A Plating‐PCR Technique for Detection and Quantification of the Biological Control Agent Agrobacterium vitis Strain E26 in Soil under Controlled Conditions

Agrobacterium vitis strain E26 is a promising biocontrol agent of grapevine crown gall, an economically important disease of grape worldwide. In this report, we developed a Plating‐PCR method that allows specific detection and quantification of E26 by combining classical microbiological techniques with molecular tools. Random amplified polymorphic DNA fingerprints were used to differentiate E26 from other A. vitis strains. A differentially amplified fragment from E26 was sequenced and characterized as a sequence characterized amplified region (SCAR) marker. Two primer pairs were then designed and evaluated for their specificity against E26. One of the two SCAR primer pairs, 740F/R, was further selected for specific detection of strain E26. A plating assay coupled to PCR with the SCAR primers 740F/R allowed the assessment of population dynamics of E26 in non‐sterile grape rhizosphere soil under controlled conditions.     

Identification of a universally primed-PCR-derived sequence-characterized amplified region marker for an antagonistic strain of Clonostachys rosea and development of a strain-specific PCR detection assay

We developed a PCR detection method that selectively recognizes a single biological control agent and demonstrated that universally primed PCR (UP-PCR) can identify strain-specific markers. Antagonistic strains of Clonostachys rosea (syn. Gliocladium roseum) were screened by UP-PCR, and a strain-specific marker was identified for strain GR5. No significant sequence homology was found between this marker and any other sequences in the databases. Southern blot analysis of the PCR product revealed that the marker represented a single-copy sequence specific for strain GR5. The marker was converted into a sequence-characterized amplified region (SCAR), and a specific PCR primer pair was designed. Eighty-two strains, isolated primarily from Danish soils, and 31 soil samples, originating from different localities, were tested, and this specificity was confirmed. Two strains responded to the SCAR primers under suboptimal PCR conditions, and the amplified sequences from these strains were similar, but not identical, to the GR5 marker. Soil assays in which total DNA was extracted from GR5-infested and noninoculated field soils showed that the SCAR primers could detect GR5 in a pool of mixed DNA and that no other soil microorganisms present contained sequences amplified by the primers. The assay developed will be useful for monitoring biological control agents released into natural field soil.     

Identification of a Universally Primed-PCR-Derived Sequence-Characterized Amplified Region Marker for an Antagonistic Strain of Clonostachys rosea and Development of a Strain-Specific PCR Detection Assay

We developed a PCR detection method that selectively recognizes a single biological control agent and demonstrated that universally primed PCR (UP-PCR) can identify strain-specific markers. Antagonistic strains of Clonostachys rosea (syn. Gliocladium roseum) were screened by UP-PCR, and a strain-specific marker was identified for strain GR5. No significant sequence homology was found between this marker and any other sequences in the databases. Southern blot analysis of the PCR product revealed that the marker represented a single-copy sequence specific for strain GR5. The marker was converted into a sequence-characterized amplified region (SCAR), and a specific PCR primer pair was designed. Eighty-two strains, isolated primarily from Danish soils, and 31 soil samples, originating from different localities, were tested, and this specificity was confirmed. Two strains responded to the SCAR primers under suboptimal PCR conditions, and the amplified sequences from these strains were similar, but not identical, to the GR5 marker. Soil assays in which total DNA was extracted from GR5-infested and noninoculated field soils showed that the SCAR primers could detect GR5 in a pool of mixed DNA and that no other soil microorganisms present contained sequences amplified by the primers. The assay developed will be useful for monitoring biological control agents released into natural field soil.     

Development of a SCAR marker and a strain‐specific genomic marker for the detection of the biocontrol agent strain CPA‐8 Bacillus amyloliquefaciens (formerly B. subtilis)

In this work, reliable tools were developed to detect and identify the biocontrol strain CPA‐8 using DNA amplification techniques. As a first approach, the RAPD (random amplified polymorphic DNA) technique was applied to a collection of 77 related Bacillus species. Among the primers tested, the primer pair OPG1/OPG6 amplified a 668 bp specific product to the strain CPA‐8 that was sequenced and used to design SCAR (sequence‐characterised amplified regions) primer pairs. The SCAR‐4 marker amplified a semi‐specific fragment of 665 bp not only for the strain CPA‐8 but also for other 12 strains whose morphology was completely different from CPA‐8. Another approach was developed to obtain a strain‐specific genomic marker related to ecological adaptations of Bacillus amyloliquefaciens species. The primer pair F2/R2 obtained from RBAM 007760, a gene involved in surface adhesion, amplified a 265 bp fragment unique for strain CPA‐8. Our results revealed that these two molecular markers, SCAR‐4 and RBAM 007760 F2/R2 provide suitable monitoring tools to specifically identify the biocontrol CPA‐8 when applied against brown rot caused by Monilinia spp. in stone fruit. Moreover, our findings demonstrate that the strain CPA‐8 is affiliated with B. amyloliquefaciens species that was formerly designated as Bacillus subtilis.     

