《分子生物学》课程教学资源（参考文献）基因功能研究 Differential expression of Rs-eng-1b in two populations of Radopholus similis（Tylenchida：Pratylecnchidae）and its relationship to pathogenicity

Differential expression of Rs-eng-1b in two populations of Radopholus similis (Tylenchida:Pratylecnchidae) and its relationship to pathogenicity Chao Zhang.Hui Xie.Chun-Ling Xu Xi Cheng-Ke-Mei Li.Yu Li 825202omc161ac302 Abstract We constructed a ssh (suppression sub was approximately 2.7 times as much as the expre tractive hybridization)library based on two popula tions (Rs-C and Rs-P)of Radopholus similis from different host plants and exhibiting differences in path paradisiaca and Anthu from the R-Cpopulationw ogenicity on Musa nression differ es amo four different develor plants.In order to screen the clones with ment stages The order of Rs-eng-lb relativ significant ex ssion differences from the SSH li ssion abundance from high to lo was females ry.a total of 2400 clones was randomly selected iuveniles males and e We furthe used RNAi to and reverse northern blotting was performed on them test whether Rs Out of the 2,400 clones,89 clones showed significant sponsible for nathogenicity which was the first rnai expression differences.Out of sequencing these 89 work about Rs-eng-1b.The RNAi results showed that clones distinct sequences from 87 clones were Rs-eng-b exnression had a nositive correlation to obtained.Aligning the 87 distinct sequences against pathogenicity of the population.The longer the the non-redundant nucleotide database (nr)in nCBl RNAi treatment,the less pathogenic the nematode we found that five sequences were highly conserved population was.Non-endogenous gfp dsRNA had no with Rs-eng-/6.Two of five sequences with lengths of significant influence on the expression of Rs-eng-/b 467 base pairs (bp)(GW395922)and 742 bp and pathogenicity of R.similis Rs-C population.In (GW395923)were further emploved to perform 5 conclusion,all our evidence indicated Rs-eng-/b RACE-PCR and 3'RACE-PCR,respectively might be a crucial pathogenicity-related gene in R. Subsequently,the complete length of Rs-eng-/b similis (EU414839)was obtained (1,427 bp).Our qPCR re sult showed that expression of Rs-eng-Ib in the pop Keywords Banana burrowing nematode.SSH ulation Rs-C with high pathogenicity on host plants B-1.4-endoglucanase.Real-time PCRRNAi Introduction ratory of Plant Nematology and Research Cente The banana burrowing nematode,Radopholus similis is one of the most damaging pests of bananas and 510642.People's Republic of China severely harms pepper,omamental plants,and many -mail:xichui@scau.edu.cn other agronomic and horticultural crops.R.similis is Springe

Differential expression of Rs-eng-1b in two populations of Radopholus similis (Tylenchida: Pratylecnchidae) and its relationship to pathogenicity Chao Zhang & Hui Xie & Chun-Ling Xu & Xi Cheng & Ke-Mei Li & Yu Li Accepted: 25 May 2012 / Published online: 16 June 2012 # KNPV 2012 Abstract We constructed a SSH (suppression sub￾tractive hybridization) library based on two popula￾tions (Rs-C and Rs-P) of Radopholus similis from different host plants and exhibiting differences in path￾ogenicity on Musa paradisiaca and Anthurium andraeanum plants. In order to screen the clones with significant expression differences from the SSH li￾brary, a total of 2,400 clones was randomly selected and reverse northern blotting was performed on them. Out of the 2,400 clones, 89 clones showed significant expression differences. Out of sequencing these 89 clones, distinct sequences from 87 clones were obtained. Aligning the 87 distinct sequences against the non-redundant nucleotide database (nr) in NCBI, we found that five sequences were highly conserved with Rs-eng-1b. Two of five sequences with lengths of 467 base pairs (bp) (GW395922) and 742 bp (GW395923) were further employed to perform 5′ RACE-PCR and 3′ RACE-PCR, respectively. Subsequently, the complete length of Rs-eng-1b (EU414839) was obtained (1,427 bp). Our qPCR re￾sult showed that expression of Rs-eng-1b in the pop￾ulation Rs-C with high pathogenicity on host plants was approximately 2.7 times as much as the expres￾sion of Rs-eng-1b in the population Rs-P with low pathogenicity on host plants. Furthermore, the gene Rs-eng-1b from the Rs-C population also showed ex￾pression differences amongst four different