Molecular Detection Of Didymella Bryoniae And Its Carbendazim-Resistant Isolates

Watermelon is an important economic crop in China,which has a large planting area throughout our country.The yield and quality of watermelon directly affects the economic income,and the occurrence of diseases can lead to the serious economic losses.Gummy stem blight?GBS?and watermelon Fusarium wilt are caused by two soil-borne pathogens,Fusarium oxysporum f.sp.niveum and Didymella bryoniae which can occur at all growing stages.The symptoms of these two diseases are very similar on the stems in the early stage,it is difficult to distinguish according to the symptoms.Besides,there is another serious disease,watermelon anthracnose,can occur not only during the growth period of watermelon,but also during the transportation and storage.GBS and watermelon anthracnose have similar symptoms on the leaves which can increase the difficulty of identification.However the occurrence of these three diseases and the main methods to prevent are not exactly the same.In the early stage of disease,if we can make a correct judgment to the pathogens,the corresponding measures can be taken to control the disease.Molecular technology such as PCR and LMAP can diagnose the diseases and reduce economic loss.In our country,GBS is mainly controlled by the application of chemical means.The benzimidazole fungicide has a good effect on the pathogens,so it was widely used for a long time.However,due to the single action site of the fungicide and the frequent use which leads to the severe resistance to the D.bryoniae.According to analyzing the result of monitoring the resistance of carbendazim to the D.bryoniae,we found that the resistant isolates in the field have become dominant populations and the level of resistance was high.The traditional detection method based on the mycelial growth inhibition or spore germination was time-consuming and complicated.Loop-mediated isothermal amplification?LAMP?is a novel nucleic acids amplification technique with high rapidity and specificity.Besides,LAMP products can be visualized by naked eyes after adding the metal indictor hydroxynaphthol blue?HNB?.Throughout the process of detection we don't need to use expensive equipment.LAMP assay is useful for assessing carbendazim resistance in the field.The results of research as follows:1.Triplex PCR detection of Didymella bryoniae,Fusarium oxysporum f.sp.niveum and Colletotrichum orbiculareIn this assay,in order to develop a triplex PCR method for simultaneous detection of D.bryoniae,F.oxysporum f.sp.niveum and C.orbiculare,three sets of specific primers were designed using the RAPD-to-SCAR molecular markers and functional gene.Specificbandof 171bpcanbeamplifiedbytheprimerof Db-F/Db-R?F:5'-GGACAACGAGGACCCGAG-3';R:5'-GAGAGACCGCATACAAG TTTT-3'?,specific band of 255 bp can be amplified by the primer of Ku-F/Ku-R?F:5'-ATTGCTGGTGTCATTCCG-3';R:5'-TTCATCTGCTCCATCTACTAGT-3'?and specific band of 394 bp can be amplified by the primer of Co-F/Co-R?F:5'-AGCCCTCTACGATAACCCA-3',R:5'-GGATACCACGCTTCGACTG-3'?.The detection sensitivity of the multiplex PCR is 10 pg for D.bryoniae,1 pg for F.oxysporum f.sp.niveum,and 10 pg for C.orbiculare.Triplex PCR system was optimized by the orthogonal design method,the reaction volume was 20?L that contained with 0.8?L of DNA from each pathogen,2?L of 10×PCR Buffer,1.5 mM Mg²⁺,0.3 mM dNTP mix,1.5 U Taq DNA polymerase,0.25?M Co-F/Co-R,0.25?M Ku-F/Ku-R,0.35?M Db-F/Db-R,and 8.1?L of ddH₂O.And the performed conditions was:initial denaturation for 5 min at 95?;35 cycles of 95?for 30 sec,52?for30 sec,and 72?for 30 sec;additional extension at 72?for 10 min.The multiplex PCR developed in this study can be used to successfully detect the three pathogens simultaneously from the field.This approach allows the rapid and efficient diagnosis and prediction of disease occurrences and provides an important guideline for disease prevention and treatment on watermelons.2.Rapid LAMP detection