Use of molecular methods for identifying culture of soil fungi from tropical forests of Vietnam
Những vấn đề chung
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].
Tạp chí Khoa học và Công nghệ nhiệt đới, Số 11, 12 - 2016
15
Những vấn đề chung
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
CABI Bioscience Databases (http://www.indexfungorum.org).
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.
16
Tạp chí Khoa học và Công nghệ nhiệt đới, Số 11, 12 - 2016
Những vấn đề chung
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
and the Clustal W 1.6 [20]. Comparison and identification of nucleotide sequences was
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%.
Tạp chí Khoa học và Công nghệ nhiệt đới, Số 11, 12 - 2016
17
Những vấn đề chung
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
The
proportion of
the total
number of
strains
Amount
of
strains
Division
Class
Order
obtained, %
Botryosphaeriales
Pleosporales
Eurotiales
8
8
12
12
15
3
Dothideomycetes
Eurotiomycetes
Sordariomycetes
10
2
Ascomycota
Diaporthales
Hypocreales
Xylariales
7
11
31
5
20
3
Incertae sedis
Agaricales
2
3
Cantharellales
Hymenochaetales
Incertae sedis
2
3
Basidiomycota Agaricomycetes
1
2
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).
18
Tạp chí Khoa học và Công nghệ nhiệt đới, Số 11, 12 - 2016
Những vấn đề chung
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.
Tạp chí Khoa học và Công nghệ nhiệt đới, Số 11, 12 - 2016
19
Những vấn đề chung
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.
20
Tạp chí Khoa học và Công nghệ nhiệt đới, Số 11, 12 - 2016
Những vấn đề chung
REFERENCES
1.
2.
Christensen M., A view of fungal ecology, Mycologia, 1989, 81(1):1-19.
Lodge D. J., Nutrient cycling by fungi in wet tropical forests, in Aspects of
tropical mycology (Isaac S., Frankland J. C., Walting R. and Whalley A. J. S.,
eds), Cambridge, British Mycological Society, 1993, p.37-57.
3.
Hawksworth D. L., Why Study Tropical Fungi? in Tropical Mycology. Vol. 2.
Micromycetes (Watling R., Frankland J. C., Ainsworth A. M., Isaac S. and
Robinson C. H., eds), New York: CABI Publishing, 2002, p.1-11.
4.
5.
Mueller G. M., Schmit J. P., Fungal biodiversity: what do we know? What can
we predict, Biodiversity and Consevation, 2007, 16:1-5.
Hyde K. D., Bussaban B., Paulus B., Crous P. W., Lee S., McKenzie E. C.,
Photita W. and Lumyong S., Diversity of saprobic microfungi, Biodiversity
and Conservation, 2007, 16:7-35.
6.
7.
Evans E. G. V. and Ashbee H. R., Tropical mycoses: hazards to travellers in
Tropical Mycology. Vol. 2. Micromycetes (Watling R., Frankland J. C.,
Ainsworth A. M., Isaac S. and Robinson C. H., eds), New York: CABI
Publishing, 2002, p.145-163.
Bills G. F., Dombrowski A., Peláez F., Polishook J. D. and Zhiqiang An.,
Recent and Future Discoveries of Pharmacologically Active Metabolites from
Tropical Fungi in Tropical Mycology. Vol. 2. Micromycetes (Watling R.,
Frankland J. C., Ainsworth A. M., Isaac S. and Robinson C. H., eds), New
York: CABI Publishing, 2002, p.165-194.
8.
9.
Hibbett D. S., Ohman A., Glotzer D., Nuhn M., Kirk P. and Nilsson R. H.,
Progress in molecular and morphological taxon discovery in fungi and options
for formal classification of environmental sequences, Fungal Biology
Reviews, 2011, 25:38-47.
Seifert K. A. Progress towards DNA barcoding of fungi, Molecular Ecology
10. Alexandrova A. V., Sidorova I. I., Tiunov A. V., Microfungi of soil and litter
of the National Park Cat Tien (South Vietnam), Mikologiya i fitopatologiya,
2011, 45:12-25 (in Russian).
