Phân lập và định danh một số vi sinh vật có khả năng kích thích sinh trưởng thực vật ở vườn quốc gia cát tiên

BỘ GIÁO DỤC VÀ ĐÀO TẠO
TRƢỜNG ĐẠI HỌC NÔNG LÂM THÀNH PHỐ HỒ CHÍ MINH
BỘ MÔN CÔNG NGHỆ SINH HỌC









KHÓA LUẬN TỐT NGHIỆP




PHÂN LẬP VÀ ĐỊNH DANH MỘT SỐ VI SINH VẬT CÓ
KHẢ NĂNG KÍCH THÍCH SINH TRƢỞNG THỰC
VẬT Ở VƢỜN QUỐC GIA CÁT TIÊN




Ngành học: CÔNG NGHỆ SINH HỌC
Niên khóa : 2003-2007
Sinh viên thực hiện: PHAN TRUNG HẬU


9/2007
BỘ GIÁO DỤC VÀ ĐÀO TẠO
TRƢỜNG ĐẠI HỌC NÔNG LÂM THÀNH PHỐ HỒ CHÍ MINH
BỘ MÔN CÔNG NGHỆ SINH HỌC
**************************








PHÂN LẬP VÀ ĐỊNH DANH MỘT SỐ VI SINH VẬT CÓ
KHẢ NĂNG KÍCH THÍCH SINH TRƢỞNG THỰC
VẬT Ở VƢỜN QUỐC GIA CÁT TIÊN








Giáo viên hƣớng dẫn: Sinh viên thực hiện:

PGS.TS. BI VĂN LỆ PHAN TRUNG HẬU
ThS. KIỀU PHƢƠNG NAM













T
9/2007

iii
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
, . 


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
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:
PGS.
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
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, 
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




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
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




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.
 




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 


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
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, 




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
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
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
.
 29 


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



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




.
, 

 , 

 , , tin





9/2007


iv


Phân lập và định danh một số vi sinh vật có khả năng kích thích
sinh trƣởng thực vật ở vƣờn quốc gia Cát Tiên”      


Kết quả:

-        
             


- Squa 
in vitro  in
vivo),              
 6012a, 6019a, 6019b, 6021a, 6027a, ON16a, ON20a,
ON28a ON29b.
 
 
Methylobacterium. 
6019a, 6019b, 6021a, ON16a, ON20a, ON28a, ON29b 

-   19b, ON16a, ON20a, ON29b      
Pichia.
- Cocidiascusc.
- Rhodotorula.
- Endomycopsis.

v
MỤC LỤC

CHƢƠNG..................................................................................... ...............TRANG


 ................................................................................................................... i





 .............................................................................................................. iii
t .................................................................................................................. iv
 ................................................................................................................... v






 ..................................................................................... viii






 ................................................................................................. ix
Danh  .............................................................................. xi






 ................................................................................................ xii

Chƣơng 1: MỞ ĐẦU ............................................... Error! Bookmark not defined.
1.1.  .......................................................... Error! Bookmark not defined.
1.2.  ............................................................. Error! Bookmark not defined.

 ............................................................... Error! Bookmark not defined.
Chƣơng 2: TỔNG QUAN TÀI LIỆU .................... Error! Bookmark not defined.
2.1.  ................................... Error! Bookmark not defined.
2.2.  ........... Error! Bookmark not
defined.
2.2.1 
 .................................................................................... 7
2.2.1.1.  ...... Error!
Bookmark not defined.
2.2.1.2.  ..... Error! Bookmark not defined.
2.2.2.  ..... Error!
Bookmark not defined.
2.3.  ............. Error! Bookmark not defined.
2.3.1.  .............................. Error! Bookmark not defined.

vi
2.3.1.  ...................................... Error! Bookmark not defined.


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Chƣơng 3: VẬT LIỆU - PHƢƠNG PHÁP ........... Error! Bookmark not defined.
3.1.  ......................................... Error! Bookmark not defined.
3.2.  ............................................................... Error! Bookmark not defined.
 ............................................... Error! Bookmark not defined.
3.2.2. T .............................................. Error! Bookmark not defined.
3.2.3.  .......................................................... Error! Bookmark not defined.
3.2.3.1. 
 ............................................. Error! Bookmark not defined.
3.2.3.2. 
O