develop￾ment stages. The order of Rs-eng-1b relative expression abundance from high to low was females, juveniles, males, and eggs. We further used RNAi to test whether Rs-eng-1b of Rs-C population was re￾sponsible for pathogenicity which was the first RNAi work about Rs-eng-1b. The RNAi results showed that Rs-eng-1b expression had a positive correlation to pathogenicity of the population. The longer the RNAi treatment, the less pathogenic the nematode population was. Non-endogenous gfp dsRNA had no significant influence on the expression of Rs-eng-1b and pathogenicity of R. similis Rs-C population. In conclusion, all our evidence indicated Rs-eng-1b might be a crucial pathogenicity-related gene in R. similis. Keywords Banana burrowing nematode . SSH . β-1 . 4-endoglucanase . Real-time PCR . RNAi Introduction The banana burrowing nematode, Radopholus similis is one of the most damaging pests of bananas and severely harms pepper, ornamental plants, and many other agronomic and horticultural crops. R. similis is Eur J Plant Pathol (2012) 133:899–910 DOI 10.1007/s10658-012-0015-4 C. Zhang : H. Xie (*) : C.-L. Xu : X. Cheng : K.-M. Li : Y. Li Laboratory of Plant Nematology and Research Center of Nematodes of Plant Quarantine, College of Natural Resource and Environment, South China Agricultural University, Guangzhou 510642, People’s Republic of China e-mail: xiehui@scau.edu.cn

900 Eur J Plant Pathol (2012)133:899-910 known to attack over 250 plant species (O'Bannon populations of Globodera pallida and identified a cel- 1977).R.similis is a migratory endoparasite that lulase and an important pathogenicity factor invades the roots and feeds on the cytoplasm of Meanwhile,several parasite-related genes from cortex cells.As a result,the roots will blacken and Meloidogyne incognita were detected by SSH (Huang die which results in a reduction of plant growth et al.2004).Although mining differentially expressed and development.This destruction of crops leads genes in PPNs is not very difficult,it is crucial to to severe economic losses and consequently,many understand gene function involved in pathogenesis and countries have entered it on the list of quarantine regulation pattems.RNA interference (RNAi)is an plant pests (Cotton and Van Riel 1993;Smith and RNA-dependent gene silencing process in which the Charles 1998). guide strand(small interference RNA,siRNA)from Nematicides have been used as one of many inte short double-stranded RNA (dsRNA)molecules are grated approaches to control plant-parasitic nematodes incorporated into the RNA-induced silencing complex (PPNs).As concems have arisen over the environmen (RISC)to bind to specific mRNA molecules (target tal implications associated with over-using of some mRNA).siRNAs prevent target mRNAs from being translated into protein by either degrac ding target n use mRN om completely trans it yield loss ha Ihus,a ating the mRN. to seek additional app for PPNs contro n recent year molecular bio ing approa trol hav et a we ting diff ath 2000.Ma app che s to i 2004: PCR (R supp iRNA displa (RSDD p y app ole K Sher SSH has the d tiall 1999)SSH ad 1002. sitivity low of fals 2002 tal.2007 d infection in the plan ince more than 100 differ one ssH (vor t al B-1.4-endoglucanase (EGases)also known as cel 1997).Grenier et al.(2002) d SSH to and ie differentially pressed genes betw en two A classified as a fifth glycosy Springer

known to attack over 250 plant species (O'Bannon 1977). R. similis is a migratory endoparasite that invades the roots and feeds on the cytoplasm of cortex cells. As a result, the roots will blacken and die which results in a reduction of plant growth and development. This destruction of crops leads to severe economic losses and consequently, many countries have entered it on the list of quarantine plant pests (Cotton and Van Riel 1993; Smith and Charles 1998). Nematicides have been used as one of many inte￾grated approaches to control plant-parasitic nematodes (PPNs). As concerns have arisen over the environmen￾tal implications associated with over-using of some nematicides, many nematicides have been decreased in use. As a result, shortcomings in our ability to successfully limit yield loss have occurred. Thus, an effort to seek additional approaches for PPNs control is imperative. In recent years, molecular biology and genetic