Research ArticleDevelopment of a SCAR Marker-Based DiagnosticMethod for the Detection of the Citrus Target Spot PathogenPseudofabraea citricarpa

Yuheng Yang 12 Junhua Hu3 Fajing Chen1 Dekuan Ding4 and Changyong Zhou 3

1College of Plant Protection Southwest University Chongqing 400715 China2Institute for Bioscience and Biotechnology Research University of Maryland College Park College Park MD 20850 USA3Citrus Research Institute Southwest University Chongqing 400712 China4Citrus Institute of Chenggu County Shaanxi 723200 China

Correspondence should be addressed to Yuheng Yang yyh023swueducn and Changyong Zhou zhoucycriccn

Received 19 October 2017 Accepted 17 April 2018 Published 3 June 2018

Academic Editor Peter J Oefner

Copyright copy 2018 Yuheng Yang et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Target spot a recently observed citrus disease that is caused by Pseudofabraea citricarpa can cause substantial economic lossesin citrus production In this study a 797 bp marker specific to Ps citricarpa was identified via random amplified polymorphicDNA (RAPD) technique The primer pair Pc-SFPPc-SRP which was designed from RAPD amplicons was utilized as a sequence-characterized amplified region (SCAR) marker This marker identified Ps citricarpa with a single and distinct band of 389 bp butdid not amplify DNA from other tested fungal species The PCR assay was highly sensitive to the target DNA at picogram levelsand could reliably amplify Ps citricarpa sequences with the Pc-SFPPc-SRP primer pair The SCAR marker that was identified inthe present study can facilitate rapid decision-making and precise disease forecasting and management

1 Introduction

Target spot a new leaf-spotting disease of citrus first de-scribed in China has caused considerable economic losses inlocal citrus production [1] The target spot pathogen wasidentified as Cryptosporiopsis citricarpa based on Kochrsquospostulates and morphological and molecular phylogeneticcharacteristics [1] and then reclassified to the monotypicgenus Pseudofabraea [2] This fungal pathogen could infectboth Satsuma mandarin (Citrus unshiu) and kumquat (For-tunellamargarita) in orchards [1] Unlike diseases that usuallyoccur on the young leaves of citrus during warm and humidseasons target spot occurs during late winter and early springand causes severe leaf spotting or even defoliation (Figure 1)However target spot is difficult to diagnose accurately basedsolely on experience and subjective judgment Once thedisease becomes epidemic fungicide application was difficultto control effectivelyTherefore monitoring the disease in thecitrus orchards plays a key role in effective control of targetspot

Citrus infected by Ps citricarpa does not show any symp-toms at early stages of invasion which is difficult to deter-mine the primary infection potential and earlymolecular de-tection of this pathogen In recent decades molecular meth-ods particularly nucleic acid-based methods have beenapplied to identify and detect plant pathogens thesemethodscan overcomeuncertain diagnosis or pathogen taxonomy andenable the rapid and accurate detection and quantificationof pathogens [3 4] Sequence-characterized amplified region(SCAR) a kind of reliable PCR-based molecular marker hasbeen developed to detect plant pathogens such as Magna-porthe grisea [5] Puccinia striiformis [6] and Fusarium oxys-porum [7]The use of the SCARmarkers simplifies identifica-tion and promotes the development of prevention strategiesthat are superior to traditional methods

In the current study we developed a useful SCARmarkervia the simple random amplified polymorphic DNA (RAPD)technique [8 9] and establish a sensitive and simple PCR-based method for the rapid molecular identification and

HindawiBioMed Research InternationalVolume 2018 Article ID 7128903 5 pageshttpsdoiorg10115520187128903