Specific PCR Assays for the Detection and Quantification of DNA from the Biocontrol Strain Trichoderma harzianum 2413 in Soil

Strain identification in situ is an important factor in the monitoring of microorganisms used in the field. In this study, we demonstrated the use of sequence-characterized amplified region (SCAR) markers to detect genomic DNA from Trichoderma harzianum 2413 from soil. Two primers (SCAR A1/SCAR A1c) were tested against DNA of 27 isolates of Trichoderma spp. and amplified a 990-bp fragment from T. atroviride 11 and a 1.5-kb fragment from T. harzianum 2413, using an annealing temperature of 68°C. These fragments showed no significant homology to any sequence deposited in the databases. The primer pair, BR1 and BR2, was designed to the 1.5-kb fragment amplified from T. harzianum 2413, generating a SCAR marker. To test the specificity of these primers, experiments were conducted using the DNA from 27 Trichoderma spp. strains and 22 field soil samples obtained from four different countries. PCR results showed that BR1 and BR2 amplified an 837-bp fragment unique to T. harzianum 2413. Assays in which total DNA was extracted from sterile and nonsterile soil samples, inoculated with spore or mycelium combinations of Trichoderma spp. strains, indicated that the BR1 and BR2 primers could specifically detect T. harzianum 2413 in a pool of mixed DNA. No other soil-microorganisms containing these sequences were amplified using these primers. To test whether the 837-bp SCAR marker of T. harzianum 2413 could be used in real-time PCR experiments, new primers (Q2413f and Q2413r) conjugated with a TaqMan fluorogenic probe were designed. Real-time PCR assays were applied using DNA from sterile and nonsterile soil samples inoculated with a known quantity of spores of Trichoderma spp. strains.     

Detection of the nec1 virulence gene and its correlation with pathogenicity in Streptomyces species on potato tubers and in soil using conventional and real-time PCR

AIMS: To evaluate the virulence gene nec1 as a reliable marker for the detection of pathogenic Streptomyces species on potato tubers and in soil samples using conventional and real-time quantitative PCR assays. Methods AND RESULTS: Two pairs of conventional primers (outer and nested) and one set of primers/probe for use in real-time PCR were designed to detect the necrogenic protein encoding nec1 gene of Streptomyces scabiei strain ATCC 49173(T). The conventional PCR primers were also incorporated into a multiplex PCR assay to simultaneously detect the nec1 gene in conjunction with the potato pathogens Helminthosporium solani and Colletotrichum coccodes. The specificity of each PCR assay was confirmed by testing 32 pathogenic and nonpathogenic reference strains of Streptomyces representing 12 different species and 74 uncharacterized streptomycete strains isolated from diseased tubers. A clear correlation between pathogenicity and the detection of nec1 by PCR was demonstrated. The sensitivity and specificity of both the conventional and real-time PCR assays allowed the detection of nec1 on potato tubers in the absence of visible symptoms of common scab, and in seeded soil down to a level equivalent to three S. scabiei spores per gram soil. CONCLUSIONS: Reliable and quantitative PCR techniques were developed in this study for the specific detection of the virulence gene nec1 of pathogenic Streptomyces species on potato tubers and in soil samples, and the data demonstrated a clear correlation between pathogenicity in Streptomyces species and the presence of the nec1 gene. SIGNIFICANCE AND IMPACT OF THE STUDY: Together with the DNA extraction protocols, these diagnostic methods will allow a rapid and accurate assessment of tuber and soil contamination by pathogenic Streptomyces species.     