en￾gineering approaches to solve the problem of PPNs control have been developed quickly. In order to have more appropriate biological controls, understanding the molecular mechanisms involved in pathogenicity by mining pathogenicity genes has become extremely important (Chen et al. 2005). It is an effective method to screen for pathogenicity genes by comparing gene expression profiles between different populations of a species exhibiting different levels of pathogenicity on host species. There are a series of approaches to iden￾tify differentially expressed genes, such as mRNA differential display reverse transcription PCR, repre￾sentational difference analysis (RDA), suppression subtractive hybridization (SSH), and reciprocal sub￾traction differential RNA display (RSDD). Of those approaches, SSH has been extensively applied in mo￾lecular genetics and molecular biology (Kuang and Ashorn 1993; Tchernitsa et al. 1999; Shen and Liu 2004). SSH has super sensitivity to recognize the differen￾tially expressed genes in low expression abundance (Diatchenko et al. 1999). SSH advantages include high sensitivity, low occurrence of false positives, high effi￾ciency, and low cost. Commonly, SSH is better than other approaches to detect differentially expressed genes, since more than 100 differentially expressed genes can be enriched in one SSH (von Stein et al. 1997). Grenier et al. (2002) used SSH to investigate differentially expressed genes between two different populations of Globodera pallida and identified a cel￾lulase and an important pathogenicity factor. Meanwhile, several parasite-related genes from Meloidogyne incognita were detected by SSH (Huang et al. 2004). Although mining differentially expressed genes in PPNs is not very difficult, it is crucial to understand gene function involved in pathogenesis and regulation patterns. RNA interference (RNAi) is an RNA-dependent gene silencing process in which the guide strand (small interference RNA, siRNA) from short double-stranded RNA (dsRNA) molecules are incorporated into the RNA-induced silencing complex (RISC) to bind to specific mRNA molecules (target mRNA). siRNAs prevent target mRNAs from being translated into protein by either degrading target mRNAs or inhibiting ribosomes from completely trans￾lating the mRNA. RNAi has been found in many eukar￾yotes including animals and plants. RNAi was initially discovered and developed to application on potato (Kawchuk et al. 1991) and Caenorhabditus elegans (Guo and Kemphues 1995; Fire et al. 1998). RNAi can be used as a simple and effective alternative gene￾knockout tool to obtain function-less or function-loss mutants, because of its high sequence-specificity and effective interference activity. Meanwhile, with its sim￾ple operation, short cycle and low cost, it has become an extremely important tool and most popular research topic in the fields of gene identification, genetic analy￾sis, gene function, gene therapy and genomics (Barstead 2001; Kamath et al. 2000; Maine 2001; Cheng et al. 2003; Rangasamy et al. 2004; Tijsterman et al. 2004). Although it is difficult to study gene function of PPNs by constructing mutants because PPNs are obligatory parasites and do not grow in artificial culture medium; with the advantage above, RNAi could be used for screening mutants, identifying gene function, or control of PPNs (Bakhetia et al. 2007). However, little research has been carried out on the pathogenicity genes of PPNs, and their functions; and, so far, research has also concentrated on the sedentary endoparasitism nemato￾des, such as Meloidogyne sp., Heterodera sp. and Globodera sp. (Goddijn et al. 1993; Brindley et al. 1997; Vercauteren et al. 2002; Shingles et al. 2007). The molecular mechanism of parasitic-related-gene-me￾diated infection in the plant host-parasite interaction is still unclear. β-1, 4-endoglucanase (EGases), also known as cel￾lulase, functions in degrading plant cellulose and is classified as a fifth glycosyl hydrolase family 900 Eur J Plant Pathol (2012) 133:899–910