system of detection of D.bryoniaeTo realize the rapid detection of D.bryoniae in the field,a loop-mediatedisothermal amplification?LAMP?detection system was established.Based on the ITS sequence of D.bryoniae,the LAMP primers were designed unsing primer Explorer Version 4.0,and the primer sequences were F3:5'-TGTTCGAGCGTCATTTGT-3';B3:5'-GTTCAGCGGGTATCCCTACC-3';FIP:5'-AATCGTTTTGAGGCGAGTCTGCGC-CCTTCAAGCTTTGCTTGGTG-3';BIP:5'-GAGCGCAGTACATCTCGCGCTT-GATCCGAGGTCAAGAGTGT-3',respectively.Hydroxynaphthol blue?HNB?was used as a coloring indicator,the color change of the HNB indicator after the reaction was observed according to the naked eye,and the LAMP detection result was visualized.The color change of HNB after LAMP reaction can be visually observed to visualize the disease detection results.The optimized 10?L optimized reaction system contained with 10×Isothermal Amplification Buffer 1?L,Bst DNA polymerase 0.16 U/?L,0.8 mmol/L dNTPs,1mmol/L of Mg²⁺,0.8 mol/L of betaine,100?mol/L of HNB,0.4?mol/L of F3/B3,1.6?mol/L of BIP/FIP,and 0.5?L of DNA.LAMP reaction was performed at 60?for 60min.The sensitivity of the LAMP was up to 1 ng/?L.These primers can only be amplified in the DNA of D.bryoniae,which makes the color of HNB is changed from violet to sky blue,and produces distinct trapezoidal bands with good specificity.Using this LAMP detection system could detect D.bryoniae in the plant tissues from field within 2 hours.3.LAMP detection system for carbendazim resistant mutant strains of D.bryoniaeTo shorten the monitoring time of the carbendazim resistant strains of D.bryoniae in the field,the specific LAMP primers were designed based on the?-tubulin sequence of the carbendazim-resistant strain.According to the specificity of the inner and outer primers of LAMP,the mutation point was placed at the 3'end of the FIP,and a single base mutation was artificially introduced near the mutation point to obtain the LAMP ofthecarbendazimresistantstrain.TheprimersequenceswereRF3:5'-TACAACGCCACCCTCTCC-3';RB:5'-TGAGCTGACCGGGGAAAC-3;RFIP:5'-TGTCGTAGAGGGCCTCGTTGTC-TTGTCGAGAACTCTGACGCC-3';RBIP:5'-ACAACCCCTCTTACGGTGACCT-GCAGGTGGTTACACCAGAC-3',respectively.The optimized 10?L optimized reaction system contained with 10×Isothermal Amplification Buffer 1?L,Bst DNA polymerase 0.16 U/?L,0.8 mmol/L of dNTPs,1 mmol/L of Mg²⁺,0.8 mol/L of betaine,100?mol/L of HNB,0.4?mol/L of RF3/B3,1.6?mol/L of RBIP/FIP,and 0.5?L of DNA.LAMP reaction was performed at 60?for 50 min.The sensitivity of the LAMP was up to 1 ng/?L.To further verify the reliability of LAMP primers,the traditional detection method of fungicide was combined used to monitor the pathogens isolated from the Wulong and Beibei of Chongqing in 2017 and 2018,respectively.The results of traditional detection method showed that the frequency of carbendazim resistance was 92.16%,and the results of LAMP detection system showed that was 88.89%.The results of the resistance frequencies obtained by these two methods were almost the same,indicating that the LAMP method has certain reliability.In a word,these LAMP primers have the advantages of fastness,simplicity,and strong operability,which can provide corresponding medication guidance for the resistance management of D.bryoniae in the field.In summary,this study established a triple PCR system that can simultaneously detect and distinguish between D.bryoniae,F.oxysporum f.sp.niveum and C.orbiculare in the infected tissue which can reduce time and cost.Moreover,the LAMP detection system was established according to the specific ITS sequence of D.bryoniae.This method can detect the D.bryoniae from the infected tissue more easily and quickly.In addition,specific LAMP primers for detecting carbendazim resistant strains of D.bryoniae was obtained by introducing an artificial mismatched base.Compared with the traditional method,LAMP technology can shorten the identification time and improve work efficiency,which provides an important detection way for disease monitoring.