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The development and molecular characterization of a rapiddetection method for Rice root-knot nematode (Meloidogynegraminicola)

ChoCho Htay & Huan Peng & Wenkun Huang &

Lingan Kong & Wenting He & Ricardo Holgado &

Deliang Peng

Accepted: 11 March 2016 /Published online: 19 March 2016# Koninklijke Nederlandse Planteziektenkundige Vereniging 2016

Abstract The root-knot nematode Meloidogynegraminicola is a major constraint in rice production inthe world. Using rDNA-ITS sequences data alignments,the genetic variation among twenty-one populations ofM. graminicola (sixteen from Myanmar and five fromChina) was investigated. The results showed that all thepopulations were clearly separated from other speciesand that there was a low level of genetic variationamong the isolates. A set of species-specific primerswas designed to develop a species-specific moleculartool for the precise identification ofM. graminicola. Theprimer reliability, specificity and sensitivity testsshowed that the primer set (Mg-F3 and Mg-R2) ampli-fied the expected fragment size of 369 bp from thetemplate DNA of target nematode populations but notfrom non-target organisms. A duplex PCR test allowsfor saving diagnostic time and costs by amplifying thespecies of interest from a nematode mixture. Therefore,this species-specific primer set may be a powerful toolto improve taxonomic identification by non-specialists

and the design of successful management practices aswell.

Keywords Rice root-knot nematode .Meloidogynegraminicola . rDNA-ITS . Species-specific primers

Introduction

Rice (Oryza sativa) is one of the most widely consumedstaple foods by a large portion of the world’s humanpopulation, particularly in Asia. The rice root-knot nem-atode Meloidogyne graminicola (Golden andBirchfield) is the most damaging root-knot nematodeof rice and is widely distributed in all rice-growingecosystems, including upland, lowland, deepwater andirrigated, in the world (Le et al. 2009), and in particular,in S.E. Asia. and the USA (Pankaj and Prasad, 2010).M. graminicola is the most damaging Meloidogynespecies for rice. In M. graminicola infested lowlandrainfall rice, nematicide application resulted in a yieldincrease of 16 %–20 % in Bangladesh (Padgham et al.2004), and in simulations of intermittently flooded rice,yield losses from M. graminicola ranged from 11 % to73 % (Soriano et al. 2000).

M. graminicola is a biotrophic, sedentary endo-parasitic nematode with a broad host range (Whitehead1998), including many of the common weeds of ricefields (MacGowan and Langdon 1989). BecauseM. graminicola is particularly well adapted to intermit-tent flooding, it is predicted that, in view of the loomingwater shortage, the importance of M. graminicola as a

Eur J Plant Pathol (2016) 146:281–291DOI 10.1007/s10658-016-0913-y

ChoCho Htay & Huan Peng contributed equally to this work.

C. Htay :H. Peng :W. Huang : L. Kong :W. He :D. Peng (*)State Key Laboratory for Biology of Plant Diseases and InsectPests, Institute of Plant Protection, Chinese Academy ofAgricultural Sciences, Beijing 100193, Chinae-mail: [email protected]

R. HolgadoNorwegian Institute of Bioeconomy Research, Høgskoleveien 7,1430 Ås, Norway

http://crossmark.crossref.org/dialog/?doi=10.1007/s10658-016-0913-y&domain=pdf

constraint in rice production will substantially increasein the years ahead (De Waele and Elsen 2007).M. graminicola has a short life cycle, which can becompleted in 15 days at 27–37 °C (Jaiswal and Singh2010). As a result, the presence of even a small numberof M. graminicola at planting can increase to a highpopulation density during a single crop cycle. Accurateand sensitive identification of M. graminicola is abso-lutely necessary for effective nematode managementand precise research.

The morphological features of adult females (perine-al pattern), males and juveniles (J2) have traditionallybeen used to identifyMeloidogyne spp. (Eisenback et al.1981). Still, diagnosticians are challenged to identifythis species correctly due to extremely similar morpho-logical features between species, wide host ranges, lifestages in different habitats, indistinct species bound-aries, sexual dimorphism, species with a potentialhybrid origin and polyploidy (Blok and Powers2009). Moreover, species and race identificationof Meloidogyne based on phenotypic characterizationis time-consuming because adult forms are hardly ac-cessible, males are usually rare and females are mal-formed (Besnard et al. 2014). Morphological identifica-tion also requires a great deal of skill (Hooper 1990).

