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Diagnostic tools using DNA bar codingfor the identification of pathogen racesand related species: a reviewZafar Iqbal a , Saeed Rauf b , Imran Hamid a , Salman Ahmad a &Muhammad Akbar Anjum ba Department of Plant Pathology, University College ofAgriculture, University of Sargodha, Sargodha, Pakistanb Department of Plant Breeding & Genetics, University College ofAgriculture, University of Sargodha, Sargodha, PakistanVersion of record first published: 25 Mar 2013.

To cite this article: Zafar Iqbal , Saeed Rauf , Imran Hamid , Salman Ahmad & MuhammadAkbar Anjum (2013): Diagnostic tools using DNA bar coding for the identification of pathogenraces and related species: a review, Archives Of Phytopathology And Plant Protection,DOI:10.1080/03235408.2013.774551

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Diagnostic tools using DNA bar coding for the identification ofpathogen races and related species: a review

Zafar Iqbala, Saeed Rauf b*, Imran Hamida, Salman Ahmada andMuhammad Akbar Anjumb

aDepartment of Plant Pathology, University College of Agriculture, University of Sargodha,Sargodha, Pakistan; bDepartment of Plant Breeding & Genetics, University College ofAgriculture, University of Sargodha, Sargodha, Pakistan

(Received 3 February 2013; final version received 4 February 2013)

In the past, disease diagnostic has been an art which was based on visual scoring ofdisease symptoms. However, visual scoring was cumbersome and often confusingfor the pathologists due to intermingling of the visual symptoms with other diseases,delayed appearance of symptoms or symptoms matching with nutrient deficiencies.As an alternative of visual scoring, several other methods were proposed such asmicroscopy to visualise the pathogen directly or to quantify the pathogen throughenzyme linked immunosorbent assay. These techniques were associated with slowrate of analysis per sample and often were culture based. This led to the develop-ment of culture-independent DNA-based molecular techniques. These DNA-basedmolecular techniques have the potential for rapid, sensitive detection and accuratequantification of pathogens. Since a plant may be infected by the multiple pathogens,these techniques could also identify the number of pathogens by multiplex assaytechnique. In this manuscript, four different DNA-based techniques are reviewedwhich show that these are now routinely being used in diverse crop species fordiversity, detection and diagnostic analyses of pathogens.

Keywords: microarray; molecular diagnostics; molecular markers; multiplexing;ribosomal sequences

Abbreviations: AFLP, amplified fragment length polymorphism; ISSR, intergenomicsimple sequence repeats; PCR, polymerase chain reaction; RAPD, random amplifiedpolymorphic DNA; RFLP, restriction fragment length polymorphism; SCAR,sequence characterised amplified region; SSCP, single strand conformation polymor-phism; SSR, simple sequence repeats

Introduction

The true management of plant diseases depends upon correct identification of the patho-gens. With the advent of new molecular techniques, it is possible to give up laboriousprocedures previously involved in disease diagnostics. Some diseases may be diagnosedby visual examinations, but still most of the pathogens need to be detected by suitablelaboratory techniques. In case of high value cash or fruit crops, proper pathogen identi-fication is essential to avoid unnecessary use of pesticides or repetition of non-specificor untimely disease treatments. Global and evolutionary changes have led to new andrevised taxonomic definitions of virulent plant pathogens. Early and timely diagnosis of

*Corresponding author. Email: agri@uos.edu.pk

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plant pathogens is essential to forecast about outbreaks and devise suitable managementstrategies to combat the disease incursions. On one side diagnostic expertise is neces-sary and on the other side, capacity building is also of prime importance. Large-scaleadoption of standard protocols and diagnostic laboratory accreditation may serve tobuild trust and confidence among institutions (Miller et al. 2009).

