| Powdery Mildew of rubber tree had occurred in the country's major planting area.Oidium heveae B A.Steinmann is an obligate parasitic fungus,it cause powdery mildew in rubber tree.At present,the detection and identification of rubber tree powdery mildew based on their symptoms,morphological,physiological and biochemical reactions.But the molecular detection techniques reported is few in rubber tree powdery mildew.In order to be detected in the leaf tissue incubation period of rubber tree powdery mildew fungus,the study established about rubber tree powdery mildew PCR,nested-PCR,LAMP molecular detection techniques.It can provide a scientific basis for early diagnosis,as well as set up to develop prediction model of disease prevention and treatment programs.The main results as follows:On the ITS sequences,we designed several rubber tree powdery mildew primers,but those primers selected to verify specificity were not strong.We found a special specificity conservative sequence of rubber tree powdery mildew through the comparative analysis(Patent application no:201510577343.9).Based on powdery mildew of rubber tree(Oidium heveae)unique conserved sequence region,specific primers and optimization of the reaction system.The optimum temperature for PCR was 57 ? and nested-PCR was 57 ?,optimum temperature of LAMP was 63 ?and the optimum reaction time was 50min.We try to use PCR to select optimal primers of LAMP.We also try to make the primers from HPLC purification way down to PAGE,although the purity of a primer on the decline but the impact of the proven LAMP reaction is almost negligible.In the specific test,only Oidium heveae got a specific bands amplified and correct alignment sequence.In the sensitivity test,on the Oidium heveae DNA level,conventional PCR,nested-PCR,LAMP detection system respectively reached 10 pg,0.01 fg,10 fg.This shows,Only in terms of sensitivity,the detection of nested-PCR system is the most sensitive,LAMP followed,PCR sensitivity minimum.Through pot experimental in leaf tissue at different time,we inoculate different spore concentration of rubber tree powdery mildew on healthy rubber tree and set different time points after inoculation sampling.The experimental results showed that:different inoculation amount,the time that use three different detection techniques of detecting target pathogen is different.Case2 ×102 spores/leaves at the minimum inoculation,nested-PCR can detect Oidium heveae target band after 24h inoculation,and LAMP can detect target band after 2 days inoculation,and 4 days after inoculation conventional PCR can detect the target band.The results shows that in the incubation period nested-PCR,conventional PCR and LAMP have good detection effect,among them,the detection of nested-PCR system is the most sensitive,LAMP followed,PCR sensitivity minimum.It coincide with the test results in the sensitivity test.After inoculation three times,we initially established a relationship between the amount of spores and disease index.In the 12 days after inoculation,when the amount of the rubber tree powdery mildew spores was 2 × 102 spores/leaves,the disease index was 0.6%;when the spores was 2 × 103 spores/leaves,the disease index was 2.4%;when the spores was 2 x 104 spores/leaves,the disease index was 37.9%;when spores was 2 × 105 spores/leaves,the disease index was 66%.So,with the increase of inoculum,powdery mildew hazard rubber tree has become increasingly serious.Thus,when detecting an amount 2 × 104 spores/leaves,it is the best prevention time.Therefore in terms of the field in terms of practicality,LAMP used to detect the shortest time,does not require high-end equipment,and have better detection effect.If only detect the existence of powdery mildew spores,nested-PCR advantage more pronounced.Different detection methods can be applied to different detection purposes.All in all,three different methods that apply to rubber tree powdery mildew detection have different advantages,they can provide a good foundation for forecasting the rubber tree powdery mildew. |
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