Therefore, the use of biochemical and moleculartools has been introduced to help nematode taxonomistsidentify nematode species as well as to investigate thephytogeography and population dynamics of nematodes(Blok and Powers 2009; Gilabert and Wasmuth 2013).Many different DNA-basedmethods have been reportedfor the identification of a large number of Meloidogynespp. (Blok and Powers 2009). The analysis of codingand non-coding regions of ribosomal DNA (rDNA)is a popular method for nematode identification(ZijIstra et al. 1995). The internal transcribed spac-er region (ITS) is variable and thus one of themost frequently used genetic markers for nematodeidentification. Unfortunately, a species-specific mo-lecular tool for immediate diagnosis of M. graminicolais lacking.

In this work, phylogenetic analysis was carried outbased on the ITS-rDNA region of individual juveniles(J2) to investigate the genetic diversity of rice root-knotisolates originating from different locations in Myanmarand China. A set of species-specific primers designedfor fast and reliable diagnosis ofM. graminicola accord-ing to the ITS-rDNA sequences obtained from this studywill be designed to specifically identify the target

nematode species from a mixture of species in a singlestep PCR.

Materials and methods

Nematode isolates

The infected soil and plant roots, especially rice root-knot nematodes, were collected from rice fields in dif-ferent locations in Myanmar and China. A total of 21populations of M. graminicola and several othernematode isolates were used in this work(Table 1). Nematodes were extracted from soiland root samples by the Whitehead tray method(Whitehead and Hemming 1965). All nematodeswere stored at 4 °C for molecular and morpholog-ical identification.

DNA Extraction

A single nematode (J2) was picked up by hand and putinto a sterile PCR tube containing about approximately14 μl of ddH2O. After freezing in liquid nitrogen for 1–2 min, the nematode was crushed with a sterilized glassrod (75 % alcohol). Then, 3 μl of 10 × PCR buffer(Promega, USA) and of proteinase K solution(600 mg/mL) (Takara, Dalian, China) were added tothe tube and centrifuged for 1 min. The samples werethen frozen at −20 °C for 2 h. Thereafter, the tube wasincubated at 65 °C for 1.5 h followed by incubation at95 °C for 10 min (Williamson et al. 1997). For thespecificity test, minor modifications were made to thegenomic DNA extraction method to obtain enough tem-plate DNA, using 8 μl of ddH2O, 1.5 μl of 10 x PCRBuffer, and 1.5 μl of proteinase K instead of the amountdescribed above. Finally, the samples were centrifugedfor 1 min at 12,000 rpm and the DNA supernatants werestored at −20 °C for future use.

PCR amplification of ITS-rDNA

Amplification reactions were performed in total vol-umes of 25 μl containing 2 μl of extracted DNA,2.5 μl 10 × PCR Buffer (Mg2+), 2 μl dNTP (2.5 mM),0.2 μl rTaq (5 U/μL), and 0.5 μl each of the TW81 andAB28 primers (Joyce et al. 1994). The PCR programmewas as follows: 94 °C for 4 min, followed by 35 cyclesof 30 s at 95 °C, 30 s at 55 °C, and 30 s at 72 °C, with a

282 Eur J Plant Pathol (2016) 146:281–291

final extension at 72 °C for 5 min. The amplified prod-ucts were separated by electrophoresis on 1.5 % agarosegels in 0.5 × TAE buffer (Sambrook et al. 1989) with the

marker D2000 and stained with ethidium bromide (EB).The gels were visualized under a UV transilluminatorand photographed.

Table 1 Nematode populations used in the present study and PCR amplified products obtained during testing of the specific primercombinations