Strain-specific SCAR markers for the detection of Trichoderma harzianum AS12-2, a biological control agent against Rhizoctonia solani, the causal agent of rice sheath blight

In order to identify a specific marker for T. harzianum AS12-2, a strain capable of controlling rice sheath blight caused by Rhizoctonia solani, UP-PCR was performed using five universal primers (UP) both separately and in pairwise combinations. The application of two UP primers resulted in the amplification of unique fragments from the genomic DNA of T. harzianum AS12-2, clearly distinguishing it from other Trichoderma strains. The unique fragments had no significant sequence homology with any other known sequence available in databases. Based on the sequences of the unique fragments, 14 oligonucleotide primers were designed. Two primer sets amplified a fragment of expected size from the DNA of strain T. harzianum AS12-2 but not from any other examined strains belonging to T. harzianum, to other Trichoderma species assayed, or to other common fungi present in paddy fields of Mazandaran province, Iran. In conclusion, SCAR (sequence characterized amplified regions) markers were successfully identified and rapid, reliable tools were provided for the detection of an effective biocontrol Trichoderma strain, which can facilitate studies of its population dynamics and establishment after release into the natural environment.     

Development of strain specific molecular markers for the Sclerotinia minor bioherbicide strain IMI 344141

The plant pathogenic fungus, Sclerotinia minor IMI 344141, has been developed as a bioherbicide for broadleaf weed control in turfgrass and a means to differentiate this biocontrol agent from like organisms is required. A strain specific molecular marker was developed to detect and monitor the Sclerotinia minor IMI 344141 bioherbicide strain. The method was based on polymerase chain reaction (PCR) amplification of two sequence-characterized amplified regions (SCAR) primer pairs for a first round PCR, and another two sets of nested primers was used for a second round PCR if higher sensitivity was needed. Sclerotinia minor IMI 344141 was successfully traced from both pure cultures and environmental samples originating from bioherbicide-released field trials. DNA of the S. minor bioherbicide isolate IMI 344141 was detected in the soil 2 months after application, but was not detected in the 3- and 9-month samples after application. When applied as a bioherbicide, S. minor (IMI 344141) did not persist into the following spring season in turf environments. This molecular detection method provides a mechanism to distinguish this isolate from related organisms and a tool to monitor behavior of the biocontrol agent S. minor IMI 344141 in nature, particularly in soil.     

Development of a strain-specific genomic marker for monitoring a Bacillus subtilis biocontrol strain in the rhizosphere of tomato

A strain-specific molecular marker enabling the detection and tracking of the biological control agent Bacillus subtilis 101, when released into the environment, was developed. Random amplified polymorphic DNA (RAPD) technique was used to differentiate this from other B. subtilis strains. A differentially amplified fragment obtained from RAPD profiles was sequenced and characterized as sequence-characterized amplified region (SCAR) marker, and four primer pairs were designed and evaluated for their specificity towards this strain. The sensibility of the selected SCAR primer pair was evaluated by qualitative PCR and Southern blotting, and the detection limit was assessed around 10(2) CFU (g dry wt soil)(-1), thus providing a reliable tool for the traceability of this B. subtilis strain in greenhouse or field trials. A plating assay coupled to PCR with the SCAR primer pair was then used as a detection method in microcosm experiments for monitoring the population of B. subtilis 101 in the rhizosphere of tomato, grown under two different soil conditions, i.e. nonsterile peat-based substrate and sandy-loam agricultural soil, respectively. The data of rhizosphere colonization indicated that the soil conditions significantly affected the rhizosphere establishment of strain 101.     

绿僵菌M189菌株特异性SCAR标记的建立及田间监测应用

为了监测生防真菌绿僵菌菌株在田间释放后的回收,有必要建立菌株DNA分子标记,以此将应用的菌株与其他菌株或田间的土著分离株鉴别开来。作者采用16条随机引物扩增了51株绿僵菌菌株的基因组DNA,得到81个多态性位点。其中M189菌株多态性位点30个,分析得到1个特异性位点,并将该位点的DNA片段测序后转化成为特异性SCAR标记。检验确定了该标记的敏感性,可以从供试的51株菌株中准确鉴定出目的菌株M189。并用该标记检测了从田间回收的3个分离株,确定其中1个与应用菌株M189一致。     

