Molecular Detection Of Phytophthora Infestans And Alternaria Solani Based On Different PCRs And LAMP Assay

Early blight and late blight,two severe diseases of potato,are widely distributed,found ubiquitously where potatoes are grown.The terms "early"and "late" refer to the relative time of their appearance in the field,although both diseases can arise at the same time.Early blight of potato is caused by the fungus,Alternaria solani,which can cause disease in potato,tomato,other members of the potato family,and some mustards.While Late blight of potato is a severe disease caused by Phytophthora infestans.It affects potato,tomato and,occasionally,eggplant and other members of the potato family.Late blight is the worst potato disease.Many species of fungi can cause disease in plants,animals and humans.Accurate and robust detection and quantification of fungi is necessary for diagnosis,modeling and surveillance.Also direct detection of fungi enables a deeper understanding of natural microbial communities,because many fungi are difficult or impossible to cultivate.In the last decade,effective amplification platforms,probe development and various quantitative PCR technologies have revolutionized study on fungal detection and identification.Examples of the latest technology in fungal detection and differentiation are discussed here.Presently in many diagnostic laboratories,the polymerase chain reaction(PCR),particularly real-time PCR,is considered the most sensitive and accurate technique for plant pathogen detection.However,laboratory-based PCRs typically require expensive laboratory equipment and skilled personnel.However,in this whole study,Late blight and Early blight pathogens of potato are used to demonstrate the potential for on-site molecular detection by using different detection strategies including conventional PCR,nested PCR,real-time qPCR,real-time PCR based on TaqMan Probe,duplex PCR and LAMP assay to find out most specific and accurate detection method.Firstly,we performed a loop-mediated isothermal amplification(LAMP)assay,conventional polymerase chain reaction(PCR),nested PCR,and real-time PCR to verify and compare the sensitivity and specificity of the reaction based on the Ypt1(Ras-related protein)gene of P.infestans documented in GenBank(accession number JN678988).In comparison with the PCR-based assays,the LAMP technique led to higher specificity and sensitivity,using uncomplicated equipment with an equivalent time frame.All 43 P.infestans isolates,yielded positive detection results using LAMP assay showing no cross reactivity with other Phytophthora spp.,oomycetes or fungal pathogens.The LAMP assay yielded the lowest detectable DNA concentration(1.28 × 10-4 ng ?L-1),being 10 times more sensitive than nested PCR(1.28 × 10-3 ng ?L-1),100 times more sensitive than real-time PCR(1.28 × 10-2 ng ?L-1)and 103 times more sensitive than the conventional PCR assay(1.28 × 10-1 ng ?L-1).In the field experiment,the LAMP assay outperformed the other tests by amplifying only diseased tissues(leaf and stem),and showing no positive reaction in healthy tissues.Overall,the LAMP assay developed in this study provides a specific,sensitive,simple,and effective visual method for detection of the P.infestans pathogen,and is therefore suitable for application in early prediction of the Late blight disease to reduce the risk of epidemics.Secondly,we conducted a loop-mediated isothermal amplification(LAMP)assay,as well as conventional polymerase chain reaction(PCR),nested PCR,and quantitative real-time PCR(RT-qPCR)assays to determine which of these techniques was less time consuming,more sensitive,and more accurate.We based our assays on sequence-characterised amplified regions of the histidine kinase gene with an accession number(FJ424058).The LAMP assay provided more rapid and accurate results,amplifying the target pathogen in less than 60 min at 63?,with 10-fold greater sensitivity than conventional PCR.Nested PCR was 100-fold more sensitive than the LAMP assay and 1000-fold more sensitive than conventional PCR.qPCR was the most sensitive among the assays evaluated,being 10-fold more sensitive than nested PCR for the least detectable genomic DNA concentration(100 fg).The LAMP assay was more sensitive than conventional PCR,but less sensitive than nested PCR and qPCR;however,it was simpler and faster than the other assays evaluated.Despite of the sensitivity,LAMP assay provided higher specificity than qPCR.The LAMP assay amplified A.solani artificially,allowing us to naturally infect young potato leaves,which produced early symptoms of EB.Hence,this technique has greater potential for developing quick and sensitive visual detection methods than other conventional PCR strategies for detecting A.solani in infected plants and culture,permitting early prediction of disease and reducing the risk of epidemics.Thirdly,A sensitive TaqMan(?)probe based real-time polymerase chain reaction(PCR)assay was established for the quantification of Phytophthora infestans,the causal agent of foliar and tuber late blight in potato.The consistency of the technique was confirmed on serially diluted fractions containing plasmid DNA with Ypt1 sequences at concentrations ranging from 108 to 10 copies per ?L and also serially diluted total DNA ranging from 126 ng to 12.6 fg using primer pairs.The real-time PCR assay we describe is highly sensitive and specific to P.infestans without showing cross reactivity with other Phytophthora spp and closely related fungi,and has several advantages over conventional PCR assays used for P.infestans detection to confirm positive inoculum level in potato leaves and elsewhere.The assay had a detection limit of 100 copies of plasmid DNA and 10 pg of Phytophthora infestans genomic DNA,which was 100-fold more sensitive than the conventional PCR.Forty five(100%)of 45 infected leaf samples from two field trials were tested to be positive by qPCR assay,whereas,by conventional PCR,only(88%)(40/45)were tested to be positive.The new qPCR assay can be used as an alternative to current diagnostic methods for P.infestans,especially when dealing with certificating a large number of healthy plants and determining disease incidence accurately in commercial fields.In our study,in case of Late blight LAMP assay proved to be highly sensitive(1.28 × 10-4 ng ?L-1)and specific,being 10 times more sensitive than nested PCR(1.28 × 10-3 ng ?L-1),100 times more sensitive than real-time PCR(1.28 ×10-2 ng ?L-1)and 103 times more sensitive than the conventional PCR assay(1.28 × 10-1 ng ?L-1).But in case of Alternaria solani,LAMP didn't prove to be so sensitive amongst all other assays,being 10-fold sensitive than conventional PCR,and 100-fold less sensitive than nested PCR,followed by 1000 fold less sensitive than real-time qPCR but specific amongst all.The real-time PCR based on TaqMan Probe had a detection limit of 100 copies of plasmid DNA and 10 pg of Phytophthora infestans genomic DNA,which was 100-fold more sensitive than the conventional PCR.So,therefore on-site diagnosis of plant diseases is a useful tool for growers for timely decisions enabling the earlier implementation of disease management strategies that decrease the impact of the disease.