Morphological techniques are now least effective to identify pathogens accuratelydue to continuous changes in pathogens population structure, lack of vast taxonomicknowledge, conventional laborious procedures and contradictions in morphologicallyidentified propagules. Timely identification of plant pathogens may help to monitor theinfected plants and devise suitable strategies for the disease management. DNA technol-ogies have their implications in almost all the disciplines of modern agriculture. Here,we review the recent advances in plant pathogen diagnostics.

Molecular diagnostics

Plant pathogen diagnosis emphasises some technical aspects, that is, clarity, specificity,sensitivity, robustness, accuracy and reproducibility (Mullis & Faloona 1987; McCartneyet al. 2003; Lievens et al. 2005; Lievens & Thomma 2005). The most importantadvantage of molecular detection techniques over conventional diagnostic methods istheir suitability to differentiate closely related strains and races. Molecular pathogenidentification techniques rely on specific conserved regions within genome of specificpathogen. Reviews of these conserved regions are as under:

Selective and randomly amplified PCR based molecular markers

Selection of target sequences for the detection of plant pathogens involves the screeningof random or selective parts of the genome (Lievens & Thomma 2005). For this pur-pose, several techniques including random amplified polymorphic DNA (RAPD),restriction fragment length polymorphism (RFLP), amplified fragment length polymor-phism (AFLP), sequence characterised amplified region (SCAR), and simple sequencerepeats (SSR) (Williams et al. 1990; Vos et al. 1995; Fraaije et al. 1999; Papp et al.2003) are used. As compared to traditional polymerase chain reaction (PCR) analysis,RAPD does not require any specific knowledge of the DNA sequence of the targetorganism. The identical 10-mer primers may or may not amplify a segment of DNAdependent on the positions that are complementary to the primers sequence.

The AFLP technique is based on the selective PCR amplification of restriction frag-ments from a total digest of genomic DNA. This technique has three steps: (i) restric-tion of the DNA and ligation of oligonucleotide adapters, (ii) selective amplification ofsets of restriction fragments, and (iii) gel analysis of the amplified fragments. Restric-tion fragments are amplified by using the adapter and restriction site sequence as targetsites for primer annealing. The technique has been proved to be a powerful DNA fin-gerprinting tool for DNAs of any origin or complexity (Vos et al. 1995). SSR marker isbased on the polymorphism of SSR in transcribing and non-transcribing genome. Thesesimple sequences may be di, tri or tetra (etc.) repeats depending on the species underinvestigation. It has been noted that SSR markers are highly polymorphic; however,they are specific to particular species (Papp et al. 2003).

Molecular markers have been utilised to determine the extent of genetic variation invarious isolates obtained from variety of hosts locations and geographical areas. Reviewof various reports showed that AFLP, SSR or RAPD markers were extensively used in

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these studies (Table 1). These markers revealed extensive polymorphism within orbetween isolates showing high degree of genetic diversity. Studies have indicated thathigher degree of genetic diversity was present within specific isolates rather thanbetween isolates collected from various locations (Messner et al. 1996; Schnieder et al.2001). However, higher degree of diversity was also present between the isolates

Table 1. Genetic diversity estimates within various isolates of pathogens collected from varioushosts and areas.

PathogenMolecularmarker Conclusion Reference

Cladosporium fulvum andPyrenopeziza brassicae

AFLP/4primercombination

AFLP were useful fordetecting fungal geneticvariation

Majer et al.(1996)

34 isolates of Verticilliumdahliae Kleb. from ninedifferent genera ofdicotyledonous host plants

RAPD/4primer

Genetic diversity wasobserved among the isolatescollected from various hostrather than geographical areas

Messner et al.(1996)

Ceratocystis fimbriata 11 ISSR Markers clearly distinguisheddifferent geographical andhost- specific populations ofD. fimbriata

Barnes et al.(2001)

6 isolates of Mycosphaerellagraminicola

AFLP/4primercombination

4% of the genetic variationbetween the local populationsbut 96% of the geneticvariation within thepopulations. Plant hostresistance had no obviouseffect on the populationstructure and diversity

Schnieder et al.(2001)

