植物保护
植物保護
식물보호
Plant Protection
2015年
5期
110-115
,共6页
桉树焦枯病%桉树焦枯病原菌%快速检测%PCR 扩增
桉樹焦枯病%桉樹焦枯病原菌%快速檢測%PCR 擴增
안수초고병%안수초고병원균%쾌속검측%PCR 확증
eucalyptus dieback%Calonectria morganii%rapid detection%PCR amplification
桉树焦枯病是威胁桉树生长的首要病害,建立准确、有效的桉树焦枯病的 PCR 快速检测技术是桉树焦枯病前期诊断的必要手段。试验以桉树焦枯病原菌(Calonectria morganii )DNA 为模板,分别以 ITS 和 factor 1-alpha序列为靶区域,针对 Calonectria 属和 C .morganii 设计了 CYS1/CYS2和 EF-S-1/EF-A-1两对特异性引物,建立了基于属和种的双重 PCR 快速检测技术。利用引物 CYS1/CYS2可以从全部 Calonectria 属的供试菌株中扩增出一条351 bp 大小的条带,单独用特异性引物 EF-S-1/EF-A-1进行 PCR 扩增,仅病原菌扩增出197 bp 的条带,同时使用2对引物时,病原菌可扩增出两条明亮条带。当体系退火温度为53℃时,DNA 灵敏度检测限度达到450 fg/μL。野外田间时效检测结果显示,该体系能准确检测出不同发病程度桉树组织上的病原菌,完全符合田间检测的要求。这是关于桉树焦枯病快速检测的首次报道。
桉樹焦枯病是威脅桉樹生長的首要病害,建立準確、有效的桉樹焦枯病的 PCR 快速檢測技術是桉樹焦枯病前期診斷的必要手段。試驗以桉樹焦枯病原菌(Calonectria morganii )DNA 為模闆,分彆以 ITS 和 factor 1-alpha序列為靶區域,針對 Calonectria 屬和 C .morganii 設計瞭 CYS1/CYS2和 EF-S-1/EF-A-1兩對特異性引物,建立瞭基于屬和種的雙重 PCR 快速檢測技術。利用引物 CYS1/CYS2可以從全部 Calonectria 屬的供試菌株中擴增齣一條351 bp 大小的條帶,單獨用特異性引物 EF-S-1/EF-A-1進行 PCR 擴增,僅病原菌擴增齣197 bp 的條帶,同時使用2對引物時,病原菌可擴增齣兩條明亮條帶。噹體繫退火溫度為53℃時,DNA 靈敏度檢測限度達到450 fg/μL。野外田間時效檢測結果顯示,該體繫能準確檢測齣不同髮病程度桉樹組織上的病原菌,完全符閤田間檢測的要求。這是關于桉樹焦枯病快速檢測的首次報道。
안수초고병시위협안수생장적수요병해,건립준학、유효적안수초고병적 PCR 쾌속검측기술시안수초고병전기진단적필요수단。시험이안수초고병원균(Calonectria morganii )DNA 위모판,분별이 ITS 화 factor 1-alpha서렬위파구역,침대 Calonectria 속화 C .morganii 설계료 CYS1/CYS2화 EF-S-1/EF-A-1량대특이성인물,건립료기우속화충적쌍중 PCR 쾌속검측기술。이용인물 CYS1/CYS2가이종전부 Calonectria 속적공시균주중확증출일조351 bp 대소적조대,단독용특이성인물 EF-S-1/EF-A-1진행 PCR 확증,부병원균확증출197 bp 적조대,동시사용2대인물시,병원균가확증출량조명량조대。당체계퇴화온도위53℃시,DNA 령민도검측한도체도450 fg/μL。야외전간시효검측결과현시,해체계능준학검측출불동발병정도안수조직상적병원균,완전부합전간검측적요구。저시관우안수초고병쾌속검측적수차보도。
Eucalyptus dieback is the primary threat to the growth of eucalyptus.It is necessary to establish an ac-curate and effective PCR rapid detection technique for the early diagnosis of this disease.The duplex PCR tech-nique,for rapid detection of genera and species of eucalyptus dieback disease pathogen was established with the total DNA as templates,and two pairs of specific primers (CYS1/CYS2 and EF-S-1/EF-A-1 )for Calonectria de Not and C .morganii based on their ITS sequence and factor 1-alpha gene were designed,respectively.The results showed that all tested strains belonging to Calonectria genera were amplified a product of 351 bp using primers CYS1/CYS2,while only C .morganii strains were amplified a product of 197 bp using primers EF-S-1/EF-A-1,and the duplex PCR with these two pairs of primers could amplify the two products.Furthermore,at the annealing temperature of 53 ℃,the minimum amount of detected DNA was 450 fg/μL.The infected eucalyptus tissue with different levels could be accurately detected by the duplex PCR technique,indicating that the duplex PCR technique fits entirely in the field test.This was the first report about the rapid detection technique on eucalyptus dieback caused by C .morganii .
