopathogens which cause rot and leaf spot many grassy and woody plants [21]. 
On the other side, the species of this genus are very reactive - they produce a wide 
variety of metabolites that may be involved in the decomposition of complex 
organic substances [20]. 
Types of genera Annulohypoxylon, Hypoxylon and Xylaria are from the wood-
destroying group of fungi, many of which form rather large fruiting bodies on wood, 
some of them can also be pathogenic for trees. On nutrient media they often produce 
stroma, but usually, do not form mature fruiting bodies required for identification. 
The next in the richness of species among sterile cultures are orders 
Botryosphaeriales and Pleosporales, all of the identified species are saprotrophs on 
plant residues and potential phytopathogens: 
Botryosphaeriales: Endomelanconiopsis endophytica E.I. Rojas & Samuels - 
№ 127 (КР074972); Lasiodiplodia theobromae (Pat.) Griffon et Maubl. - 161 
(KP074976), 276 (KP074984), 285 (KP074985); L. pseudotheobromae A.J.L. 
Phillips, A. Alves & Crous - 124 (КР074970), № 125 (КР074971), № FV-13 69 
(KP747702); Microdiplodia sp. - FV-13 341 (KP747699). 
Pleosporales: Leptosphaeria spegazzinii Sacc. & P. Syd. - № 372 (KP075001); 
Leptosphaeria sp.1 - № FV-13 39 (KP747704); Leptosphaeria sp.2 - № FV-13 147 
(KP747710); Lewia infectoria (Fuckel) M.E. Barr & E.G. Simmons - № 28 
(KP074967); Paradendryphiella salina (G.K. Sutherl.) Woudenberg & Crous - 
№336 (KP074993); Phoma tropica R. Schneid. etBoerema - № 298 (KP074989); 
Pseudocochliobolus eragrostidis Tsuda et Ueyama - № 293 (KP074986). 
Sterile cultures from order Hypocreales: Emericellopsis sp. - № 259 
(KP074981); Gibberella baccata (Wallr.) Sacc - № 226 (KP074977); Nectria 
mauriticola (Henn.) Seifert et Samuels - № 233 (KP074978). The last two of them 
are phytopathogens. 
Representatives of the order Diaporthales are also not form sporulation in culture: 
Diaporthe eucalyptorum Crous & R.G. - № FV-13340 (KP747690); D. neotheicola 
A.J.L. Phillips & J.M. Santos Shivas - № Ba8 (KP747693) are phytopathogens. 
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Sterile cultures related to the division Basidiomycota belong to two species of 
wood-agaricoid fungi: Gymnopilus sp.1 - № 121 (KP074969), Gymnopilus sp.2 - 
№374 (KP075002); two types of sponk wood-destroying fungi are: Oxyporus 
corticola (Fr.) Ryvarden - № FV-13279 (KP747703), Phellinus noxius (Corner) G. 
Cunn. - № FV-13219 (KP747708). № FV-13 30 (KP747689), FV-13243 (KP747696) 
- strains Thanatephorus cucumeris (A.B. Frank) Donk also identified. This is a very 
interesting fungus, better known as the asexual stage (Rhizoctonia solani Kühn), it can 
be phytopathogen for culture plants, causing root rot, in natural biocenoses it often 
acts as mycorrhiza forming species with a variety of plants, including orchids [21]. 
Among the strains that require clarification species identification by molecular 
methods were 13 strains of the genera Penicillium and Trichoderma. 
Six species belong to genus Penicillium: Penicillium citreonigrum Dierckx - 
№ 268 (KP074982), 269 (KP074983); P. commune Thom - № 344 (KP074995); 
P.melinii Thom - № 334 (KP074991); P. sacculum E.Dale - № 339 (KP074994); 
Penicillium sp. - № 252 (KP074980), 347 (KP074996), FV-13214 (KP747701); 
Penicillium sp.1 - № 364 (KP075000). At the present time the description of many 
new species of this genus is conducted exceptionally on the basis of molecular 
characteristics [22], so it is not possible to accurately identify Penicilllium species 
without using of a molecular method. 