Eur J Plant Pathol (2012)133:899-910 901 Oniacgeman et al 2008)EGcne tanster con Nematodes 2007 ed RNAi to silence EGases in lod Two populations of R similis:Rs-C and Rs-P,which ath d Philoe odes with silen ntly les Rs. Rs.E sbeen wide th。 the ole in tw arg ned to pop natodes.The ltured on ex nm ion of p studied by of 6 cm at 25C in al (2008).and m seful in eidentified The four EGase Nematode extraction es in R.similis had been cloned by Haegeman et al.(2008).Their res The carrot callus was mashed with a blender The only focused on tisst ion and tim mashed solution was filtered through combined sieves (Hae n et al 2008)Currently t ie no bort or with aperture of 0.147 mm and 0.026 mm.Nematodes whether the function of EGases is involved in pathoge were collected from the 0.026 mm ap re si nicity of r.similis on plant Roots of anthurium were cut into I cm frag ent n this study.a differential ge ession libran These fra nts were mashed in a blender and furthe was constructed by SSH.based on tw onulation of filtered by nested sie R similis from different and different hosts des wer om the EGases in R.similis (Rs- 0.026mm eve.This first nematode collec ened from a ssh library. In tion was labelled as NI.Meanwhile.the 0.147 m ne function.a series of experiments including pore sieve subiected to the baermann funnel sepa RNAi.real-time PCR (aPCR).carrot callus culture ing the nematodes number was labelled as n2 The and artificial infection,were performed to investigate total nematodes in roots were calculated as the sum of the relationship between the expression of Rs-eng-/b NI and N2 Nematodes in soil were isolated from and pathogenicity of R.similis on plant. 200 ml of mixed soil from the host plant pot using a Baermann funnel method Extraction from sieve or Baermann funnel were adjusted to 10 ml of nematode Materials and methods suspension.and I ml of the suspension was pipetted into a glass dish with a diameter of 6 cm.and the Plant materials number of nematodes were counted under the stereo microscone while the number of females males and Anthurium,Anthurium andraeanum plants were iuveniles were counted respectively.The same work bought from the Flowers and plants research center was done 10 times until all nematode suspensions had Guangzhou,Guangdong.The roots of anthurium seed- e o been counted.Total nematode lings were washed with sterile water and nematodes collected on nested 0.147 mm and 0.026 mm pore (NI N2)and soil. sieves.Baermann funnels (Viglierchio and Schmitt 1983)were used to separate nematodes from the pre RNA extraction cipitation and microscope inspection was employed.If there were nematodes in roots.the roots were treated About 20.000 mixed-stage nematodes from each pop- to remove nematodes (Tsang et al.2004).All non- ulation separated from carrot disks were respectively contaminated seedlings were grown in sterilized soil collected in an Eppendorf tube and washed with dieth medium for 15 days for later use. ypyrocarbonate (DEPC)water three times.Cleaned Springe

(Haegeman et al. 2008). EGases was first thought to originate through horizontal gene transfer (HGT) (Jones et al. 2005). Chen et al. (2005) and Bakhetia et al. (2007) employed RNAi to silence EGases in Globodera rosto￾chiensis and Heterodera glycines, respectively. Their investigation showed that the nematodes with silenced EGases had significantly less pathogenicity on their host plants. It has been widely accepted that EGases plays a key role in pathogenicity in the two nematodes. Therefore, EGases is viewed as a prospective target gene that could be developed to provide resistance to the nematodes. The first high throughput molecular characterization of R. similis was studied by Jacob et al. (2008), and much useful information was discovered; some genes involved in parasitism, including EGases were identified. The four EGases genes in R. similis had been cloned by Haegeman et al. (2008). Their research only focused on tissue-expression and time-expression (Haegeman et al. 2008). Currently, there is no report on whether the function of EGases is involved in pathoge￾nicity of R. similis on plant. In this study, a differential gene expression library was constructed by SSH, based on two populations of R. similis from different areas and different hosts. EGases in R. similis (Rs-eng-1b) was selectively screened from a SSH library. In order to clarify the gene function, a series of experiments including RNAi, real-time PCR (qPCR), carrot callus culture, and artificial infection, were performed to investigate the relationship between the expression of Rs-eng-1b and pathogenicity of R. similis