) . Error! Bookmark not
defined.
3.2.3.3.  Error! Bookmark not defined.
3.2.3.4.  .................................... Error! Bookmark not defined.
3.3.  .................................... Error! Bookmark not defined.
3.3.1. n ..................................... Error! Bookmark not defined.
3.3.1.1.  .......................... Error! Bookmark not defined.
3.3.1.2.  ......................................... Error! Bookmark not defined.
3.3.1.3. 
... Error! Bookmark
not defined.
3.3.2.  .......................................................... Error! Bookmark not defined.
3.3.2.1. in
vitro in vivo ........................................................... Error! Bookmark not defined.
3.3.2.2.  .............................................. Error! Bookmark not defined.
3.3.2.3.  ......................................... Error! Bookmark not defined.
3.3.3.  ......................................... Error! Bookmark not defined.
3.3.3.1.  .... Error! Bookmark not
defined.
3.3.3.2.   ........................................... Error! Bookmark not defined.
3.3.3.3.  ........................................ Error! Bookmark not defined.

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3.3.3.4.  ................. Error!
Bookmark not defined.
3.3.3.5.  ....................... Error! Bookmark not defined.
3.3.4.  ........................................ Error! Bookmark not defined.
3.3.4.1.  ... Error! Bookmark not defined.
3.3.4.2.  ....... Error! Bookmark not
defined.

Chƣơng 4: KẾT QUẢ - THẢO LUẬN .................. Error! Bookmark not defined.
4.1.  ........................... Error! Bookmark not defined.
4.2.  .............. Error!
Bookmark not defined.
4.2.1.  VSV trong
in vitro ....................................................... Error! Bookmark not defined.
4.2.2.  .... Error! Bookmark
not defined.
4.3.  ......................................... Error! Bookmark not defined.
4.3.1.  ............................... Error! Bookmark not defined.
4.3.2. 
4.3.2.1. 
......................................70

7


Chƣơng 5: KẾT LUẬN – ĐỀ NGHỊ ....................... Error! Bookmark not defined.2
5.1  .................................................... Error! Bookmark not defined.2
5.2  ....................................................... Error! Bookmark not defined.


ix
TÀI LIỆU THAM KHẢO .. 

PHỤ LỤC

viii
DANH SÁCH CÁC CHỮ VIẾT TẮT

Bp Base pair
EDTA Ethylene-Diamine-Tetraacetic-Acid
MS 
CMS 2% casein
MMS 
Gram (-) 
Gram (+) 
PPFM Pink-Pigment Facultative Methylotrophic
PCR Polymerase chain reaction
MSo 
DNA Deoxyribonucleotide Acid
Taq Tag polymerase
LDC Lysine decarboxylase
PHB Poly-beta-hydroxybutyrate

ix
DANH MỤC CÁC HÌNH

TRANG
 (
 Methylobacterium
 .......................................................................... 6
 ................................................. 10
Azospirillum brasilense  ............................................... 11
 .................................................... 41

 ............................................................................................. 49

 .............................................................................................................. 49


 ..................................................................................... 52
 
 .......................................................................... 55
 
 .......................................................................................... 57
 ..................................... 60
 
 ........................................................................................... 63
K
 .................................................................................................................... 66

 ................................................................................................................... 67
  

nm.
............................................................................. 68
  ..................................................... 71

x
 ..................................................... 72
.............................................. 73
 (520F, 920R).............................................. 74

Methylobacterium 
 ................................... 76


xi
DANH MỤC CÁC SƠ ĐỒ VÀ BIỂU ĐỒ

TRANG
  ............................................................................ 24

 ................................... 47

  .......................... 48

 So ..................................................... 51

So .............................................. 51

610nm

 .......................................................................................................... 61
 ........... 61

 ..................... 62
 
 .................................... 62

xii
DANH MỤC CÁC BẢNG

TRANG
 
14GNR ............................................................................................... 37

  MS (Murashine & Skoog, 1962) ............... 47


 in vitro So .................... 50

 ................................................................................... 53
4.4: 
So ...................................................... 55
 
 ......................................................... 58
 ............................. 60
 .................. 64
 4.8: 
66

 ................................................................................................. 69

  ........................................................ 71

Methylobacterium  .................................................................... 75
 .......................................... 78
cose .......................................................... 80
 ............................ 80



1


Chƣơng 1
MỞ ĐẦU
1
1.1 Đặt vấn đề



t       


 
          
  















2

Phân lập và định danh một số vi sinh vật có khả năng kích thích sinh
trƣởng thực vật ở vƣờn Quốc gia Cát Tiên