Genus Trichoderma is represented by 3 species: Trichoderma gamsii Samuels 
& Druzhin - № Psp6 (KP075006), Trichoderma koningiopsis Samuels, C. Suárez & 
HC Evans - № Psp4 (KP075007), Trichoderma reesei EG Simmons - № MDL1 
(KP074990). Fungi of this genus are very common in nature, it could be found in the 
soil, on plant residues and in the wood. They are used as producers of cellulolytic 
enzymes and antibiotic substances, as an agent for the biological control of 
pathogenic fungi of plants [23]. 
4. CONCLUSION 
As a result of this work, the authors could succeed in species identifying by 
the molecular-genetic method for only one-third of the cultures in the research. The 
rest of the strains with no sufficiently close conformity could be the non-information 
species in the GenBank database, or they may be the new for science and 
undescribed species. 
In reality the study on identification of fungi culture allows to perform more 
focused search for producers of biologically active metabolites. The molecular 
genetic method is usefull to identify or clarify the species attachment for 
microscopic fungi when it is difficult or even not possible by morphological 
features. However, its use is restricted by the shotage and incompleteness of data of 
the fungi in the GenBank database. 
The work was supported by the RNF Fund, the project № 14-50-00029. 
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TÓM TẮT 
SỬ DỤNG PHƯƠNG PHÁP SINH HỌC PHÂN TỬ TRONG ĐỊNH LOẠI 
CÁC CHỦNG NẤM ĐẤT CỦA RỪNG NHIỆT ĐỚI VIỆT NAM 
Vi nấm đất luôn hiện diện trong tất cả các quần xã sinh vật và là một trong 
những thành phần cơ bản của lưới thức ăn mùn bã, góp phần vào chu trình dinh 
dưỡng. Chúng đóng vai trò quan trọng trong việc phân hủy các chất hữu cơ trong 
các quá trình hình thành đất và các quần xã thực vật. Hiện chưa có thông tin về 
thành phần loài vi nấm đất trong nhiều vùng khác nhau do có sự khó khăn trong 
nghiên cứu đa dạng loài của của chúng - không thể quan sát chúng trong tự nhiên, 
mà chỉ có thể phân lập bằng môi trường nhân tạo. Việc định loại vi nấm được tiến 
hành dựa trên các đặc điểm hình thái của chúng kết hợp với sự hình thành bào tử. 
Tuy nhiên, không phải tất cả các loài có thể hình thành bào tử trong môi trường nuôi 
cấy, vì thế không thể định loại hết được bằng các phương pháp truyền thống. Việc 
phát triển các kỹ thuật sinh học phân tử trong ngành nấm học có thể giúp giải quyết 
vấn đề này. Bài báo đề cập đến việc nghiên cứu nấm đất trong rừng nhiệt đới Việt 
Nam. Từ kết quả nghiên cứu, đã ghi nhận được 1.149 chủng, trong đó có 252 chủng 
không có khả năng hình thành bào tử (chủng vô sinh). Các trình tự của gen rDNA 
ITS 1 - ITS 2 đã được xác định bằng phương pháp phân tử đối với 184 chủng trong 
số 1.149 chủng thu thập được và chỉ có 65 chủng từ 184 chủng nêu trên được tìm 
thấy trong cơ sở dữ liệu của GenBank, trong đó 42 chủng được định loại tới loài, 23 
chủng được định loại tới chi. Đã bổ sung 30 loài vi nấm cho khu hệ vi nấm đất Việt 
Nam, đưa tổng số loài hiện tại của khu hệ vi nấm đất Việt Nam lên 379 loài thuộc 
117 chi. 
Từ khóa: Vi nấm đất, rừng nhiệt đới, vùng đệm được sao mã, ITS, microscopic 
soil fungi, tropical forests, internal transcribed spacer. 
Nhận bài ngày 04 tháng 11 năm 2016 
Hoàn thiện ngày 06 tháng 12 năm 2016 
(1)Moscow State Lomonosov University, Faculty of Biology,119234, Russia, Moscow, 
Leninskiye gory St., 1/12, info@mail.bio.msu.ru, *kri2012@yandex.ru 
(2) Joint Vietnam-Russian Tropical Research and Technological Centre, Nguyen Van 
Huyen, Nghia Do, Cau Giay, Hanoi, Vietnam