LAMP Detection And Pathogenicity Differences Of Berkeleyomyces Basicola

As a common filamentous,hemibiotrophs soil-borne plant pathogen,Berkeleyomyces basicola can cause black root rot of 137 different plants,such as tobacco,cotton,carrots and so on,which is distributed all over the world.B.basicola threatened the quality and yields of the crops all above seriously.However,there is no relevant report on the LAMP detection of the pathogen,few reports revealed about the pathogenicity differences,and the utilization of carbon and nitrogen sources of B.basicola isolates from different hosts.For making clear the projects of above,and in order to provide a basis for those kind disese early diagnosis and disese control,this study was carried out systematically,the results showed as follows:1.Four specific primers were designed for the target gene based on the 28s rDNA sequence,and the LAMP detection system for B.basicola was successfully established.The optimized detection system(25?L)contained:1?L of MgSO₄(100 mM),2.5?L of 10×Thermopol Buffer,1.5?L of d NTP mixed solution(10 mM),3?L of betaine(5 M),and internal primers(FIP,BIP)40?M each 1?L,5?M external primers(F3,B3)1?L each,BstDNA polymerase(8U/?l)1?L,DNA template 1?L,sterile dd H₂O11.75?L.Each component was reacted in a 0.2 m L PCR tube at 62?for 60min,then inactivated at 80?for 20min,and stained with 1?L SYBR Green?to observe the color change.2?L of LAMP reaction product was coagulated on 2%agarose perform electrophoresis detection on gel electrophoresis.The detection of the disease samples showed that the primers had strong specificity,and the optimized reaction system had high sensitivity.The lowest detection concentration was 162 fg/?L.This method was easy to operate and the detection results were visible to the naked eye.2.To clarify using an improved wound inoculation method,the pathogenicity differences of B.basicola isolates from different hosts were observed.The results showed that the pathogenicity of B.basicola from different hosts to the same grafted plant was different,and the same strain also had different pathogenicity to different host plants.Among them,the HLL5-2 strain has a low pathogenicity to other test plants except for its high pathogenicity to tobacco,10HU-7B has a moderate pathogenicity to all test plants,and SYSB-03 has a low pathogenicity to other test plants The pathogenicity of the test plants is relatively high.At the same time,this study also found that B.basicola are more pathogenic to their hosts than to other hosts.3.The metabolism of 180 carbon sources and 90 nitrogen sources of the three isolates with different pathogenicity of above were analyzed by Biolog phenotypic chip technology.That's showed the utilization of D-mannitol,D-fructose,maltose,fructose,maltotriose,D-cellobiose and kelp polysaccharide was higher in SYSB-03than in HLL5-2 and 10HU-7B strains.The metabolism of quinic acid,L-sorbose and D-glucosamine in 10HU-7B is higher than in SYSB-03 and HLL5-2.The metabolism of L-methionine,D-glucosamine and D,L-a-amino-N-butyric acid in SYSB-03 is stronger than that in 10HU-7B.It has been shown that D-Mannitol,maltose and fructose can promote the resistance of fungi,the germination rate of spores and the growth of mycelium,especially maltose may be related to the secretion of some extracellular enzymes,which is the reason why SYSB-03 has strong pathogenicity..