Dịnh danh và phân nhóm nấm Trichoderma spp. phân lập tại việt nam

BỘ GIÁO DỤC VÀ ĐÀO TẠO
TRƢỜNG ĐẠI HỌC NÔNG LÂM TP. HCM
------






KHÓA LUẬN TỐT NGHIỆP




ĐỊNH DANH VÀ PHÂN NHÓM NẤM
Trichoderma spp. PHÂN LẬP
TẠI VIỆT NAM






Ngành học: CÔNG NGHỆ SINH HỌC
Niên khóa: 2003 – 2007
Sinh viên thực hiện: NGUYỄN THỊ KHẢ TÚ


Thành phố Hồ Chí Minh
Tháng 9/2007

iii
BỘ GIÁO DỤC VÀ ĐÀO TẠO
TRƢỜNG ĐẠI HỌC NÔNG LÂM TP. HCM
BỘ MÔN CÔNG NGHỆ SINH HỌC
-------








ĐỊNH DANH VÀ PHÂN NHÓM NẤM
Trichoderma spp. PHÂN LẬP
TẠI VIỆT NAM






Giáo viên hƣớng dẫn Sinh viên thực hiện
TS. LÊ ĐÌNH ĐÔN NGUYỄN THỊ KHẢ TÚ

Thành phố Hồ Chí Minh
Tháng 9/2007


iii






           






 


            


            
 












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TÓM TẮT
        Trichoderma     
      




Mục đích
Trichoderma 
Trichoderma spp..
Trichoderma spp. (Trichoderma

Phƣơng pháp thực hiện

 rDNA (Internal Transcribed Space  
tef1 (4
th
large intron) (EF   

 
            tef1   
Trichoderma            
TreeView 1.6.6.
 tef1  Trichoderma


Kết quả đạt đƣợc
Trichoderma.
Trichoderma.
Trichoderma T. longibrachiatum, T. asperellum,
T. atroviride, T. harzianum, T. virens T. asperellum 



v
T. asperellum 

Kết luận
  
tef1 
 Trichoderma.
 tef1 
Trichoderma 


vi
ABSTRACT

Trichoderma spp. colle
carried out by Nguyen Thi Kha Tu, from 19 Mar, 2007 to 19 Aug, 2007 at Plant
Protection Pepartment, Argonomy Faculty, Nong Lam University and
Phytobiotechnology Laboratory, Reseach Institute Biological and Enviromental
Technology, Nong Lam University.
This thesis was based on molds identified results then using Clustal 1.83 and
Treeview 1.6.6 software to compare ITS  rDNA (Internal Transcribed Space 
rDNA) stable and apart of tef1 (EF   1 alpha)
funtional regions with their data on NCBI to identifying species and grouping of 11
Trichoderma genus. Finally, ITS  rDNA and apart of tef1 regions of 11

Trichoderma genus were compared with their data at different ecological,
geographic areas on over the world to clear the genetic relationship.
The results show that confirming exactly the species of 10 Trichoderma genus,
T68 genus was identified T. atroviride. Establishing and clearing the genetic
relationship among 11 Trichoderma genus. In which, 11 Trichoderma genus in Viet
Nam of 5 species T. longibrachiatum, T. asperellum, T. atroviride, T. harzianum ,
T. virens (except T. asperellum genus (T6)) have a rather far genetic relationship
from others in the world. 10 genus orginated from locality and
T. asperellum (T6) is the one migrated in our country. Genetic relationship among 5
species is stable and suitable with morphological characteristics.



vii
MỤC LỤC

 ................................................................................................................. iv
ABSTRACT .............................................................................................................. vi
 ................................................................................................................. vi
 ....................................................................... x
 ..................................................................................... xii
 ...................................................................................... xii
 ..................................................................................................................... 0
 ..................................................................................................................... 0
1.1.  ...................................................................................................... 0
1.2.  ........................................................................................................ 0
1.3.  .......................................................................................................... 0
 ..................................................................................................................... 2
 ........................................................................................... 2
2.1. Trichoderma ..................................................................... 2

2.1.1. ................................................................................................... 2
2.1.2.  ................................................................................................ 2
2.1.3.  ................................................................................... 3
2.1.4.  ....................................................... 4
2.1.5.  ....................................... 4
2.2. Trichoderma ........................................................................... 6
2.3. Trichoderma ................................ 7
2.4.  rDNA ........................................... 9
2.5. tef1 ................................................................................. 10
2.6.   ....................... 10
2.7. Trichoderma  .................. 11
2.8.  Trichoderma ... 13
 ................................................................................................................... 15


viii
 ............................................................................ 15
3.1.  .................................................. 15
3.1.1.  ......................................................... 15
3.1.2. u ............................................................................ 15
3.2.  ....................................................................................................... 16
3.3.  ....................................................................................... 17
3.4.  ................................................................. 17
3.5. Trichoderma ............................................................ 17
3.6.  ............................................................. 18
3.7.  ............................................... 18
3.8.  .............................................................................................. 19
3.9.  ................................................................................. 21
3.10. Trichoderma ................................................... 22
3.11.  .................................................................. 24