on plant. Materials and methods Plant materials Anthurium, Anthurium andraeanum plants were bought from the Flowers and Plants Research Center, Guangzhou, Guangdong. The roots of anthurium seed￾lings were washed with sterile water and nematodes collected on nested 0.147 mm and 0.026 mm pore sieves. Baermann funnels (Viglierchio and Schmitt 1983) were used to separate nematodes from the pre￾cipitation and microscope inspection was employed. If there were nematodes in roots, the roots were treated to remove nematodes (Tsang et al. 2004). All non￾contaminated seedlings were grown in sterilized soil medium for 15 days for later use. Nematodes Two populations of R. similis: Rs-C and Rs-P, which were collected from roots of ornamental plants Calathea makoyana and Philodendron cv Green Emerald, respectively, and the internal transcribed spacer (ITS) regions of Rs-C and Rs-P were se￾quenced. The different pathogenicity of the two nem￾atode populations was certified on Musa paradisiaca by a member of our group (personal communication). These populations were cultured on excised carrot (Daucus carota) disks in Petri dishes with a diameter of 6 cm at 25 °C in incubator (Fallas and Sarah 1994). Nematode extraction The carrot callus was mashed with a blender. The mashed solution was filtered through combined sieves with aperture of 0.147 mm and 0.026 mm. Nematodes were collected from the 0.026 mm aperture sieve. Roots of anthurium were cut into 1 cm fragments. These fragments were mashed in a blender and further filtered by nested sieves with apertures 0.147 mm and 0.026 mm. Nematodes were collected from the 0.026 mm aperture sieve. This first nematode collec￾tion was labelled as N1. Meanwhile, the 0.147 mm pore sieve subjected to the Baermann funnel separat￾ing the nematodes number was labelled as N2. The total nematodes in roots were calculated as the sum of N1 and N2. Nematodes in soil were isolated from 200 ml of mixed soil from the host plant pot using a Baermann funnel method. Extraction from sieve or Baermann funnel were adjusted to 10 ml of nematode suspension, and 1 ml of the suspension was pipetted into a glass dish with a diameter of 6 cm, and the number of nematodes were counted under the stereo￾microscope, while the number of females, males and juveniles were counted respectively. The same work was done 10 times until all nematode suspensions had been counted. Total nematode population size was calculated as the sum of nematodes isolated from roots (N1 + N2) and soil. RNA extraction About 20,000 mixed-stage nematodes from each pop￾ulation separated from carrot disks were respectively collected in an Eppendorf tube and washed with dieth￾ypyrocarbonate (DEPC) water three times. Cleaned Eur J Plant Pathol (2012) 133:899–910 901

902 Eur J Plant Pathol (2012)133:899-910 nematodes were ground in liquid nitrogen.Total RNA non-redundant protein database(nr)and a non- was extracted from the nematodes using TRIZOL redundant nucleotide database (nt)in NCBL following manufacturer instructions (Invitrogen)and further treated with DNase I(Promega)for 15 min at Obtaining complete sequence of Rs-eng-/b 37 C.The RNA was verified by 1.0 agarose gel electrophoresis and was stored-80C for later use. The fragment candidate of Rs-eng-Ib was screened Only 500 mixed stages nematodes separated from out of the library with alignment analysis.To obtain carrot disks were treated by RNAi.Therefore,a the complete sequence of Rs-eng-/b,3RACE primers MicroElute total RNA kit (OMEGA)was used to (NEST-S1 and NEST-S2)(Table 1)and 5'RACE extract the total RNA according to the kit operation primers (NEST-Aland NEST-A2)(Table 1)were protocol. designed to amplify the 3'end and 5'end of Rs-eng /b using a SMART RACE cDNA amplification kit SSH library (Clontech),respectively.Finally,we spliced three frag ments of Rs-eng-Ib(5'end,middle fragment,and 3 A SMARTer PCR DNA Synthesis Kit (Clontech) end)into the complete sequence of Rs-eng-/6.Two tran tota RNA ron A-C an specific primers (cds-F and cds-R)(Table 1)from to cDN ording t complet sequences of Rs-eng-Ib were anu cture designed to form the complete sequence of Rs-eng-/b went using a PCR-S on ki Expression analysis of Rs-eng-Ib and qPCR secondary I amplifie produ nt pGEM-T easy ega and qPCR was used to asse the var on in the e expres tran plet Escherich a coli