Development Of LAMP Techniques To Detect Leptosphaeria Species In Oilseed Rape

Blackleg and stem canker of oilseed rape are caused by Leptosphaeria biglobosa(Lb)and Leptosphaeria maculans(Lm),respectively.So far,there are at least two phylogenetic subgroups of Lb,namely L.biglobosa‘brassicae’(Lbb)and L.biglobosa‘canadensis’(Lbc),and one subgroup of Lm,namely L.maculans‘brassicae’,infecting oilseed rape.In China,only Lbb and Lbc have been found,whereas Lm has not been found,in oilseed rape-producing areas.Therefore,Lm is officially treated as a quarantine plant pathogenic fungus for our country.Symptoms caused by Lbb,Lbc and Lm in oilseed rape and the morphological characteristics of these fungi are pretty similar.Therefore,it is imperative to develop a rapid,sensitive and high throughput method to detect and identify Lbb,Lbc and Lm,This study was done to establish an easy-to-use detection system based on Loop-Mediated Isothermal Amplification(LAMP).The main results obtained are as follows:(1)Based on the ITS sequences,specific LAMP primers for Lb(including Lbb and Lbc)and Lm were designed.One loop primer was retained for the LAMP of Lb and two loop primers were retained for the LAMP for Lm.Differential DNA bands from the assay of random amplified polymorphic DNA(RAPD)for Lbb were obtained and successfully used to design the specific LAMP primers for Lbb.The artificial nucleotide mismatch in ITS-rDNA was successfully used to design the specific LAMP primers for Lbc.(2)The factors(dNTPs,Mg~+,F3/B3,FIP/BIP,LF/LB)affecting LAMP performance for detection of Lb were optimized.At the same time,the reaction time and temperature for LAMP were determined.The optimal temperature was 65℃and the duration time was 40 min.LAMP performance for Lbb,Lbc and Lmb was tested in terms of specificity,sensitivity and practicability.The results showed that the four groups of primers had a high specificity and could be specifically used to detect the target fungi.At the same time,they showed a high sensitivity and the threshold(the lowest amount of template DNA)reached fg level and the sensitivity was higher by 100 to 1000times than the conventional PCR.The LAMP reaction could be used to detect the target fungi in diseased stems showing the blackleg symptoms.(3)The method for extraction of fungal DNA was simplified by using TE buffer and NaOH solution to lyse fungal hyphae for releasing DNA.The entire extraction lasted only two minutes.Using the procedures of LAMP and DNA extraction,Lbb,Lbc and Lmb in diseased stems of oilseed rape were detected and the whole reaction could be finished within 1 h.The LAMP system established in this study can be used to detect and identify Lbb,Lbc and Lm.