Trichoderma 
 ............................ 24
g 4 ................................................................................................................... 25
 ................................................................................. 25
4.1.  ................................................................................... 25
4.2.   tef1 ........... 26
4.3.  ........................................................................... 27
4.4.  ..................................................... 35
4.4.1.  tef1
................................................................................................................ 35
4.4.2.  rDNA ........ 37
4.5.  ...... 39
4.6.  
tef1 ................................................................. 41
 ................................................................................................................... 44


ix
  ....................................................................................... 44
5.1.  ........................................................................................................ 44
5.2.  ......................................................................................................... 44



x
DANH SÁCH CÁC CHỮ VIẾT TẮT




dNTP: Deoxyribonucleotide triphosphate.
DNA: Deoxyribonucleic acid
EDTA: Ethylene diamin tetracetic acid.
PCR: Polymerase chain reaction.
RAPD: Random amplified polymorphism DNA.

RNA: Ribonucleic acid.
SDS: sodium dodecyl sulfate.
Taq: Thermus aquaticus.
TAE: Tris-glacial acetic acid- ethylenne diamine tetra acetic acid.
TE: Tris ethylene diamine tetra acetate.
Tm: Melting Tempereture .
.














xi
DANH SÁCH CÁC BẢNG
  ........... 15
  ............................... 20
  ....... 21
  ................................................................... 21
  Trichoderma  ....... 22
 tef1 Trichoderma  ..................... 23
  ............ 28

  ............ 28
  ............ 29
  ............ 29
  ............ 30
  .............. 30
  ............ 31
  ............ 31
  ............ 32
  ............. 32
  ........... 33
Trichoderma  ............... 33












xii
DANH SÁCH CÁC HÌNH
 T. harzianum   PDA
sau 2  3 ng nui c  ................... 3
 
 ............................................................................... 9
 

 . 20
 tef1 
EF1   986F .............................. 20
Trichoderma 
 .............................................................. 26
Trichoderma 
 V, 400 A trong 20 p ............ 26
 V, 400 
 rDNA 
 ...................................................................................... 27

 
EF1   ............................................................. 27
 
tef1 (B). ...................................................................................................... 36
 rDNA (A)  rDNA (B)
................................................................................................................... 38
  rDNA 
 ............................................................................. 40
  rDNA Trichoderma .......
 ........................................... 43
 tef1 
 ........................................... 42

MỞ ĐẦU
1.1. Đặt vấn đề
Trichoderma 

Trichoderma 







Trichoderma 
, 
ng (Gary J. Samuels, 2004).


        tef1 (4
th
large intron)  
              
                
Trichoderma 
1.2. Mục đích


 
Trichoderma  .
Trichoderma 

1.3. Yêu cầu
 Trichoderma .


1
Trichoderma.
Trichoderma 

              
 tef1   986R
Trichoderma 

 tef1 

Trichoderma
 tef1 
talX 1.83 

1.6.6.
  
tef1 Trichoderma 
              
TreeView 1.6.6.













2


TỔNG QUAN TÀI LIỆU
2.1. Giới thiệu về nấm Trichoderma
2.1.1. Phân loại
Trichoderma            



Trichoderma  g
Ascomycota, Euascomycetes, Hypocreales, Hypocreaceae,
 Trichoderma             
Trichoderma Trichoderma 
Trichoderma, Longibrachiatum, Saturnisporum, Pachybasium và Hypocreanum.
   Saturnisporum        

Hypocreanum 
Trichoderma, Pachybasium, Longibrachiatum 
Hypocrea

2.1.2. Nguồn gốc
Trichoderma 
Trichoderma 

 
(Gary J. Samuels, 2004). Trichoderma 
Trichoderme 
arman, 2000).


3
2.1.3. Đặc điểm hình thái (Gary J. Samuels, 2004)

 Trichoderma 

 


Hình 2. 1 T. harzianum  2  3 

Trichoderma   l
                

T. koningii-

 
Trichoderma 
               

Trichoderma 
T. virens), 
 
 m.
           