43 isolates of Fusariumoxysporum

RAPDs andAFLP

AFLP was found to be moreinformative as it differentiat-ed more number of pathogenisolates

Sivaramakrishnanet al. (2002)

20 isolates of Magnaporthegrisea

RAPD/33primer

High polymorphism amongthe isolates

Chadha andGopalkrishna(2005)

Phytophthora infestans SSR/100primers

10% of the primers wereshown to be polymorphic.Considerable SSR diversityamong the isolates

Lees et al. (2006)

Colletotrichum falcatum SCAR Specificity of SCAR primerswas evaluated by multiplesamples containingColletotrichum falcatum andits related species whichshowed that SCAR primerwere highly specific for thespecie

Nithya et al.(2012)

Tilletia foetida SCAR SCAR distinguish the Tilletiafoetida from its relatedspecies

Zhang et al.(2012)

Abbreviations: Sequence characterised amplified regions (SCAR); Random amplified polymorphic DNA(RAPD); Amplified fragment length polymorphism (AFLP); Simple sequence repeats (SSR); Intergenomicsimple sequence repeats (ISSR).

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collected from various hosts indicating some degree of differentiation due to adaptationto specific host (Messner et al. 1996; Barnes et al. 2001).

Ribosomal sequences

Conventional morphological and cultural methods do not provide desired sensitivity andspecificity as compared to advanced nucleic acid based techniques (De Boer et al. 1996;Cuppels et al. 2006). Therefore, conserved known genes carrying sequence variation areexploited. The nuclear ribosomal DNA (rDNA) has been widely used in molecular phy-logenetic studies (White et al. 1990). A diagnostic assay can be designed with the helpof rDNA sequence data, available in public databases. These extensive sequence dataallow comparison of sequences that help to determine diagnostic regions harbouring therequired specificity. On the basis of these specific sequences, differences among variouspathogen strains could be determined with the help of some specific markers that is,RFLP (Tooley et al. 1996). The rDNA is found as a structured unit consisting of threeribosomal RNA subunit genes that are separated by internal transcribed spacers (ITS).This ITS region plays an important role in pathogen diagnostics as it contains alternatingareas of high conservation and variability (Weiland & Sundsbak 2000). This variabilityallows classification over diversity of taxonomic levels (White et al. 1990), sometimeseven at subspecies level (Atkins et al. 2003). Summary of some studies showing differ-ences between various pathogenic species of a genus due to polymorphism in rDNA isgiven in Table 2. A review of the table reveals that primers were designed from thespecific conserved part of the rDNA genes that is, ITS of 16S rRNA gene, 5.8S rRNA

Table 2. Utilisation of conserved sequence for pathogen race identification and geneticrelationships.

Pathogen Gene/Primer Conclusion References

40 MLO organism 16S rDNA Sequence variability was used toclassify pathogens through RFLP

Lee et al.(1993)

Phytoplasma 16S-23SIntergenicspacerregions

Phytoplasmas group specificprimer was designed

Harrisonet al.(1996)

Various species of genusPhytophthora

5.8 S rDNAITS 5 and 4

Polymorphism was detected aftertreating the amplified DNA withrestriction enzymes MspI, andHaeIII

Ristainoet al.(1998)

Phytophthora ramorum 5·8S rDNAITS-1

Unique pattern of SSCP withinrDNA was used to identify thespecies

Konget al.(2004)

Phytoplasma 16S rDNA RFLP was used to differentiateinto 28 groups

Wei et al.(2007)

Fusarium solani 10 primersTEF-1αITS1, ITS2

The designed primers efficientlyidentified and discriminated thepathogens of genus Fusariumand did not cross react withclosely related species

Arif et al.(2012)

Ralstonia solanacearum,Xanthomoans axonopodis pv.vesicatoria, and Xanthomonasoryzae pv. oryzae

PCR-SSCPUniversalprimer 16SRNA

PCR-SSCP tool may be appliedfor the diagnostic of plantpathogens of economicimportance

Srinivasaet al.(2012)

Abbreviations: PCR-Single strand conformation polymorphism (PCR-SSCP); Internal transcribed spacer (ITS).