Species code species Populations origin Plant Host Length of PCR fragment

369 bp ~766 bp

M01 Meloidogyne graminicola Yezin, Mandalay, Myanmar Oryza sativa L. + +

M02 M. graminicola KanGyiDaunt, Pathein, Myanmar Oryza sativa L. + +

M03 M. graminicola KanGyiDaunt, Pathein, Myanmar Oryza sativa L. + +

M04 M. graminicola KhaYangGwin, Pathein, Myanmar Oryza sativa L. + +




M08 M. graminicola NyungChaung, Pathein, Myanmar Oryza sativa L. + +



M11 M. graminicola Hlagu, Yangon, Myanmar Oryza sativa L. + +

M12 M. graminicola TikeGyi, Yangon, Myanmar Oryza sativa L. + +





GD1 M. graminicola Guangzhou, Guangdong, China Oryza sativa L. + +

GD2 M. graminicola Guangzhou, Guangdong, China Oryza sativa L. + +

Fj M. graminicola Fujian, China Oryza sativa L. + +

Hn M. graminicola Haikou, Hainan, China Oryza sativa L. + +

CM M. graminicola Chengmai, Hainan, China Oryza sativa L. + +

Mi-01 M. incognita Langfang, Hebei, China Solanum lycopersicum L. − +

Mi-02 M. incognita Fujian, China Cucumis sativus − +

Mi-03 M. incognita Fujian, China Daucuscarota subsp. Sativus − +

Me-01 M. enterolobii Hainan, China Solanum lycopersicum L. − +

Me-02 M. enterolobii Yunnan, China Solanum lycopersicum L. − +

Mh-01 M. hapla Yunnan, China Solanum lycopersicum L. − +

Mh-02 M. hapla Beijing, China Rosa chinensis − +

Mj M. javanica Yunnan, China Solanum lycopersicum L. − +

Ma M. arenaria Yunnan, China Nicotiana tabacum − +

Ha Heterodera avenae Daxin, Beijing, China Triticum spp. − +

Hf H. filipjevi XuChang, Henan, China Triticum spp. − +

Pz Pratylenchus zeae GuangXi, China Zea mays − +

Ho-01 Hirschmanniella oryzae TikeGyi, Yangon, Myanmar Oryza sativa L. − +

Ho-02 H.a oryzae LiuYang, China Oryza sativa L. − +

Hm H. mucronata PingJiang, China Oryza sativa L. − +

Ta Tylenchorhynchus annulatus XiangZi, China Oryza sativa L. − +

+presence of amplified fragment; −: absence of amplified fragment

Eur J Plant Pathol (2016) 146:281–291 283

Cloning and sequencing

The ITS amplified fragments for three individual nem-atodes for each populationwere purified using the TIANgel Purification Kit according to the manufacturer’sprotocol, followed by ligation into pGEM-T Easy vectorand transformation into competent cells (E. coli,DH5α). Positive colonies with the expected insert sizewere sent for sequencing. The sequences were editedand analysed using the software packages Chromas 2.00(Technelysium, Helensvale, QLD, Australia) andDNAstar 7.1. The ITS sequences obtained in this studywere deposited at NCBI (National Center forBiotechnology Information). The sequences were com-pared with previously published sequence data inGenBank using the Blastn programme to analyse intra-specific differences among the specimens.

Phylogenetic analysis

To reduce experimental error, the resulting sequences weremanually edited with EditSeq and aligned with ClustalW.Maximum likelihood (ML) based on 1000 bootstrap rep-licates was used to test the node support of the generatedtrees with MEGA 5 software (Tamura et al. 2011). Thecomplete-deletion option was used to eliminate all posi-tions containing gaps and missing data. Pairwise geneticdistance among the isolates of the same specieswas carriedout by the Maximum-Composite Likelihood model. Asreferences, three ITS sequences of M. graminicola(Taiwan, India, Bangladesh), M. hapla, M. arenaria,M. incognita, and M. javanica and two outgroups(Hirschmanniella oryzae and Pratylenchus penetrans) ex-tracted from GenBank were used.

Designing species-specific primers

Through sequence alignments, a set of species-specificprimers was prepared using the Primer Premier 5 softwarebased on the unique regions that could be utilized asmolecular tools for the diagnosis of M. graminicola. Thealignment consisted of ITS sequences from this study andother Meloidogyne species from GenBank, including M.graminicola from Taiwan (KJ572383), M. naasi(JN157859), M. chitwoodi (JN241864), M. incognita(KJ641591), M. hapla (JX024147), M. arenaria(LC030350), M. javanica (JQ917440) and M. enterolobii(JF309157). The selected potential species-specificprimers were compared with the Nucleotide (Nt) dataset

using primer-blast to examine the specificity. The PCRconditions described above were optimized for specificityby performing a temperature gradient PCR to determinethe optimum annealing temperature.

Primer reliability test

To test the reliability of the primers, DNA from 21populations of M. graminicola that provided templateDNA for sequencing were amplified with the optimizedconditions: initial denaturation at 95 °C for 4 min,followed by 35 cycles of 95 °C for 30 s, 51 °C for30 s, 72 °C for 30 s and a final extension at 72 °C for5 min. Additional primer reliability was tested withdifferent life stages of nematodes, including eggs, J2,J3, J4, female and male. PCR without DNAwas used asa negative control.