JM1 eng-Ib be etween Rs-Ca -P ne cells.Final single were ran omly selecte ato ent for later PCR detecting and sequencing. eggs carrot callus Reverse Northern blotting juvenile Total RNAs from Rs-Cand Rs-P nematode population ively wer R h synthesize P fro PCR de populati prime DNA la samples as three biol dete on th cDNA ed CR-R by ano-Dr DNA abe pro kit and P om SSH d Rs-p nd c Sequencing and alignment analysis d in trinlic CFX-96 (Bio-Rad)qPCR machir SYBR GO After hybridization the results of the hybridized sis lus kit (TOYOBO) the nale analysed by UV transilluminator (Alpha and 60C for 30eA0 verse orthern blotting signals wer elected for cing.After CEX-96 ted ct valu and adapter ea uences, alig extrap olated relative levels of PCR products from Springer

nematodes were ground in liquid nitrogen. Total RNA was extracted from the nematodes using TRIZOL following manufacturer instructions (Invitrogen) and further treated with DNase I (Promega) for 15 min at 37 °C. The RNA was verified by 1.0 % agarose gel electrophoresis and was stored −80 °C for later use. Only 500 mixed stages nematodes separated from carrot disks were treated by RNAi. Therefore, a MicroElute total RNA kit (OMEGA) was used to extract the total RNA according to the kit operation protocol. SSH library A SMARTer PCR cDNA Synthesis Kit (Clontech) was used to transcribe total RNAs from Rs-C and Rs-P nematode populations into cDNA according to the manufacturer’s protocol. Resulting cDNAs under￾went Suppression Subtractive Hybridization (SSH) using a PCR-Select cDNA subtraction kit (Clontech). The secondary PCR amplified product was cloned into the vector pGEM-T easy (Promega) and was then transformed into complete Escherichia coli JM109 cells. Finally, single clones were randomly selected for later PCR detecting and sequencing. Reverse Northern blotting Total RNAs from Rs-C and Rs-P nematode populations were transcribed into cDNA according to operation in￾struction of a ReverTra Ace qPCR RT kit (TOYOBO). To synthesize Probe-C and Probe-P from Rs-C and Rs-P nematode populations, a DIG high prime DNA labelling and detection starter kit I (Roche) was used to label the cDNA templates. The concentration of probes was quantified by a Nano-Drop spectrophotometer. According to operation protocol of DIG high prime DNA labelling and detection starter kit I (Roche), the PCR products from SSH were hybridized with Probe-C and Probe-P. Sequencing and alignment analysis After hybridization, the results of the hybridized sig￾nals were analysed by UV transilluminator (Alpha Innotech). Blots that had two-fold differences in re￾verse northern blotting signals were selected for se￾quencing. After removing vector sequences and adapter sequences, sequences were aligned against a non-redundant protein database (nr) and a non￾redundant nucleotide database (nt) in NCBI. Obtaining complete sequence of Rs-eng-1b The fragment candidate of Rs-eng-1b was screened out of the library with alignment analysis. To obtain the complete sequence of Rs-eng-1b, 3′RACE primers (NEST-S1 and NEST-S2) (Table 1) and 5′ RACE primers (NEST-A1and NEST-A2) (Table 1) were designed to amplify the 3′ end and 5′ end of Rs-eng- 1b using a SMART RACE cDNA amplification kit (Clontech), respectively. Finally, we spliced three frag￾ments of Rs-eng-1b (5′ end, middle fragment, and 3′ end) into the complete sequence of Rs-eng-1b. Two specific primers (cds-F and cds-R) (Table 1) from spliced complete sequences of Rs-eng-1b were designed to form the complete sequence of Rs-eng-1b. Expression analysis of Rs-eng-1b and qPCR qPCR was used to assess the variation in the expres￾sion levels of Rs-eng-1b between Rs-C and Rs-P nem￾atode populations, and among different development stages of Rs-C: females, males, juveniles and eggs. Rs￾C and Rs-P were isolated from infected carrot callus for total RNA extraction. One hundred females, males, juveniles and eggs respectively were used for RNA extraction. The RNA was then quantified and qualified using a Nano-drop spectrophotometer, and then stored at −80 °C for further analysis. All the RNA used for qPCR was prepared from three different samples as three biological replicates. Based on the complete sequence of Rs-eng-1b, spe￾cific primers qPCR-F and qPCR-R (Table 1) were designed to represent Rs-eng-1b expression. According to the