杉木种子随体染色体SCAR标记的建立

回收、纯化由引物OPB07(5’-GGTGACGCAG-3’) OPB18(5’-CCACAGCAGT-3’)扩增而得的杉木(Cunninghamia lanceolata(Lamb.)Hook)种子随体染色体特异性RAPD(随机扩增的DNA多态性分析)片段OPB07-18907,将该片段克隆至pUCm-T载体,转化受体菌后筛选出阳性克隆,并对插入片段进行测序,根据序列特点设计两对SCAR(序列特异性扩增区)引物,PCR结果显示,这两对引物的4种组合都可以扩增出属于随体染色体的特征带,适宜退火温度为57℃。成功将特异RAPD标记OPB07-18907转化为稳定的SCAR标记。开发随体染色体SCAR标记目的是:一方面能在分子水平上鉴定微分离的杉木随体染色体,另一方面,也可以将杉木已构建的遗传图谱中连锁群与染色体进行对应。探讨了该SCAR标记对杉木核型分析的作用。     

Molecular Detection of Colletotrichum lindemuthianum by Duplex PCR

A duplex PCR technique was developed to detect the pathogenic fungus Colletotrichum lindemuthianum infection in the tissues of common bean. Based on the differences of 24 internal transcribed spacer, DNA sequences of Colletotrichum spp. retrieved from GeneBank database, one pair of specific primers of CY1/CY2 (CY1: 5'-CTT TGT GAA CAT ACC TAA CC-3'; CY2: 5'-GGT TTT ACG GCA GGA GTG-3'), was designed. The CY1/CY2 primers amplified a single PCR product of 442 bp only from C. lindemuthianum and Colletotrichum orbiculare , not from any other tested species. By using random amplification of polymorphic DNA technique, a product closely associated with C. lindemuthianum was generated. This product was cloned, sequenced and used for designing a species-specific primers of CD1/CD2 (CD1: 5'-ACC TGG ACA CAT AAG TCA AAG-3'; CD2: 5'-CAA CAA TGC CAG TAT CAG AG-3'). The CD1/CD2 primers could distinguish C. lindemuthianum from C. orbiculare by a 638 bp PCR band. A duplex PCR method, combining both primers of CY1/CY2 and CD1/CD2, was used to detect C. lindemuthianum infection. The sensitivity of the detection with this PCR method was 1 pg of pure genomic DNA from the pathogen. Therefore, the PCR-based methods could be used for accurate and rapid detection of C. lindemuthianum from common bean.     

Development of SCAR markers for the DNA-based detection of the Asian long-horned beetle,Anoplophora glabripennis (Motschulsky)

DNA markers were identified for the molecular detection of the Asian long-horned beetle (ALB), Anoplophora glabripennis (Mot.), based on sequence characterized amplified regions (SCARs) derived from random amplified polymorphic DNA (RAPD) fragments. A 2,740-bp DNA fragment that was present only in ALB and not in other Cerambycids was identified after screening 230 random primers in a PCR-based assay system. Three pairs of nested 22-mer oligonucleotide primers were designed on the basis of the sequence of this fragment and were used to perform diagnostic PCR. The first pair of primers (SCAR1) amplified a single 745-bp fragment of ALB DNA, but this did not differentiate ALB from other species. The other two pairs of SCAR primers (SCAR2 and SCAR3) amplified bands of 1,237- and 2,720-bp, respectively, that were capable of differentiating ALB from other closely related non-native and native Cerambycids, such as A. chinensis (Forster), A. malasiaca (Thomson), A. nobilis (Ganglbauer), Monochamus scutellatus (Say), Plectrodera scalator (Fab), Saperda tridentata (Olivier), and Graphisurus fasciatus (Degeer). The latter two SCAR markers could be amplified using DNA extracted from body parts of ALB such as the wing, the leg, and the antennae as well as tissues from all the developmental stages including the egg, larva, pupa, and adult. These markers were also capable of identifying ALB using the DNA extracted from frass. Our results demonstrate that the SCAR markers we have identified can be used for unambiguously identifying ALB from other closely related Cerambycids using a simple PCR procedure.     