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genes and 23S were used as primers (Table 2). These primers allowed specific amplifica-tion of rDNA of various species related to a pathogen. Afterward, amplified DNA wastreated with restriction enzymes such as MspI and HaeIII to show polymorphism amongthe species (Table 2). These differences at the marker level allowed to establish phyloge-netic relationships or to measure genetic distance between various species.

Multiplexing

The plant or their organs are attacked by pathogens of diverse nature. Multiplex assayscan quantify different pathogens simultaneously. DNA array technology is used for mul-tiplex detection of plant pathogens which consists of many discretely located pathogen-specific sequences (Livak 1999). To produce a macro or microarray, the detectorsequences are immobilised on nylon filter or glass slide, respectively. The target DNAis amplified using consensus primers, which locate a genomic region containing thepathogen-specific sequences, and is labelled simultaneously or subsequently. By thismethod, a large number of organisms can be differentiated using a single PCR. Thistechnique has been applied for identification of oomycetes, nematode, bacterial and fun-gal DNA from pure cultures (Lévesque et al. 1998; Uehara et al. 1999; Fessehaie et al.2003; Lievens et al. 2003) as well as for the identification of a number of viruses.

At present, the real-time PCR is the most reliable culture-independent technique toquantify the detected pathogens as well as the disease progress (Schaad & Frederick2002; Brouwer et al. 2003; Gachon et al. 2004). It combines sensitivity of conventionalPCR and also generates a specific fluorescent signal that allows the quantification ofspecific DNA targets. Schena et al. (2004) grouped real-time PCR into four main chem-istries in plant pathology. These included amplicon sequence non-specific (SYBR GreenI) and sequence-specific (TaqMan, Molecular beacons, and Scorpion-PCR) methods.Amplicon sequence non-specific method relies on a dye that emits fluorescent lightwhen intercalated into double-stranded DNA. On the other hand, amplicon sequence-specific method is based on oligonucleotide probes labelled with a donor fluorophoreand an acceptor dye (quencher). The fluorescent signal eliminates the requirement forpost-amplification processing steps, such as gel electrophoresis and ethidium bromidestaining. This significantly reduces time and labour required for the analysis and greatlyincreases the throughput of PCR testing as an automated diagnostic system, making itsuitable for large-scale analyses. Furthermore, the use of different fluorescent dyesfacilitates the detection of several target microorganisms in a single reaction (multiplex-PCR). Real-time PCR makes possible an accurate, reliable and high-throughput quantifi-cation of target pathogenic DNA in various environmental samples, including hoststissues, soil, water and air; thus, opening new research opportunities for the study ofdiagnosis, inoculum threshold levels, epidemiology and host–pathogen interactions.According to Schaad and Frederick (2002), real- time PCR will revolutionise the plantpathogen diagnosis. However, it will depend on the availability of DNA sequence datato design highly specific primers and fluorescent probe to yield target amplicons tounique regions of a pathogen’s genome. Similarly, Brouwer et al. (2003) used thistechnique to quantify inoculums of fungal species pathogenic for Arabidopsis andconcluded that host DNA does not interfere with the quantification of pathogen DNA.Furthermore, this technique is host resistance independent that is, may be used to quan-tify pathogen DNA both in susceptible and in resistance genotypes. Bates et al. (2001)used this technique to check the health of barley seed for the attack of Pyrenophoraspp., as the technique can quantify the pathogen even the incidence of disease was

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lower than 2%. The test was performed in three parts; (i) quantification of infectionusing Pyrenophora-specific PCR primers; (ii) test of any negative samples from (i) withbarley-specific PCR primers to check the DNA extraction process; (iii) test of positivesamples from (i) for the presence of Pyrenophora graminea using P. graminea-specificPCR primers. Summary of various reports for the utilisation of real time PCR forvarious pathogen diagnoses is given in Table 3.