2 BioMed Research International

Figure 1 Symptoms of citrus target spot caused by Pseudofabraea citricarpa

differentiation of Ps citricarpa from other fungal pathogensof citrus

2 Materials and Methods

21 Fungal Pathogens Ps citricarpa strains were isolatedfrom citrus leaves or shoots with disease symptoms Thediseased plant materials were obtained from local orchardsFive fungal pathogens of citrus leaveswere collected fromCit-rus Research Institute Southwest University The pathogensincluded Alternaria alternata Colletotrichum gloeospori-oides Diaporthe citri Botrytis cinerea and Phyllosticta citri-carpa Three fungal pathogens of citrus fruit were collectedfrom the College of Food Science Southwest UniversityThe pathogens included Oospora citri-aurantii Penicilliumitalicum and Pe digitatum Except for Ps citricarpa whichwas cultured at 20∘C all tested strains were cultured at 25∘Con potato dextrose agar media until the mycelium coveredapproximately three-quarters of the plates

22 DNA Isolation Approximately 1 g of fresh fungal my-celium and approximately 03 g of field-infected citrus tis-sues were snap-frozen in liquid nitrogen and ground to afine powder with a mortar and pestle Genomic DNA wasextracted via the CTAB method [10] DNA samples weredissolved in 01x TE buffer quantified and adjusted to a finalconcentration of 100 ng120583L for PCR amplification

23 RAPD Analysis RAPD amplification was conductedwith 15 120583L of reaction mixture with 40 random primers(Table S1) Each reaction tube contained 100 ng of DNA 1Uof rTaq DNA polymerase (Takara Co China) 100 120583molLof each dNTP 15 120583L of 10x Taq DNA polymerase bufferwith 15mmolL MgCl

2 and 10 120583L of random primer

(10mmolL) PCR amplification was performed in a DNAthermocycler (Bio-Rad S1000) with the following condi-tions 94∘C for 5min 35 cycles at 94∘C for 30 s 36∘C for 30 sand 72∘C for 90 s with a final extension at 72∘C for 10minTheamplified PCR products were resolved on 15 agarose gelsfollowed by GoldView staining and visualization under UVlight

24 Amplicon Cloning and Sequencing The amplicon whichwas specific to Ps citricarpa but absent in the other eightspecies was identified and purified with a gel extractionminikit (Tiangen Biotech Co China)The purifiedDNAproductswere cloned into a pGEM-T Easy vector (Promega Co USA)and introduced into the competent cells of Escherichia colistrain DH5120572 in accordance with manufacturerrsquos instructionsSubsequently the positive cloneswere sequenced by ShanghaiBiotech Co

25 Primer Design and Establishment of Detection SystemBased on the sequenced RAPD amplicons the specific SCARprimers (Table 1) Pc-SFP (specific forward primer) and Pc-SRP (specific reverse primer) were designed using PrimerPremier 6 software (Premier Biosoft International USA) A20120583L reaction systemwas developed to simplify the detectionsystem The system contained 10 120583L of Premix Taq Version20 plus dye (Takara Co China) 10 120583L of forward primer(10mmolL) 10 120583L of reverse primer (10mmolL) and100 ng of genomic DNA Amplifications were conducted ina DNA thermocycler (Bio-Rad S1000) with the followingconditions 94∘C for 5min 35 cycles at 94∘C for 30 s 55∘Cfor 30 s and 72∘C for 60 s with a final extension at 72∘C for10min

26 Specificity and Sensitivity of the SCAR Marker All DNAsamples including those from six foliar pathogens and threepostharvest pathogens of citrus were amplified via PCRwith the Pc-SPF and Pc-SPR primers (Table 1) to verify thespecificity of the SCAR marker To test detection sensitivity50 ng120583L to 5 fg120583L serial dilutions of the DNA of Ps citri-carpa strainwere used as theDNA templates for PCR amplifi-cation under the above thermocycling conditions

27 Validating SCAR Marker in Citrus Tissues Collected fromOrchards To confirm the effectiveness of the primer pairsPc-SPF and Pc-SPR for detecting Ps citricarpa in the fieldthe primers were used to amplify DNA samples from symp-tomatic and asymptomatic citrus tissues that were collecteddiseased orchards DNAwas extracted from leaves and shootsin accordancewith themethod described abovePs citricarpaDNA was used as positive control and the DNA of healthy

BioMed Research International 3

Table 1 Pseudofabraea citricarpa-specific SCAR primers designed from sequenced RAPD amplicons

