Development And Application Of LAMP Method For The Identification Of Heterodera Avenea In Diseased Plant Tissues

The cereal cyst nematode is one of the most important pathogenic nematodes on cereal crops worldwide, It caused serious economic losses every year for the production of wheat.In China, H.avenae is a new kind of potentially threatening nematode disease,it is seriously threat to wheat production safety in our country.The avenae group is known contains12effective biological species and some unkown species, different types of H.avenae species may have different pathogenicity for wheat.So for accurate identification of Heterodera avenae species are not only engaged in H.avenae research foundation, but also in preventing cereal cyst nemaotode disease is of great significance.In this study,we take H.avenae and H.filipjevi as the research object, LAMP primers were designed according to the specificity of RAPD fragments,respectively,to establish LAMP detection system.The reaction system of betaine,Mg2+,dNTPs,reaction time and temperature were optimized,and the specificity and sensitivity were test.Meanwhile,the actual sample were identificated by this detection system.After optimization,the H.avenae LAMP reaction system as follows:0.4μmol/L outer primers,2.8μmol/L inner primers,0.4umol/L loop primers,3.2mmol/L dNTPs and3mmol/L Mg2+; H.filipjevi LAMP reaction system as follows:0.4μmol/L outer primers,2.8μmol/L inner primers,0.4μmol/L loop primers,1.6mmol/L dNTPs,5mmol/L Mg2+; Reaction procedures was:63℃for75min,85℃for10min.To dilute the single cereal cyst nematode(Heterodera avenae and Heterodera filipjevi) DNA according to10concentration gradient, and comparing with ordinarly PCR method. In this study,LAMP detection sensitivity reached1/20000single nematode DNA,and none of common plant parasitic nematodes of H.glycines,H.goettingiana,H.elachista,R. Similes,M. Enterolobii,etc. showed cross reaction.It suggested that this system with a strong specificity.At the same time,The LAMP detection technology established in this study can be detect cereal cyst nemotode,H.avenae directly from diseased wheat root and soil. It is of great significance for early diagnosis of cereal cyst nematode disease.The results indicate that the LAMP was of strong specificity, high sensitivity, it is an effective method for identification of cereal cyst nematodes. And LAMP method is simple, requires simple equipment, low cost, is suitable for the application of departments at the grass-roots level.