4
T. harzianum               
hlamydospores 
Trichoderma           

2.1.4. Đặc điểm sinh lý, sinh hóa, sinh học
Trichoderma 

 Trichoderma 
25  30
0

0

0
C
             i
Trichoderma 
35
0


0

Trichoderma           
           

Trichoderma. Trichoderma 

Trichoderma 


Trichoderma
 

(Gary E. Harman, 2000).
2.1.5. Cơ chế đối kháng với nấm gây bệnh cây trồng (Gary J. Samuels, 2004)
Trichoderma    Pythium

spp., Phytophthora spp., Rhizoctonia spp., Sclerotinia spp., Botrytis spp., Fusarium
 Crinipellis spp. 



5


-1,3-
Tiết kháng sinh: T. virens  , 
Rhizoctonia solani  Pythium spp. Isonitriles 
T. hamatum, harzianum, viride, koningii, polysporum 
 T. atroviride  T. viride -pentyl alpha-pyrone
 pyrones) 
Phytophthrora cinnamomea 
Botrytis cinnerea. Peptaibols do T. polysporum, harzianum, koningii 


            T. virens, koningii,
viride sesquiterpenes olyketides T. harzianum Steroids
 
T. virens.
Ký sinh: Trichoderma          

                
Trichoderma  

Trichoderma 
             glucanase,
 

  
Trichoderma.    Trichoderma     t  
 
               
T. asperellum  cellulase,         Phytophthora


6
Pythium spp. .
Cạnh tranh: Trichoderma c      
 
 Trichoderma c
  Botrysis spp. Sclerotina 
(
)Trichoderma 
       Trichoderma      
    Botrysis spp.  Sclerotina spp..   
Trichoderma c Phytium spp. do 
, Phytium spp.
Trichoderma 
Phytium spp. Phytium spp.
 Trichoderma 
  
  .
Trichoderma 

  
  
Trichoderma 
Trichoderma 



2.2. Cấu tạo tế bào Trichoderma
Trichoderma
 
  glucan, mannoprotein (Siu-Wai Chiu
 


7

 

Trichoderma 


2.3. Các phƣơng pháp định danh tên loài của Trichoderma
Trichoderma 
.  
          Trichoderma.    

Hypocrea GliocladiumTrichoderma.  
Trichoderma
    


T. asperellum T. viride


    

 
      





T. longibrachiatum T. sinensis


0
C.
    
0
  

       

0
).
T. atroviride T. koningii
 


8


B

 

      

0
).


T. atroviride T. viride
 
    




       Trichoderma     
  .  (ITS1, ITS2, RAPD) 





Trichoderma 

Trichoderma/ Hypocrea 
 
h danh Trichoderma
 T. viride 
T. koningii T. atroviride tef1 
            
(Genealogical Concordance Phylogenetic Species Recognition
Trichoderma 


  
- rDNA, mitssuDNA,
tef1 ech42 
Trichoderma 
Trichoderma 


9

 







Trichoderma 
2003).

Hình 2. 2  


2.4. Giới thiệu về vùng rDNA và vùng ITS – rDNA




  




10
                
 

 
 

 

             



          

      
     N. crassa, Schizosaccharomyces pombe S. cerevisiae,
Vicia faba       S. prombe, S. cerevisiae   Oryza
sativa
N. crassa, 

2.5. Giới thiệu về vùng tef1
  


Trichoderma 
           Trichoderma (Gary J.

Samuels, 2005).
2.6. Phƣơng pháp định lƣợng nồng độ DNA bằng phân tử Mass
      


11



                

Bảng 2.1 
Nồng độ gốc Nồng độ sử dụng (2,5 µ l/ giếng)
 1000 bp 100 ng 1000 bp
 700 bp 70 ng 700 bp
 500 bp 50 ng 500 bp
 200 bp 20 ng 200 bp
 100 bp 10 ng 100 bp



               

2.7. Các nghiên cứu có liên quan đến Trichoderma và vùng rDNA
  rDNA (Small Subunit  rDNA)
                 
18S  

Hình 2. 3 
 (Bio- Rad)

         
        
            





12

  rDNA,
       Penicillium spp., Aspergillus spp. 
Paecilomyces spp.  Trichocomaceae       
 Acrasiomycota, Myxomycota,

Oomycota  Fungi            
Schizosaccharomycetales Myxomycota 
         rDNA (Large Subunit  rDNA, 24S),
P. carinii Basidiomycete trong

                
SSU  S. apiospermum

and S. prolificans 


ng T.
harzianum  rDNA.
  Trichoderma           
Trichoderma 




Trichoderma 


Trichoderma 

             
 Trichoderma 