Primer specificity test

Investigations of the potential of specific primer setswere undertaken with duplex PCR containing two setsof primers in a single test. The first primer set, D2D3(D2A -5′ACAAGTACCGTG AGGGAAAGTTG-3′and D3B 5′-TCGGAAGGAACCAGCTACTA-3′),which amplifies the D2-D3 expansion regions of the28S rRNA gene (Nunn 1992), was used as an internalcontrol to confirm the presence of DNA in the sampleand the success of the PCR. The second set includedspecies-specific primer sets that targeted nematodeDNA sequences of interest. For this PCR, 2 μl ofDNAwas added to 23 μl of a PCR mixture containing2.5 μl 10 x Ex Taq PCR Buffer (Mg2+), 2 μl dNTP(2.5 mM), 0.3 μl Ex Taq (5 U/μL), 0.5 μl of each primer(100 μM) and 16.2 μl ddH2O. The amplification condi-tions were as described above except for the annealingtemperature, which was increased from 51 °C to 53 °Cto improve the utility and flexibility of the primers. Anegative control containing the PCR mixture withoutany DNA template was also included in every PCRreaction. To verify the data, the experiments were re-peated at least twice.

Primer sensitivity test

To investigate primer sensitivity, different numbers of J2DNA (n = 1, 2, 3, 4 and 5) were used to determine theminimum amount of J2 detectable in the PCR assays.Sensitivity was also performed in a PCR tube containing


M. graminicola genomic DNA alone or in combinationwith DNA from non-target root-knot nematode species(M. enterolobii and M. incognita) with the ratio of 1:1.M. enterolobii-specific primers (Me-F 5′-AACTTTT

GTGAAAGTGCCGCTG-3′ and Me-R 5′- TCAGTTCAGGCAGGATCAACC-3′) and M. incognita-specificprimers (Mi-F 5′- GTGAGGATTCAGCTCCCCAG-3′and Mi-R 5′-ACGAGGAACATACTTCTCCGTCC-3′)

Fig. 1 Maximum-likelihood tree showing the phylogenetic rela-tionships of Meloidogyn ebased on ITS1–5.8S-ITS2 rDNA se-quences. Hirschmanniella oryzae and Pratylenchus penetrans

ITS-rDNA sequences have been used as outgroups. The numbersindicated on the nodes are the bootstrap values for each clusterbased on 1000 permutations


(Long et al. 2006; Meng et al. 2004) were used to amplifythe specific sequence from related species. Duplex PCRusingMg-F3/Mg-R2 primers and Mi-F/Mi-R primers wascarried out with a primer annealing temperature of 57 °Cand theMg-F3/Mg-R2 primers andMe-F/Me-R primers at53 °C. PCR without DNAwas used as a negative control.

Results

Sequence and phylogenetic analysis

In this study, PCR amplification of the ITS-containingregion yielded a single fragment with a length of

approximately 579 bp in all populations tested by usingthe general primers set. All sequences were submitted toGenBank under accession No. (KR 604730 – KR604749 and KU646999). The blast results showed thatall nucleotide sequences of ITS sequences were 98–100 % similar to those M. graminicola sequences iso-lated from GenBank. Pairwise genetic distances amongisolates of the same species ranged from 0 to 0.016 andthe highest distance was observed between M 08 and M11. The maximum-likelihood tree (Fig. 1) generated onthe basis of the Tamura-Nei model with 1000 bootstrapvalues demonstrated that all theM. graminicola isolatesformed an independent cluster with a high bootstrapsupport value of 99 % (Fig. 1). All the Chinese isolates,

Fig. 2 Multi-alignmentgenerated with ClustalW of thedifferent ITS sequences ofM. graminicola and otherMeloidogyne species fromGenBank (see Materials andMethods for sequence accessionnumber) used to develop thespecies-specific primer set. Theposition of the primers MgF3 andMgR2 are shown with a blackarrow and a gap with a dash. TheRoman numeral I indicates thejunction between 18S and ITS1, IIindicates between ITS1 and 5.8S,III indicates between 5.8S andITS2, and IV indicates betweenITS2 and 28S


five Myanmar isolates and JN241866.1 clustered to-gether with a bootstrap value of 82 % and 71 % forthe rest of the Myanmar isolates. M. minor served as asister group with a high bootstrap value of 98 %. Thethree mitotic parthenogenesis species M. arenaria, M.incognita andM. javanica clustered together with 99 %bootstrap support. A facultative meiotic parthenogenesisspecies, M. hapla, was clearly separated during phylo-genetic analysis. Hirschmanniella oryzae andPratylenchus penetrans were considered as outgroups.