CHARACTERIZATION OF GENETIC MARKERS LINKED TO SEX DETERMINATION IN THE HAPLOID‐DIPLOID RED ALGA GRACILARIA CHILENSIS1

Bulk segregant analysis, random amplified polymorphic DNA (RAPD), and sequence characterized amplified region (SCAR) methods were used to identify sex‐linked molecular markers in the haploid‐diploid rhodophyte Gracilaria chilensis C. J. Bird, McLachlan et E. C. Oliveira. One hundred and eighty 10 bp primers were tested on three bulks of DNA: haploid males, haploid females, and diploid tetrasporophytes. Three RAPD primers (OPD15, OPG16, and OPN20) produced male‐specific bands; and one RAPD primer (OPD12), a female‐specific band. The sequences of the cloned putative sex‐specific PCR fragments were used to design specific primers for the female marker SCAR‐D12‐386 and the male marker SCAR‐G16‐486. Both SCAR markers gave unequivocal band patterns that allowed sex and phase to be determined in G. chilensis. Thus, all the females presented only the female band, and all the males only the male band, while all the tetrasporophytes amplified both male and female bands. Despite this sex‐specific association, we were able to amplify SCAR‐D12‐386 and SCAR‐G16‐486 in both sexes at low melting temperature. The differences between male and female sequences were of 8%–9% nucleotide divergence for SCAR‐D12‐386 and SCAR‐G16‐486, respectively. SCAR‐D12‐386 and SCAR‐G16‐486 could represent degenerated or diverged sequences located in the nonrecombining region of incipient sex chromosomes or heteromorphic sex chromosomes with sequence differences at the DNA level such that PCR primers amplify only one allele and not the other in highly specific PCR conditions. Seven gametic progenies composed of 19 males, 19 females, and the seven parental tetrasporophytes were analyzed. In all of them, the two SCAR markers segregated perfectly with sexual phenotypes.     

Novel SCAR primers for specific and sensitive detection of Agrobacterium vitis strains

An Agrobacterium vitis-specific DNA fragment (pAVS3) was generated from PCR polymorphic bands amplified by primer URP 2R. A. vitis specificity of this fragment was confirmed by Southern hybridization with genomic DNA from different Agrobacterium species. Sequence-characterized amplified region (SCAR) markers were developed for A. vitis specific detection, using 24-mer oligonucleotide primers designed from the flanking ends of the 670 bp insert in pAVS3. The SCAR primers amplified target sequences only from A. vitis strains and not from other Agrobacterium species or other bacterial genera. First round PCR detected bacterial cells between 5×10² and 1×10³ cfu/ml and the detection sensitivity was increased to as few as 2 cfu/ml by nested PCR. This PCR protocol can be used to confirm the potential presence of infectious A. vitis strains in soil and furthermore, can identify A. vitis strains from naturally infected crown galls.     

Molecular identification of isolates of <Emphasis Type="Italic">Peronosclerospora sorghi</Emphasis> from maize using PCR-based SCAR marker

The fungus Peronosclerospora sorghi [Weston and Uppal (Shaw)] infects both sorghum and maize and incites downy mildew disease. Pathogenic and molecular variability among isolates of P. sorghi from sorghum and maize has been reported. In the present study we developed a DNA sequence characterized amplified region (SCAR) marker for identification of isolates of P. sorghi from maize by using polymerase chain reaction (PCR). The random amplified polymorphic DNA (RAPD) primer OPB15 consistently amplified a 1,000 base pairs (bp) product in PCR only from DNA of P. sorghi isolates from maize and not from isolates of sorghum. The PCR-amplified 1,000-bp product was cloned and sequenced. The sequence of the SCAR marker was used for designing specific primers for identification of maize isolates of P. sorghi. The SCAR primers amplified a 800 bp fragment only from genomic DNA of maize isolates of P. sorghi. The SCAR primers developed in this study are highly specific and reproducible, and proved to be powerful tool for identification of P. sorghi isolates from maize.     

Generation of a Sequence Characterized Amplified Region Probe for Authentication of Mainland Serow (Capricornis sumatraensis)

Mainland serow is an endanged artiodactyl of southern Anhui province, China, that is often subject to poaching. To provide an easy, rapid and reliable marker for identification of bushmeat, skin and other tissues of the species, we developed a sequence characterized amplified region (SCAR) based on a species-specific random amplified polymorphic DNA (RAPD) marker. Initially, a 1012-bp species-specific DNA fragment of mainland serow was detected by a RAPD primer S1193. Then, a serow-specific primer pair (SCF/SCR) was designed according to the specific RAPD fragment, resulting in a 438-bp SCAR for the species. Finally, the reliability of the SCAR primers was tested by a common multiplex polymerase chain reaction using the combination of the SCAR and cyt b universal primers. The results that all mainland serow samples presented two target bands but the others failed to produce the SCAR indicated that the designed primers were highly diagnostic. Therefore, the SCAR probe developed in this study will be useful for quick authentication of mainland serow tissue samples for conservation biology and bushmeat regulation.     

