應(yīng)用pcrrflp和巢式pcr檢測黃瓜尖鐮孢菌fusariumoxysporumf.sp.cucumarinum

1、東北農(nóng)業(yè)大學(xué)碩士學(xué)位論文應(yīng)用PCR-RFLP和巢式PCR檢測黃瓜尖鐮孢菌（Fusariumoxysporum f.sp.cucumarinum）姓名：陳微申請學(xué)位級別：碩士專業(yè)：植物病理學(xué)指導(dǎo)教師：文景芝20060618D e t e c t i o n o f F u s a r i u m o x y s p o r u mf ．s p ．c u c u m a r i n u mb y P C R —R F L P a n dN

2、e s t e d —P C RA b s t r a c tT h e5 9p u r ec u l t u r a l i s o l a t e s ，i n c l u d i n g3 F u s a r i u mo x y s p o r u mf ．s p c u c u m a r i n u m ．2 6o t h e rF u s a r i u m i s o l a t e s a n d2 0 f u n g

3、 i ，6 b a c t e r i a ，7 a c t i n o m y c e t e s f r o mc u c u m b e rr h i z o s p h e r e ，h a v eb e e n d e t e c t e d b y P C R —R F L P a n d N e s t e d - P C R I t s h o w s t h a t P C R —R F L Pi S s p e c

4、i a l f o r t h ep a t h o g e n o fC u c u r b i t s F u s a r i u m W i l t ，a n dN e s t e d - P C R i s s p e c i a l f o r F u s a r i u m o x y s p o r w nf ．s pC l l c u l n Q r i H u l T l N e s t e d ．P C R ；S m

5、 o r e s p e c i a l t h a n P C R —R F L P i n d e t e c t i o n o fF u s o r i u mo x y s p o r u m f ．s pc u c u m a r i n u mT h e p r i m e r sa n dt h ed e t e c t i o np r o g r a m sh a v eb e e nu s e dt od e t

6、e c tt h ep a t h o g e n si n t h ei n o c u l a t e d —d i s e a s e dc u c u m b e r p l a n t s T h ed e t a i l s a r er e p o r t e da s f o | | O W S ：l B yu s i n gt h ep r i m e r sI T S l ／I T S 4 ．o n l yF u s

7、 w 。i u mi s o l a t e sc o u l db ea m p l i f i e dt o p r o d u c eas p e c i a l f r a g m e n t o f 7 5 0 b p ．T h i sp r o d u c t g e n e r a t e d 4 8 0 b p ，2 0 0 b p a n d 1 0 0 b pf r a g m e n t s a f t e rd

8、i g e s t e d b yr e s t r i c t i o n e n z y m eH i n fI o n l yf r o m t h ep a t h o g e n o fC u c u r b i t s F u s a r i u m W i l t ．B yu s i n g t h e p r i m e r s B I K 1 ／B I K 4 a n d B I K 2 ／B I K 3 o f N

9、e s t e d ·P C R ，o n l y F u s a r i u m o x y s p o r u mf s pc u e u m a r i n m n c o u l db ea m p l i f i e dc of o r ma s p e c i a lf r a g m e n to f g O O b p ．o t h e ri s o l a t e s h a v e n ot h i s f

10、 r a g m e n t ．T h a t i n d i c a t e st h a t N e s t e d —P C R i s m o r es p e c i a l t h a n P C R ‘R F L Pi n d e t e c t i o no fF u s a J ‘i u mo x y s p o t 。u mf ．s p ．c u c u m a r i n u m2 P C R ．R F L P a

11、 n dN e s t e d —P C R d e t e c t e dS u c c e s s f u I l yt h ep a t h o g e n sj nt h ed i s e a s e dc u c u m b e rp l a n t s i n o c u l a t e dw i t hF u s a r i u mo x y s p o l 4 t i mf ．s pc u c u m a r i n u

12、 m ，t h ec h a r a c t e r i s t i c7 5 0 b pf r a g m e n tw a s o b t a i n e db yP C R - R F L P a n da l s ot h ec h a r a c t e r i s t i c 4 8 0 b p ，2 0 0 b pa n d 1 O O b pf r a g m e n t sa f t e rd i g e s t e

13、db yr e s t r i c t i o ne n z y m eH i n f I T h e s p e c i a l8 0 0 b pf r a g m e n tw h i c hr e p r e s e n t sF u s o r i u mo x y s p o r u m f ．s pc u c u m a r i n u m g e n e r a t e d b y u s i n g N e s t e

14、d ·P C R I t i n d i c a t e s t h a t t h e r e e x i s t sF u s a t ·i u m o x ) , s T a o l ·l t l n f ．s pC u m l m a l ·Y l U l l 7 i n t h e d e t e c t e d i n o c u l a t e d t i s s u e so fc

15、 u c u m b e r , a n dt h i sP C R —R F L Pa n dN e s t e d - P C Rp r o g r a m c o u l d e x a c t l ya n ds e n s i t i v e l y d e t e c tF u s a r i u mo x y s p o ，’Ⅲ”f i s p ．c u e u m a l i n u r ei n t h ei n o

16、c u l a t e dt i s s u e so f c u c u m b e r3 P C R —R F L Pc o u l dd e t e c tF u s a ／·i u m o x y s p o t —u m f ．s p ．c u c u m w 。i n u m i n t h e i n o c u l a t e dt i s s u e so f s u s c e p t i b l e c

17、u c u m b e rp l a n t so n t h e3 t hd a y N e s t e d —P C Rc o u l dd e t e c tt h e p a t h o g e n so n t h e5 t hd a y I t i n d i c a t e st h a t P C R ．R F L Pi s m o r es e n s i t i v e t h a n N e s t e d —P

18、C Ri n d e t e c t i o n o fF u s a r i u mo x ) , s p o l 。u m f ．s pC u c u m a r i n u m ．4 N e s t e d —P C Rc o u l dd e t e c tF u s a r i u mo x y s p o ，u mf ．s p ．c u c u m a r i n u mj n t h ei n o c u l a t e

19、dt i s s u e so f s u s c e p t i b l ec u c u m b e r p l a n t s0 n t h e5 f hd a y , h o w e v e rr e s i s t a n tc u c u m b e r p l a n t s w e r e o nf h e7 f hd a y I t i n d i c a t e st h a tp a t h o g e n si

20、n v a d em o r e e a r l i e r l yi n s u s c e p t i b l ep l a n t st h a ni n r e s i s t a n tp l a n t sK e y w o r d s ：C u c u m b e r F u s a r i u m W i l t ，F(xiàn) u s a r i u m o x y s p o r u m f ．s pc u c u m o r