RAPD primer SCAR markerNumber ofbase pairs

(bp)Nucleotide sequence G + C content () Annealing

temperature

CS38 Pc-SFP 20 51015840-GCTGATTGAGTGCCCATAGA-31015840 50 55∘CPc-SRP 22 51015840-ACTCCAACCAACGAGATGATAG-31015840 45

Figure 2 Random amplified polymorphic DNA (RAPD) profiles of Pseudofabraea citricarpa and other citrus fungal pathogens obtainedwith random primer CS38 M DNA ladder 2000 lane 1 Ps citricarpa lane 2 Alternaria alternata lane 3 Colletotrichum gloeosporioides lane4 Diaporthe citri lane 5 Botrytis cinerea lane 6 Oospora citri-aurantii lane 7 Phyllosticta citricarpa lane 8 Penicillium italicum lane 9 Pedigitatum The dotted box represents the location of the Ps citricarpa-specific band

Figure 3 Specific DNA sequence of Pseudofabraea citricarpaobtained with the RAPD primer CS38The gray region indicates thesequence that was amplified by the primer pair Pc-SFPPc-SRP (thesequence of the primer pairs were in bold) The first 10 nucleotidesof the obtained sequence completely matched the correspondingRAPD primer CS38

citrus leaves obtained from greenhouse were used as negativecontrol PCR amplification was performed with the primersPc-SPF and Pc-SPR under the above conditions

3 Results

31 Screening and Sequencing of RAPD Markers for Pscitricarpa Of the 40 screened RAPD primers CS38 (51015840-TGCTGACGAC-31015840) consistently amplified a single intenseband of over 750 bp from Ps citricarpa This band was absentin the eight other pathogens (Figure 2)This differential bandwas selected to develop a species-specific SCAR marker andsubsequently was cloned and sequenced The sequencingresult showed that the length of the specific amplicon was797 bp with 50G+C content (119860 = 188119879 = 212119862 = 176 and119866 = 221) (Figure 3) BLAST result revealed that no significantsimilar sequence had been found at different levels

32 Specific SCAR Marker Design and Amplification Theprimer pair Pc-SFPPc-SRP (Table 1) was designed usingPrimer Premier 60 software (Premier Biosoft International)

Figure 4 The specificity of PCR product for the detection ofPseudofabraea citricarpa using the primer pair Pc-SFPPc-SRP MDNA ladder 2000 lane 1 Ps citricarpa lane 2 Alternaria alternatalane 3 Colletotrichum gloeosporioides lane 4 Diaporthe citri lane5 Botrytis cinerea lane 6 Oospora citri-aurantii lane 7 Phyllostictacitricarpa lane 8 Penicillium italicum lane 9 Pe digitatum

based on the sequence of the specific amplicon When Pc-SFP and Pc-SRPwere used to amplify genomicDNA from thenine selected pathogens a single and distinct band of 389 bpwas only observed in Ps citricarpa (Figure 4) Sequencinganalysis showed the amplicon was the expected Ps citricarpafragment indicating that the designed SCAR marker is spe-cific for the citrus target spot pathogen

33 Sensitivity Test of the Detection System To test thesensitivity of the specific marker for detecting Ps citricarpaserial dilutions of Ps citricarpa DNA were used as templatesin the PCRassaywith Pc-SFP andPc-SRPprimersThe resultsrevealed that the SCAR marker could detect Ps citricarpaDNA at levels as low as 50 pg120583L (Figure 5)

34Detection of Ps citricarpa inOrchards To test the reliabil-ity of the Ps citricarpa-specific SCARmarker Pc-SFP and Pc-SRP citrus leaves and shoots without any visible symptomswere collected from diseased orchards and were used for theverification test The expected 389 bp bands were obtainedfrom portions of the selected samples (Figure 6) No PCR

4 BioMed Research International

Figure 5 The PCR sensitivity of the primer pair Pc-SFPPc-SRPwith a serial dilution of Pseudofabraea citricarpa DNA M DNAladder 2000 lane 1 50 ng120583L lane 2 5 ng120583L lane 3 500 pg120583L lane4 50 pg120583L lane 5 5 pg120583L lane 6 500 fg120583L lane 7 50 fg120583L lane8 5 fg120583L