method described by Jacob et al. (2007), the primers Actin-F and Actin-R were synthesized (Table 1) to amplify the reference gene, β-actin. qPCRs were performed on mixed life stages of Rs-C and Rs-P and females, males, juveniles, and eggs of Rs￾C, and reactions were performed in triplicate on CFX-96 (Bio-Rad) qPCR machine, using SYBR Green qPCR Master Mix-plus kit (TOYOBO) according to the man￾ufacture’s protocol with the following reaction condi￾tions: 95 °C for 15 s and 60 °C for 30 s (40 cycles). Initial data analysis was carried out using the Bio-Rad CFX-96 manager software, which created Ct values and extrapolated relative levels of PCR products from 902 Eur J Plant Pathol (2012) 133:899–910

Eur J Plant Pathol (2012)133:899-910 903 Table 1 Primers used for RACE of cndoglucanase Rs it-/6 Primer Sequence Source hain rea Actin- S-GAAAGAGGGCCGGAAGAG-3 Jacob et al.(2007 Actin-R 5-AGATCGTCCGCGACATAAAG-3 Jacob et al.(2007) on in Rs lation NEST S 5-ACGAGACCTACAATGAGC-3 NEST S2 5-AGCCTGCCCGTGTTCGTGAC-3 NEST Al S-GGCAGGTACTCGGTCACG-3 (ds)RNA of Rs-eng-Ib NEST A2 5-CATTGTAGGTCTCGTACC-3 OPCR-E 5-AATCTCTTACGTGAACTGGGC-3 OPCR-R SGGTCGCTCCAGATTTAGTCG-3 cds-E 5.TCCGCTTTCACCGCTTTCA-3 cds-R SCAGACATTCAGCATCCA3 T7S S-TAATACGACTCACTATAGGGGCTGTI A TCATTGTAG-3 NGGTGGGCTCATTGTAG-3 G-T7S G-A 5-CGATGCGGTTCACCAGGGTGTCG-3 G-T7A Gs 5CACAAGTTCAGCGTGTCCGGCG-3 standard curves.Melt curves were done routinely.and nd G)(Table)o gp)was generated with desig necific nrimer this allowed the possibility of hoth contamination and primer dimers to be discounted.Actin was used as a cloning vector PYL 322-d1-GFPn supplied by QL positive control in all experiments.All experiments Zhu,College of Life-Science,South China Agricultural were performed in triplicate. University. Synthesis of Rs-eng-Ib dsRNA Rs-eng-/b's RNAi treatment and silence detection A fragment of about 450 bp from the ORF of Rs-eng Five hundred nematodes from the Rs-C population Ib was cloned into the vector PMD18-T (TAKARA) cultivated on carrot callus were collected and trans The constructed vector was further confirmed by se ferred in an Eppendorf tube,treated with DEPC quencing.Based on the fragment,specific primers water and soaked in 50 ul rs-eng-lb dsRNa solution (T7S.A T7A.and S)(Table 1)with a T7 promoter (2 ug/ul)at room temperature for 12 h.24 h.36 h were designed to amplify the sense and anti-sense and 48 h,respectively.Non-endogenous gfp dsRNA product.Sense and antisense rNA were transcribed solution (2 ug/ul)was used as a control.The using T7 transcription kit(TOYOBO)according to the treatment times used for the control were same as manufacturer's instructions.Sense and antisense Rs-eng-/b dsRNAs.Meanwhile,untreated nematodes transcripts were annealed for 30 min at 37 C and were used as a blank control.Treated nematodes were analyzed by agarose gel electrophoresis.The dsRNA cleaned with DEPC water three times and the total RNA was purified by equal amount of LiCl(3 mol/l) was then extracted.aPCR was used to analyze transcript ovemight and washed by 70%ethylalcohol three times suppression of Rs-eng-/b in the nematodes after the finally stored at-80C for later use.Non-endogenous RNAi treatments.All experiments were performed control dsRNA (125 bp)(green fouorescent protein three times. Springe

standard curves. Melt curves were done routinely, and this allowed the possibility of both contamination and primer dimers to be discounted. Actin was used as a positive control in all experiments. All experiments were performed in triplicate. Synthesis of Rs-eng-1b dsRNA A fragment of about 450 bp from the ORF of Rs-eng- 1b was cloned into the vector PMD18-T (TAKARA). The constructed vector was further confirmed by se￾quencing. Based on the fragment, specific primers (T7S, A, T7A, and S) (Table 1) with a T7 promoter were designed to amplify the sense and anti-sense product. Sense and antisense RNA were transcribed using T7 transcription kit (TOYOBO) according to the manufacturer’s instructions. Sense and antisense transcripts were annealed for 30 min at 37 °C and analyzed by agarose gel electrophoresis. The dsRNA was purified by equal amount of LiCl (3 mol/l) overnight and washed by 70 % ethylalcohol three times, finally stored at −80 °C for later use. Non-endogenous control dsRNA (125 bp) (green fouorescent protein gene, gfp) was generated