Primer design

Species-specific forward (Mg-F3 5′-TTATCGCATCATTTTATTTG-3 ′) and reverse (Mg-R2 5 ′-CGCTTTGTTAGAAAATGACCCT-3′) primers were de-signed (Fig. 2). The length of the expected amplifiedfragment with the specific primers is 369 bp, which con-tains part of the ITS 1 and 5.8 S regions of ribosomal DNA(Fig. 2).

Primer reliability test

The expected band size of 369 bpwas amplified by PCRwith the specific primer set from all populations of M.graminicola tested (Fig. 3). Additionally, reliability testswith different life stages of the nematode (eggs, J2, J3,

J4, female and male) showed that amplification of alldevelopmental stages gave positive results (Fig. 4).

Primer specificity test

After optimizing PCR cycling conditions, every targetnematode species of M. graminicola yielded two dis-tinct fragments: ~766 bp for 28S regions and 369 bp forthe specific primer set (Fig. 5). A single band of ~766 bpwas observed from the 28S region of non-target species.A negative control PCR mixture without any DNAtemplate gave no amplification result.

Primer sensitivity test

Primer sensitivity was investigated with a different num-ber of J2 nematodes in the PCR assays. The resultsdemonstrated that the minimum detection concentrationrequired for the PCR assay is a single juvenile (Fig. 6).In addition, the higher the juvenile numbers used in thePCR, the stronger the bands.

Moreover, specific bands were noted in the PCRtubes containing target nematode species either aloneor in a mixture with non-target nematode species(Fig. 7). The DNA mixture of M. graminicola andM. enterolobii produced two fragments, of 369 bp and~230 bp (Fig. 7a). The mixture of M. graminicola andM. incognita generated two fragments, of 369 bp and

Fig. 3 Amplification products of different isolates of M. graminicola using the species-specific primer set. Lane 1–21, twenty-onepopulations as described in Table 1; ck, negative control; Lane M, D2000 DNA ladder

Fig. 4 PCR products of differentdevelopmental stages of M.graminicola amplified with thespecies-specific primer set.Lane 1, egg masses; Lane 2, J2;Lane 3, J3; Lane, 4, J4; Lane 5,Female; Lane 6, Male; ck,negative control; Lane M, D2000Marker


~1000 bp (Fig. 7b). No fragments were generated in thePCR tube without template DNA.

Discussion

M. graminicola has a short life cycle, a wide host rangeand is well adapted to flooded conditions, which makesthis species difficult to control (De Waele and Elsen2007). To select effective management schemes, accu-rate and rapid identification of root-knot nematodes atthe species level is essential. Although the formation ofhook-like galls at the tip of the young roots is a typicalcharacteristic of M. graminicola, only soil samples areusable, more than one species may occur in the samesoil sample or low population densities might becomeproblematic, interfering with the accurate identificationof this nematode species.

In the present work, molecular identification based onITS-rDNA of twenty-one populations was undertakenusing a general ITS primer set. Nucleotide variability wasnoted in comparison to the ITS-rDNA sequences obtainedfrom the same individual nematode as well as differentnematodes from the same sample. This finding has beenreported by others (Blok and Powers 2009; De Luca et al.2011). The differences in the ITS sequences betweenclones of the same population could be due to variationsamong copies of the ITS within an individual, or due toerrors arising through PCR amplification, cloning or se-quencing (Pokharel et al. 2007).

The low level of genetic distance indicated a lack ofgenetic diversity between twenty-one M. graminicolaisolates. Similar results were reported fromM. ethiopica(Correa et al. 2013),M. enterolobii (Tigano et al. 2010),M. incognita and M. javanica (Randig et al. 2002). A

high bootstrap value in the Maximum Likelihood treesuggested a clearly separated group from different spe-cies ofMeloidogyne. Pokharel and his group (2007) alsoreported that a phylogenetic tree with a well-supportedclade was observed in NepaleseM. graminicola isolatesbased on the sequences of partial ITS of the rRNAgenes.