Figure 6 PCR amplification using DNA extracted from citrussamples that were collected from orchards with target spot M DNAladder 5000 lane 1 positive control (Ps citricarpa DNA) lanes2ndash15 citrus leaves or shoots without any visible symptoms lane 16negative control (uninfected citrus leaf DNA)

product was amplified in the negative control (uninfectedcitrus leaves) The results validated the reliability of the de-signed SCAR marker

4 Discussion

Given that knowledge on the infection cycle and diseaseepidemics of citrus target spot is limited the disease has beenmistaken as a brown spot or anthracnose for prevention andcontrol for a long time which caused poor control effects[11]The sensitivity tests showed that the SCARmarker coulddetect as low as 50 pg120583L ofPs citricarpaDNAextracted frommycelia and from citrus leaves or shoots collected diseasedorchards but not fromhealthy leaves (Figure 5)These resultsindicated that the proposed amplification system could helpillustrate the oversummering mechanism and occurrencecharacteristics of citrus target spot which will be useful forthe effective forecasting and management of this disease

RAPD analysis reveals a high degree of polymorphismeven without the DNA sequence information of the speciesmoreover RAPD is easy to perform [12] Given the advan-tages of low workload rapidity and high efficiency comparedwith traditional identification methods RAPD-based SCARmarkers are extensively used for the in planta detection ofseveral plant pathogens [5 13 14] The SCAR marker devel-oped in this study can also facilitate rapid decision-makingand precise early season disease management to reduce therisk of Ps citricarpa epidemics

Conflicts of Interest

The authors declared no conflicts of interest

Acknowledgments

This study was funded by the Chongqing Research ProgramofBasicResearchandFrontierTechnology (cstc2016jcyjA0316)theChongqingPostdoctoral Science Foundation (Xm2016124)the Fundamental Research Funds for the Central Universi-ties (XDJK2016A020) and the China Scholarship Council(201706995068) The authors thank Professor Kaifang ZengSouthwest University for providing them with the fungalpathogens of citrus fruits used in this study

Supplementary Materials

Table S1 sequence of random amplified polymorphic DNA(RAPD) PCR primers used in this study (Supplementary Ma-terials)

References

[1] L Zhu X Wang F Huang et al ldquoA destructive new disease ofcitrus in China caused by Cryptosporiopsis citricarpa sp novrdquoPlant Disease vol 96 no 6 pp 804ndash812 2012

[2] C Chen G J M Verkley G Sun J Z Groenewald and P WCrous ldquoRedefining common endophytes and plant pathogensinNeofabraea Pezicula and related generardquoFungal Biology vol120 no 11 pp 1291ndash1322 2016

[3] F Martinelli R Scalenghe S Davino et al ldquoAdvanced methodsof plant disease detection A reviewrdquo Agronomy for SustainableDevelopment vol 35 no 1 pp 1ndash25 2015

[4] M M Lopez P Llop A Olmos E Marco-Noales M Cambraand E Bertolini ldquoAre molecular tools solving the challengesposed by detection of plant pathogenic bacteria and virusesrdquoCurrent Issues in Molecular Biology vol 11 pp 13ndash46 2009

[5] LG Jesumaharaja RManikandan andT Raguchander ldquoSCARmarker specific to detect Magnaporthe grisea infecting fingermillets (Eleusine coracana)rdquo Journal of Applied Microbiologyvol 121 no 3 pp 778ndash786 2016

[6] J Zhao X J Wang C Q Chen L L Huang and Z S KangldquoA PCR-based assay for detection of Puccinia striiformis f sptritici in wheatrdquo Plant Disease vol 91 no 12 pp 1669ndash16742007

[7] Y-H Lin C-C Su C-P Chao et al ldquoA molecular diagnosismethod using real-time PCR for quantification and detection ofFusarium oxysporum f sp cubense race 4rdquo European Journal ofPlant Pathology vol 135 no 2 pp 395ndash405 2013

[8] B A McDonald ldquoThe population genetics of fungi Tools andtechniquesrdquo Journal of Phytopathology vol 87 no 4 pp 448ndash453 1997