High throughput genome-specific and gene-specific molecular markers for erucic acid genes in <Emphasis Type="Italic">Brassica napus</Emphasis> (L.) for marker-assisted selection in plant breeding

A single base change in the Bn-FAE1.1 gene in the A genome and a two-base deletion in the Bn-FAE1.2 gene in the C genome produce the nearly zero content of erucic acid observed in canola. A BAC clone anchoring Bn-FAE1.1 from a B. rapa BAC library and a BAC clone anchoring Bn-FAE1.2 from a B. oleracea BAC library were used in this research. After sequencing the gene flanking regions, it was found that the dissimilarity of the flanking sequences of these two FAE1 homologs facilitated the design of genome-specific primers that could amplify the corresponding genome in allotetraploid B. napus. The two-base deletion in the C genome gene was detected as a sequence-characterized amplified region (SCAR) marker. To increase the throughput, one genome-specific primer was labeled with four fluorescence dyes and combined with 20 different primers to produce PCR products with different fragment sizes. Eventually, a super pool of 80 samples was detected simultaneously. This dramatically reduces the cost of marker detection. The single base change in the Bn-FAE1.1 gene was detected as single nucleotide polymorphic (SNP) marker with an ABI SNaPshot kit. A multiplexing primer set was designed by adding a polyT to the 5' primer end to increase SNP detection throughput through sample pooling. Furthermore, the Bn-FAE1.1 and Bn-FAE1.2 were integrated into the N8 and N13 linkage groups of our previously reported high-density sequence-related amplified polymorphism (SRAP) map, respectively. There were 124 SRAP markers in a N8 bin in which the Bn-FAE1.1 gene-specific SCAR marker was located and 46 SRAP markers in a N13 bin into which the Bn-FAE1.2 SNP marker was integrated. These three kinds of high throughput molecular markers have been successfully implemented in our canola/rapeseed breeding programs.     

Development of a sequence-characterized amplified region marker-targeted quantitative PCR assay for strain-specific detection of Oenococcus oeni during wine malolactic fermentation

Control over malolactic fermentation (MLF) is a difficult goal in winemaking and needs rapid methods to monitor Oenococcus oeni malolactic starters (MLS) in a stressful environment such as wine. In this study, we describe a novel quantitative PCR (QPCR) assay enabling the detection of an O. oeni strain during MLF without culturing. O. oeni strain LB221 was used as a model to develop a strain-specific sequence-characterized amplified region (SCAR) marker derived from a discriminatory OPA20-based randomly amplified polymorphic DNA (RAPD) band. The 5' and 3' flanking regions and the copy number of the SCAR marker were characterized using inverse PCR and Southern blotting, respectively. Primer pairs targeting the SCAR sequence enabled strain-specific detection without cross amplification of other O. oeni strains or wine species of lactic acid bacteria (LAB), acetic acid bacteria (AAB), and yeasts. The SCAR-QPCR assay was linear over a range of cell concentrations (7 log units) and detected as few as 2.2 × 10(2) CFU per ml of red wine with good quantification effectiveness, as shown by the correlation of QPCR and plate counting results. Therefore, the cultivation-independent monitoring of a single O. oeni strain in wine based on a SCAR marker represents a rapid and effective strain-specific approach. This strategy can be adopted to develop easy and rapid detection techniques for monitoring the implantation of inoculated O. oeni MLS on the indigenous LAB population, reducing the risk of unsuccessful MLF.     

Genetic diversity of root-knot nematodes from Brazil and development of SCAR markers specific for the coffee-damaging species.

RAPD markers were used to characterize the genetic diversity and relationships of root-knot nematodes (RKN) (Meloidogyne spp.) in Brazil. A high level of infraspecific polymorphism was detected in Meloidogyne arenaria, Meloidogyne exigua, and Meloidogyne hapla compared with the other species tested. Phylogenetic analyses showed that M. hapla and M. exigua are more closely related to one another than they are to the other species, and illustrated the early divergence of these meiotically reproducing species from the mitotic ones. To develop a PCR-based assay to specifically identify RKN associated with coffee, three RAPD markers were further transformed into sequence-characterized amplified region (SCAR) markers specific for M. exigua, Meloigogyne incognita and Meloidogyne paranaensis, respectively. After PCR using the SCAR primers, the initial polymorphism was retained as the presence or absence of amplification. Moreover, multiplex PCR using the three pairs of SCAR primers in a single reaction enabled the unambiguous identification of each species, even in mixtures. Therefore, it is concluded that the method developed here has potential for application in routine diagnostic procedures.