Furthermore, sequence analysis showed that 21 iso-lates clearly divided into two groups. The first groupconsisted of 16 populations from Myanmar that splitinto two subgroups, leading to the conclusion that ge-netic variation did not correspond to geographical loca-tion. However, the second group included five popula-tions from China that belonged to same cluster. Thisresult may have arisen because a smaller population wastested or there is lower degree of sequence dissimilarityamong the Chinese isolates. To provide strong data onthe genetic diversity of M. graminicola, it would benecessary to add the isolates of nematodes originatingfrom different countries, climatic conditions or geo-graphic locations.

This study also reports a species-specific primer setthat can be used as a molecular tool in the diagnosis of

Fig. 5 Duplex PCR products using two sets of primers, D2D3 andMg-F3/Mg-R2, in a single step PCR. Lane 1–7, M01, M02, M03,M04, M05, GD1, Fj; Lane 8, Mi-01; Lane 9, Mi-02; Lane 10, Mi-03; Lane11, Me-01; Lane 12, Me-02; Lane 13, Mh-01; Lane 14,

Mh-02; Lane 15, Mj; Lane 16, Ma; Lane 17, Ha; Lane 18, Hf;Lane19, Pz; Lane 20, Ho-01; Lane 21, Ho-01; Lane 22, Hm; Lane23, Ta; ck, negative control; Lane M, D 2000 molecular marker

Fig. 6 Primer sensitivity test with different numbers of secondstage juveniles (n = 5, 4, 3, 2 and 1). M, D 2000 marker


the rice root-knot nematode M. graminicola. To theauthors’ knowledge, this is the first report of a species-specific primer pair for the detection and diagnosis ofthe rice root-knot nematode M. graminicola. In theprimer reliability test, the newly developed primer setproduced a unique specific band of 369 bp with geno-mic DNA from 21M. graminicola isolates. Similarly, aspecific PCR amplicon was produced with DNA fromdifferent developmental stages of target nematodespecies.

The primer specificity analysis with other nematodespecies showed that specific fragments were producedonly with DNA from M. graminicola. The specificproducts were not amplified when PCR was carriedout with DNA templates from non-target nematodespecies. In addition, primer sensitivity is excellent be-cause this molecular tool was able to detect the singleindividual juvenile of the target nematode. It was alsopossible to detect target nematode species in a popula-tionmixture containingM. incognita andM. enterolobii.Thus, newly developed species-specific primers mightbe used as molecular markers that satisfy the threecriteria of Hübschen and his group (Hübschen et al.2004a) to diagnosis the rice root-knot nematode M.graminicola. However, more research is needed to con-firm this primer set with other closely related species,especially M. trifoliophila, which ITS sequence is sim-ilar to M. graminicola (Birchfield 1965). Fortunately,rice is not a host forM. trifoliophila and this species onlyinfects white clover.

Duplex PCR using two sets of primers was devel-oped in a single step amplification to save on time,labour and cost required for identification of pathogens.Some faint nonspecific bands observed with the normalPCR programme were eliminated by varying PCR an-nealing temperatures and the proportion of primers inthe reaction. Therefore, optimization of the PCR cyclingconditions and components is key for successful

amplification and improvements in the sensitivity orspecificity of the test (Elnifro et al. 2000). The PCRtechnique using two or more pairs of primers for theidentification of species has been widely applied innematology (Correa et al. 2013; Hu et al. 2011;Hübschen et al. 2004a; Hübschen et al. 2004b;Oliveira et al. 2005; Ou et al. 2008; Randig et al.2002; Subbotin et al. 2001).

In conclusion, the high level of homogeneity amongthe tested isolates suggested that similar control methodsagainst nematodes could be used in the sample collec-tion locations. Furthermore, the species-specific primerset reported here appears to be a useful molecular mark-er that is reliable, specific and sensitive for the rapiddiagnosis of M. graminicola. However, further valida-tion of this newly developed primer set is required withlarge populations of the same species and closely relatedspecies.

Acknowledgments This study was supported by the Organiza-tion forWomen in Science for the DevelopingWorld (OWSD) andthe Swedish International Development Cooperation Agency(SIDA). Research facilities were provided by the Special Fundfor Agro-scientific Research in the Public Interest (No.201103018) the National Key Basic Research Program of China(973 Program, 2013CB127502).

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