[9] J G KWilliams A R Kubelik K J Livak J A Rafalski and SV Tingey ldquoDNApolymorphisms amplified by arbitrary primersare useful as geneticmarkersrdquoNucleic Acids Research vol 18 no22 pp 6531ndash6535 1990

[10] A Velegraki M Kambouris A Kostourou G ChalevelakisandN J Legakis ldquoRapid extraction of fungal DNA from clinicalsamples for PCR amplificationrdquoMedical Mycology vol 37 no 1pp 69ndash73 1999

[11] Y Liu W Tian and G Wei ldquoFeature and prevention of citrustarget spotrdquo Fruit Growerrsquos Friend vol no 5 p 36 2015

BioMed Research International 3

Table 1 Pseudofabraea citricarpa-specific SCAR primers designed from sequenced RAPD amplicons

RAPD primer SCAR markerNumber ofbase pairs

(bp)Nucleotide sequence G + C content () Annealing

temperature

CS38 Pc-SFP 20 51015840-GCTGATTGAGTGCCCATAGA-31015840 50 55∘CPc-SRP 22 51015840-ACTCCAACCAACGAGATGATAG-31015840 45

Figure 2 Random amplified polymorphic DNA (RAPD) profiles of Pseudofabraea citricarpa and other citrus fungal pathogens obtainedwith random primer CS38 M DNA ladder 2000 lane 1 Ps citricarpa lane 2 Alternaria alternata lane 3 Colletotrichum gloeosporioides lane4 Diaporthe citri lane 5 Botrytis cinerea lane 6 Oospora citri-aurantii lane 7 Phyllosticta citricarpa lane 8 Penicillium italicum lane 9 Pedigitatum The dotted box represents the location of the Ps citricarpa-specific band

Figure 3 Specific DNA sequence of Pseudofabraea citricarpaobtained with the RAPD primer CS38The gray region indicates thesequence that was amplified by the primer pair Pc-SFPPc-SRP (thesequence of the primer pairs were in bold) The first 10 nucleotidesof the obtained sequence completely matched the correspondingRAPD primer CS38

citrus leaves obtained from greenhouse were used as negativecontrol PCR amplification was performed with the primersPc-SPF and Pc-SPR under the above conditions

3 Results

31 Screening and Sequencing of RAPD Markers for Pscitricarpa Of the 40 screened RAPD primers CS38 (51015840-TGCTGACGAC-31015840) consistently amplified a single intenseband of over 750 bp from Ps citricarpa This band was absentin the eight other pathogens (Figure 2)This differential bandwas selected to develop a species-specific SCAR marker andsubsequently was cloned and sequenced The sequencingresult showed that the length of the specific amplicon was797 bp with 50G+C content (119860 = 188119879 = 212119862 = 176 and119866 = 221) (Figure 3) BLAST result revealed that no significantsimilar sequence had been found at different levels

32 Specific SCAR Marker Design and Amplification Theprimer pair Pc-SFPPc-SRP (Table 1) was designed usingPrimer Premier 60 software (Premier Biosoft International)

Figure 4 The specificity of PCR product for the detection ofPseudofabraea citricarpa using the primer pair Pc-SFPPc-SRP MDNA ladder 2000 lane 1 Ps citricarpa lane 2 Alternaria alternatalane 3 Colletotrichum gloeosporioides lane 4 Diaporthe citri lane5 Botrytis cinerea lane 6 Oospora citri-aurantii lane 7 Phyllostictacitricarpa lane 8 Penicillium italicum lane 9 Pe digitatum

based on the sequence of the specific amplicon When Pc-SFP and Pc-SRPwere used to amplify genomicDNA from thenine selected pathogens a single and distinct band of 389 bpwas only observed in Ps citricarpa (Figure 4) Sequencinganalysis showed the amplicon was the expected Ps citricarpafragment indicating that the designed SCAR marker is spe-cific for the citrus target spot pathogen