Microarray

Microarray technique was first introduced by Schena et al. in 1995. The introduction ofhigh density microarrays ensured to determine the expression levels of all or most ofthe genes in a given genome using cDNA as the probe target simultaneously. Later,oligonucleotide arrays technique was introduced. Screening of environmental sample

Table 3. Summary of reports for the utilisation of real time PCR for various pathogendiagnoses.

Pathogen Probes/Primers Remarks Reference

Ralstoniasolanacearum

Broad-range probe(RS) for all biovarsof R. solanacearum,and specific probe(B2) for biovar 2A.

The specificity and sensitivity, highspeed, robustness, reliability, offerpotential advantages in routineindexing of potato tubers and otherplant material for the presence ofpathogen

Weller et al.(2000)

Apple proliferation(AP)phytoplasma

16S rDNA geneprimer–probe

Highly specific amplification whiledetection at very low concentration

Baric andDalla-Via(2004)

Erwinia amylovora Common plasmidpEA29 primer

Real-time PCR could be used forlarge scale screening of pathogenand for resistance studies in breedingexperiments and after treatments tocontrol fireblight

Salm andGeider(2004)

Alternariabrassicae

Brassicae-specificprimers designedfrom sequence oftwo genes causingpathogenicity

The techniques was efficient andeasy for the quantification of seedinfection

Guillemetteet al. (2004)

Phytophthoraramorum

P. ramorum-specificTaqMan primers(Pram-114F andPram-190R)

Portable real-time PCR platform wasused for molecular testing of a plantpathogen entirely in the field

Tomlinsonet al. (2005)

CandidatusLiberibacter

16S rDNA-basedTaqManprimer–probe

The assays do not cross-react withother pathogens. The ratio ofpathogen DNA to host plant DNAwas estimated by to be 1:13,000(w/w)

Li et al.(2006)

Pantoea stewartii Genetic markers(wtsE, cpsA andhrpN)

PCR based genetic markers aresensitive and they provide highselective amplification of pathogenin mixed culture

Thapa et al.(2012)

Botryosphaeriaceaespecies

Four species-specificprimers

The nested multiplex PCRsuccessfully detected Lasiodiplodiatheobromae, Neofusicoccum parvum,N. mangiferae and Fusicoccumaesculi from total DNA

Ni et al.(2012)

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DNA can be performed against several hundred to several thousand oligoprobes fixedto a support. The environmental DNA can be labelled, and the resulting patterns can becompared with a reference set of known organisms. With the help of this technique, allfungi in a particular sample may be detected and quantified (Service 1998; Lipshutzet al. 1999; Wang 2000; Atkins & Clark 2004). Lievens et al. (2006) indicated thatDNA array technique was most suitable to detect, identify and quantify multiplepathogens. They utilised this technique for the detection of single- nucleotide polymor-phism among several races of pathogens. It was shown that those closely relatedpathogens that have completely changed host range differ for only single or few bases.Similarly, Boonham et al. (2003) used the array construct for the detection of severalcommon potato viruses (PVY, PVX, PVA and PVS) in single and mixed infections.The method was shown to be able to discriminate sequences with less than 80%sequence identity but was able to detect sequence variants with greater than 90%sequence identity. Thus, the method is useful for discriminating at the species level, butalso able to cope well with the intrinsic variability found within the genomes of RNAviruses. Summary of the reports related to oligo microarray analyses of pathogenicspecies is given in Table 4. These studies have indicated that microarray analysis is areliable tool for the identification of various strains of prokaryotes (Table 4).

The use of fluorescent probes has permitted direct in situ assay of organisms, eventhose that cannot be cultured. This technique is named as fluorescent in situ hybridisa-tion (FISH) and has been employed to study fungal interactions (Li et al. 1997; Spearet al. 1999; Schröder et al. 2000).

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Table 4. Summary of the reports related to oligo-microarray analyses of pathogenic species.

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