11. Kalashnikova K. A., Alexandrova A. V., Soil inhabited microscopic fungi of
the national park Bi Dup - Nui Ba (South Vietnam), Mikologiya i fitopatologiya,
2014, 48:355-364 (in Russian).
12. Kalashnikova К. А., Alexandrova А. V., Soil inhabiting microscopic fungi of
the piedmont tropical forest (Loc Bac Forest Enterprise, South Vietnam),
Mikologiya i fitopatologiya, 2015, 49:91-101 (in Russian).
Tạp chí Khoa học và Công nghệ nhiệt đới, Số 11, 12 - 2016
21
Những vấn đề chung
13. Methods of soil microbiology and biochemistry (Ed. Zvyagintsev D. G.),
Moskva: Izdatelstvo MGU, 1991, 304 p. (in Russian).
14. Fungal Biodiversity, CBS Laboratory Manual Series 1 (Crous P.W. et al., eds),
Utrecht, Netherlands: CBS, 2009, 270 p.
15. Domsch K. H., Gams W. and Anderson T. H., Compendium of soil fungi,
Second edition, IHW Verlag Ehing, 2007, 672 p.
16. Gardes M. and Bruns T. D., ITS primers with enhanced specificity for
Basidiomycetes - application to the identification of mycorrhizae and rusts,
Molecular Ecology, 1993, 2(2):113-118.
17. White T. J., Bruns T., Lee S. and Taylor J., Amplification and direct sequencing
of fungal ribosomal RNA genes for phylogenetics, in PCR Protocols: a guide to
methods and applications (Innis M. A, Gelfand D. H, Sninsky J. J, White T. J,
eds.), New York, USA: Academic Press, 1990, p.315-322.
18. Thompson J. D., Higgins D. G. and Gibson T. J., CLUSTAL W: improving the
sensitivity of progressive multiple sequence alignment through sequence
weighting, position specific gap penalties and weight matrix choice, Nucleic
Acids Res, 1994, 22:4673-4680.
19. Maharachchikumbura S. N., Guo L. D., Cai L., Chukeatirote E., Wu W., Sun
X., Crous P., Bhat D. J., McKenzie E. C., Bahkali A. and Hyde K., A multi-
locus backbone tree for Pestalotiopsis, with a polyphasic characterization of
14 new species, Fungal Diversity, 2012, 56(1):95-129.
20. Xu J., Ebada S. S. and Proksch P., Pestalotiopsis a highly creative genus:
chemistry and bioactivity of secondary metabolites, Fungal Divers, 2010,
44(1):15-31.
21. Srinivasachary, Willocquet L. and Savary S., Resistance to rice sheath blight
(Rhizoctonia solani Kühn) [(teleomorph: Thanatephorus cucumeris (A.B.
Frank) Donk.] disease: current status and perspectives, Euphytica, 2011,
178(1):1-22.
22. Visagie C. M., Houbraken J., Frisvad J. C., Hong S. B., Klaassen C. H. W.,
Perrone G., Seifert K. A., Varga J., Yaguchi T. and Samson R. A.,
Identification and nomenclature of the genus Penicillium, Studies in
Mycology, 2014, 78:343-371.
23. Druzhinina I. S., Seidl-Seiboth V., Herrera-Estrella A., Horwitz B. A.,
Kenerley C. M., Monte E. and Kubicek C. P., Trichoderma: the genomics of
opportunistic success, Nature Reviews Microbiology, 2011, 9(10):749-759.
22
Tạp chí Khoa học và Công nghệ nhiệt đới, Số 11, 12 - 2016
Những vấn đề chung
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,
(2) Joint Vietnam-Russian Tropical Research and Technological Centre, Nguyen Van
Huyen, Nghia Do, Cau Giay, Hanoi, Vietnam
Tạp chí Khoa học và Công nghệ nhiệt đới, Số 11, 12 - 2016
23
9 trang
20/04/2022
1260
Bạn đang xem tài liệu "Use of molecular methods for identifying culture of soil fungi from tropical forests of Vietnam", để tải tài liệu gốc về máy hãy click vào nút Download ở trên
File đính kèm:
- use_of_molecular_methods_for_identifying_culture_of_soil_fun.pdf