33 Sensitivity Test of the Detection System To test thesensitivity of the specific marker for detecting Ps citricarpaserial dilutions of Ps citricarpa DNA were used as templatesin the PCRassaywith Pc-SFP andPc-SRPprimersThe resultsrevealed that the SCAR marker could detect Ps citricarpaDNA at levels as low as 50 pg120583L (Figure 5)

34Detection of Ps citricarpa inOrchards To test the reliabil-ity of the Ps citricarpa-specific SCARmarker Pc-SFP and Pc-SRP citrus leaves and shoots without any visible symptomswere collected from diseased orchards and were used for theverification test The expected 389 bp bands were obtainedfrom portions of the selected samples (Figure 6) No PCR

4 BioMed Research International

Figure 5 The PCR sensitivity of the primer pair Pc-SFPPc-SRPwith a serial dilution of Pseudofabraea citricarpa DNA M DNAladder 2000 lane 1 50 ng120583L lane 2 5 ng120583L lane 3 500 pg120583L lane4 50 pg120583L lane 5 5 pg120583L lane 6 500 fg120583L lane 7 50 fg120583L lane8 5 fg120583L

Figure 6 PCR amplification using DNA extracted from citrussamples that were collected from orchards with target spot M DNAladder 5000 lane 1 positive control (Ps citricarpa DNA) lanes2ndash15 citrus leaves or shoots without any visible symptoms lane 16negative control (uninfected citrus leaf DNA)

product was amplified in the negative control (uninfectedcitrus leaves) The results validated the reliability of the de-signed SCAR marker

4 Discussion

Given that knowledge on the infection cycle and diseaseepidemics of citrus target spot is limited the disease has beenmistaken as a brown spot or anthracnose for prevention andcontrol for a long time which caused poor control effects[11]The sensitivity tests showed that the SCARmarker coulddetect as low as 50 pg120583L ofPs citricarpaDNAextracted frommycelia and from citrus leaves or shoots collected diseasedorchards but not fromhealthy leaves (Figure 5)These resultsindicated that the proposed amplification system could helpillustrate the oversummering mechanism and occurrencecharacteristics of citrus target spot which will be useful forthe effective forecasting and management of this disease

RAPD analysis reveals a high degree of polymorphismeven without the DNA sequence information of the speciesmoreover RAPD is easy to perform [12] Given the advan-tages of low workload rapidity and high efficiency comparedwith traditional identification methods RAPD-based SCARmarkers are extensively used for the in planta detection ofseveral plant pathogens [5 13 14] The SCAR marker devel-oped in this study can also facilitate rapid decision-makingand precise early season disease management to reduce therisk of Ps citricarpa epidemics

Conflicts of Interest

The authors declared no conflicts of interest

Acknowledgments

This study was funded by the Chongqing Research ProgramofBasicResearchandFrontierTechnology (cstc2016jcyjA0316)theChongqingPostdoctoral Science Foundation (Xm2016124)the Fundamental Research Funds for the Central Universi-ties (XDJK2016A020) and the China Scholarship Council(201706995068) The authors thank Professor Kaifang ZengSouthwest University for providing them with the fungalpathogens of citrus fruits used in this study

Supplementary Materials

Table S1 sequence of random amplified polymorphic DNA(RAPD) PCR primers used in this study (Supplementary Ma-terials)

References

[1] L Zhu X Wang F Huang et al ldquoA destructive new disease ofcitrus in China caused by Cryptosporiopsis citricarpa sp novrdquoPlant Disease vol 96 no 6 pp 804ndash812 2012

[2] C Chen G J M Verkley G Sun J Z Groenewald and P WCrous ldquoRedefining common endophytes and plant pathogensinNeofabraea Pezicula and related generardquoFungal Biology vol120 no 11 pp 1291ndash1322 2016

[3] F Martinelli R Scalenghe S Davino et al ldquoAdvanced methodsof plant disease detection A reviewrdquo Agronomy for SustainableDevelopment vol 35 no 1 pp 1ndash25 2015

[4] M M Lopez P Llop A Olmos E Marco-Noales M Cambraand E Bertolini ldquoAre molecular tools solving the challengesposed by detection of plant pathogenic bacteria and virusesrdquoCurrent Issues in Molecular Biology vol 11 pp 13ndash46 2009