1.2 Mục tiêu
 


 
1.3 Yêu cầu
                
 
        

 

















3

Chƣơng 2
TỔNG QUAN TÀI LIỆU
2
2.1 Vƣờn Quốc gia Cát Tiên [23]
  

              

 VQGCT
               

Nam.
 Hệ thực vật
 
 Dipterocarpaceae
Lagertroemia sp.
Saccharum
sp. Hydrocarpus sp. Ficus benjamina 

1
 
to
 Hệ động vật


4
 


 Crocodylus siamensis           

(Arborophila davidi   Polyplectron germani  
Pseudibis davisoni), nCairina
scutulataLeptoptilos javanicus



2.2 Sơ lƣợc sự tƣơng tác giữa vi sinh vật và thực vật
  
    
t. 
     
n. T,       
  trong  
  

 N
   

 
 [18].

 
+  



5
 10
9
 

 10
10
  [18].
+ Eukaryote            
   vi

6
-10
9

0
7
, 10
5
-10
6

3

2
-10
3

c

8
-10
9
 [6].
  

 
 
Các vi sinh vật biểu sinh (epiphyte): bvsv 
vsv Phyllosphere). vi sinh

             
polysaccharide, acid  
 N

 [16].
 , 
-pigmented facultatively
              
[14].    
 

               
tham g [19] 
2.1).



6

               




Hình 2.1: (A)      

methyl
(B)  [20]

Các vi sinh vật nội sinh (endophytic)
 (endophytic)  




 vsv             
            
  [14].


7
2.2.1 Phân nhóm vi sinh vật tƣơng tác với thực vật dựa vào bản chất của sự
tƣơng tác [16]

 

 




- 
- 
- 
-   
2.2.1.1 Các vi sinh vật ức chế tác hại của mầm bệnh lên thực vật: 

 i sinh 

Cạnh tranh về nơi ở và dinh dƣỡng

 dinh
        

B. cinerea .
Sự tổng hợp các loại hợp chất thứ cấp



Sporothrix floccolosa 
-


8
B. cinerea, Fusarium oxysporum sp
(2002)  Tilletiopsis
pallescens.
Enzyme phân hủy vách tế bào [16]


, 



















 :
-1,3-glucanase...




 Pichia anomala, P. membranifaciens,R. glutinis, C.
laurentii, A. pullulans, Tilletiopsis albescens, -1,3-















 : A. niger, Sphaerotheca fuliginea, P. xanthii
(Urquhart, 1994; Castoria, 1997; 

 1998; 





,
2001; 

 2002). Trong c       

   Methylobactreium         
[16].
Kích thích phản ứng phng vệ ở cây chủ






  









phenylalanine ammonia lyase phytoalexin, 

 






.
2.2.1.2 Vi sinh vật gia tăng dinh dƣỡng cho cây [16]


  

 


 
               
 


9
 Vi khuẩn cộng sinh trên cây họ đậu

u. 
 


           Rhizobium, Sinorhizobiu,
Mesorhizobium, Bradyrhizobium.
 

V              
            

i
i

M
 

 
 


ai trong 
   Mimosoideae  Papilionoideae       
 
 
                
, enzyme

10
Sự gia
tăng độ
mật
thiết của
các
tƣơng
tác
1. Vi sinh vật cộng sinh cƣ trú ở vùng gian bào
(Endosymbionts)
Ví dụ: 
2. Vi sinh vật cƣ trú ở vùng ngoại bào
(Ectosymbionts)
Ví dụ: t

3. Vi sinh vật bên trong mô cây chủ (Endophytes)
Ví dụ: Herbaspirillum sp. 
4. Vi sinh vật chỉ sống trên bề mặt cây
Ví dụ: Azospirillum  Klebsiella






Caesalpinoideae

 

        

).



Gluconacetobacter diazotrophicus    Acetobacter diazotrophicus),
Herbaspirillum spp Alcaligenes, Azospirillum, Bacillus, Enterobacter, ,
Klebsiella, Pseudomonas  Rhizobium 
          


1
- 10
7
 
 Vi sinh vật tự do cố định nitơ
  


Hình 2.2: 
 [16].



11
 do. 
nitr

Azospirillum 
 
Gluconacetobacter
diazotrophicus Herbaspirillum Azospirillum sp. 
 


B 
ate 
 tan
-ketogluconic acid,
             
               

3+
, 

2+
) [16].

Hình 2.3. Azospirillum brasilense bề mặt
của rễ lúa [20]