[5] LG Jesumaharaja RManikandan andT Raguchander ldquoSCARmarker specific to detect Magnaporthe grisea infecting fingermillets (Eleusine coracana)rdquo Journal of Applied Microbiologyvol 121 no 3 pp 778ndash786 2016

[6] J Zhao X J Wang C Q Chen L L Huang and Z S KangldquoA PCR-based assay for detection of Puccinia striiformis f sptritici in wheatrdquo Plant Disease vol 91 no 12 pp 1669ndash16742007

[7] Y-H Lin C-C Su C-P Chao et al ldquoA molecular diagnosismethod using real-time PCR for quantification and detection ofFusarium oxysporum f sp cubense race 4rdquo European Journal ofPlant Pathology vol 135 no 2 pp 395ndash405 2013

[8] B A McDonald ldquoThe population genetics of fungi Tools andtechniquesrdquo Journal of Phytopathology vol 87 no 4 pp 448ndash453 1997

[9] J G KWilliams A R Kubelik K J Livak J A Rafalski and SV Tingey ldquoDNApolymorphisms amplified by arbitrary primersare useful as geneticmarkersrdquoNucleic Acids Research vol 18 no22 pp 6531ndash6535 1990

[10] A Velegraki M Kambouris A Kostourou G ChalevelakisandN J Legakis ldquoRapid extraction of fungal DNA from clinicalsamples for PCR amplificationrdquoMedical Mycology vol 37 no 1pp 69ndash73 1999

[11] Y Liu W Tian and G Wei ldquoFeature and prevention of citrustarget spotrdquo Fruit Growerrsquos Friend vol no 5 p 36 2015

4 BioMed Research International

Figure 5 The PCR sensitivity of the primer pair Pc-SFPPc-SRPwith a serial dilution of Pseudofabraea citricarpa DNA M DNAladder 2000 lane 1 50 ng120583L lane 2 5 ng120583L lane 3 500 pg120583L lane4 50 pg120583L lane 5 5 pg120583L lane 6 500 fg120583L lane 7 50 fg120583L lane8 5 fg120583L

Figure 6 PCR amplification using DNA extracted from citrussamples that were collected from orchards with target spot M DNAladder 5000 lane 1 positive control (Ps citricarpa DNA) lanes2ndash15 citrus leaves or shoots without any visible symptoms lane 16negative control (uninfected citrus leaf DNA)

product was amplified in the negative control (uninfectedcitrus leaves) The results validated the reliability of the de-signed SCAR marker

4 Discussion

Given that knowledge on the infection cycle and diseaseepidemics of citrus target spot is limited the disease has beenmistaken as a brown spot or anthracnose for prevention andcontrol for a long time which caused poor control effects[11]The sensitivity tests showed that the SCARmarker coulddetect as low as 50 pg120583L ofPs citricarpaDNAextracted frommycelia and from citrus leaves or shoots collected diseasedorchards but not fromhealthy leaves (Figure 5)These resultsindicated that the proposed amplification system could helpillustrate the oversummering mechanism and occurrencecharacteristics of citrus target spot which will be useful forthe effective forecasting and management of this disease

RAPD analysis reveals a high degree of polymorphismeven without the DNA sequence information of the speciesmoreover RAPD is easy to perform [12] Given the advan-tages of low workload rapidity and high efficiency comparedwith traditional identification methods RAPD-based SCARmarkers are extensively used for the in planta detection ofseveral plant pathogens [5 13 14] The SCAR marker devel-oped in this study can also facilitate rapid decision-makingand precise early season disease management to reduce therisk of Ps citricarpa epidemics

Conflicts of Interest

The authors declared no conflicts of interest

Acknowledgments

This study was funded by the Chongqing Research ProgramofBasicResearchandFrontierTechnology (cstc2016jcyjA0316)theChongqingPostdoctoral Science Foundation (Xm2016124)the Fundamental Research Funds for the Central Universi-ties (XDJK2016A020) and the China Scholarship Council(201706995068) The authors thank Professor Kaifang ZengSouthwest University for providing them with the fungalpathogens of citrus fruits used in this study

Supplementary Materials

Table S1 sequence of random amplified polymorphic DNA(RAPD) PCR primers used in this study (Supplementary Ma-terials)

References

[1] L Zhu X Wang F Huang et al ldquoA destructive new disease ofcitrus in China caused by Cryptosporiopsis citricarpa sp novrdquoPlant Disease vol 96 no 6 pp 804ndash812 2012

[2] C Chen G J M Verkley G Sun J Z Groenewald and P WCrous ldquoRedefining common endophytes and plant pathogensinNeofabraea Pezicula and related generardquoFungal Biology vol120 no 11 pp 1291ndash1322 2016

[3] F Martinelli R Scalenghe S Davino et al ldquoAdvanced methodsof plant disease detection A reviewrdquo Agronomy for SustainableDevelopment vol 35 no 1 pp 1ndash25 2015

[4] M M Lopez P Llop A Olmos E Marco-Noales M Cambraand E Bertolini ldquoAre molecular tools solving the challengesposed by detection of plant pathogenic bacteria and virusesrdquoCurrent Issues in Molecular Biology vol 11 pp 13ndash46 2009

[5] LG Jesumaharaja RManikandan andT Raguchander ldquoSCARmarker specific to detect Magnaporthe grisea infecting fingermillets (Eleusine coracana)rdquo Journal of Applied Microbiologyvol 121 no 3 pp 778ndash786 2016

[6] J Zhao X J Wang C Q Chen L L Huang and Z S KangldquoA PCR-based assay for detection of Puccinia striiformis f sptritici in wheatrdquo Plant Disease vol 91 no 12 pp 1669ndash16742007

[7] Y-H Lin C-C Su C-P Chao et al ldquoA molecular diagnosismethod using real-time PCR for quantification and detection ofFusarium oxysporum f sp cubense race 4rdquo European Journal ofPlant Pathology vol 135 no 2 pp 395ndash405 2013

[8] B A McDonald ldquoThe population genetics of fungi Tools andtechniquesrdquo Journal of Phytopathology vol 87 no 4 pp 448ndash453 1997

[9] J G KWilliams A R Kubelik K J Livak J A Rafalski and SV Tingey ldquoDNApolymorphisms amplified by arbitrary primersare useful as geneticmarkersrdquoNucleic Acids Research vol 18 no22 pp 6531ndash6535 1990

[10] A Velegraki M Kambouris A Kostourou G ChalevelakisandN J Legakis ldquoRapid extraction of fungal DNA from clinicalsamples for PCR amplificationrdquoMedical Mycology vol 37 no 1pp 69ndash73 1999

[11] Y Liu W Tian and G Wei ldquoFeature and prevention of citrustarget spotrdquo Fruit Growerrsquos Friend vol no 5 p 36 2015

BioMed Research International 5

[12] W Dnyaneshwar C Preeti J Kalpana and P Bhushan ldquoDevel-opment and application of RAPD-SCAR marker for identifica-tion of Phyllanthus emblica Linnrdquo Biological amp PharmaceuticalBulletin vol 29 no 11 pp 2313ndash2316 2006

[13] K Nithya K A I M Bukhari V Valluvaparidasan V Paranid-haran and R Velazhahan ldquoMolecular detection of Colletotri-chum falcatum causing red rot disease of sugarcane (Saccharumofficinarum) using a SCAR markerrdquo Annals of Applied Biologyvol 160 no 2 pp 168ndash173 2012

[14] D Ladhalakshmi A Vijayasamundeeswari V Paranidharan RSamiyappan and R Velazhahan ldquoMolecular identification ofisolates of Peronosclerospora sorghi from maize using PCR-based SCAR markerrdquo World Journal of Microbiology and Bio-technology vol 25 no 12 pp 2129ndash2135 2009

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Virolog y Stem Cells International

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Enzyme Research

Hindawiwwwhindawicom Volume 2018

International Journal of

MicrobiologyHindawiwwwhindawicom

Nucleic AcidsJournal of

Volume 2018

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