with designed specific primers (G-T7S, G-A, G-T7A, and G-S) (Table 1) from the cloning vector PYL 322-d1-GFPn supplied by QL Zhu, College of Life-Science, South China Agricultural University. Rs-eng-1b’s RNAi treatment and silence detection Five hundred nematodes from the Rs-C population cultivated on carrot callus were collected and trans￾ferred in an Eppendorf tube, treated with DEPC water, and soaked in 50 μl Rs-eng-1b dsRNA solution (2 μg/μl) at room temperature for 12 h, 24 h, 36 h and 48 h, respectively. Non-endogenous gfp dsRNA solution (2 μg/μl) was used as a control. The treatment times used for the control were same as Rs-eng-1b dsRNAs. Meanwhile, untreated nematodes were used as a blank control. Treated nematodes were cleaned with DEPC water three times and the total RNA was then extracted. qPCR was used to analyze transcript suppression of Rs-eng-1b in the nematodes after the RNAi treatments. All experiments were performed three times. Table 1 Primers used for RACE of endoglucanase Rs-eng-1b gene sequence, quantitative polymerase chain reaction (qPCR) for Rs-eng-1b expression in Rs-C population and Rs-P population and different development stage of Radopholus similis, and to generate double-stranded (ds) RNA of Rs-eng-1b and non-endogenous gfp dsRNA control Primer Sequence Source Actin-F 5′-GAAAGAGGGCCGGAAGAG-3′ Jacob et al. (2007) Actin-R 5′-AGATCGTCCGCGACATAAAG-3′ Jacob et al. (2007) NEST S1 5′-ACGAGACCTACAATGAGC-3′ NEST S2 5′-AGCCTGCCCGTGTTCGTGAC-3′ NEST A1 5′-GGCAGGTACTCGGTCACG-3′ NEST A2 5′-CATTGTAGGTCTCGTACC-3′ qPCR-F 5′-AATCTCTTACGTGAACTGGGC-3′ qPCR-R 5′-GGTCGCTCCAGATTTAGTCG-3′ cds-F 5′-TCCGCTTTCACCGCTTTCA-3′ cds-R 5′-CAGACATTCAGCATCCA-3′ T7S 5′-TAATACGACTCACTATAGGGGCTGTT CTGGTCGCAATG-3′ A 5′-CAGAGGTGGGCTCATTGTAG-3′ T7A 5′-TAATACGACTCACTATAGGGCAG AGGTGGGCTCATTGTAG-3′ S 5′-GCTGTTCTGGTCGCAATG-3′ G-T7S 5′-GGATCCTAATACGACTCACTATAGGG CACAAGTTCAGCGTGTCCGGCG-3′ G-A 5′-CGATGCGGTTCACCAGGGTGTCG-3′ G-T7A 5′-GGATCCTAATACGACTCACTATAGGG CGATGCGGTTCACCAGGGTGTCG-3′ G-S 5′-CACAAGTTCAGCGTGTCCGGCG-3′ Eur J Plant Pathol (2012) 133:899–910 903

904 Eur I Plant Pathol (2012)133:899-910 Reproduction and pathogenicity of nematodes Data analysis Thirty female Rs-C nematodes were treated with Rs- All data in this study were subjected to analysis of eng-Ib dsRNA solution (2 ug/ul)for 12 h,24 h and variance (ANOVA)and multiple comparisons of 36 h.Controls were treated with gp dsRNA solution means were conducted by Duncan's Multiple Range (2 ug/ul)for 12 h,24 h and 36 h,or without any Test at P=0.05 using SAS (Release 8.01). treatment with dsRNA solution.Another control group of thirty female Rs-P nematodes without treat ment with dsRNA solution was also included. Results nematodes were then inoculated onto a carrot callus which were then maintained in a dark incubator Alignment and differences of the ITS sequences of two R.similis populations Rs-C and Rs-P 25 C for 56 days after which total nematodes in the carrot callus were isolated and calculated as described above.Each treatment was repeated five times The length of the two ITS sequences(Accession num- ber:JQ619539,JQ619538)of Rs-C and Rs-P were d nem atodes. both 625 bp.There were two variant nucleotides in ITS sequence at 5.8s different between Rs-C and Rs-P, at NO.373 and NO.402 respectively. solation and identification of Rs-eng-1b of R.similis Total RNA extrac d from Rs-C and Rs-P A to I of 2 P d ar ully g the ve that A6 uld b 1183h d1.183bp 4 of 750 bp (5'RACE)and 1,180 bp (3'RACE) ad Finally the t ngth of 1 400 hn The Fig.1 Anthurium andrae num root infected with Radopholu with genBank a of the ity with Rs ng-1b (Acc ouration:4=50-75 discolouration EU414839).The d that the 1 400 br dis vas Rs. eng-16 of R sin Springer

Reproduction and pathogenicity of nematodes Thirty female Rs-C nematodes were treated with Rs￾eng-1b dsRNA solution (2 μg/μl) for 12 h, 24 h and 36 h. Controls were treated with gfp dsRNA solution (2 μg/μl) for 12 h, 24 h and 36 h, or without any treatment with dsRNA solution. Another control group of thirty female Rs-P nematodes without treat￾ment with dsRNA solution was also included. All nematodes were then inoculated onto a carrot callus which were then maintained in a dark incubator at 25 °C for 56 days after which total nematodes in the carrot callus were isolated and calculated as described above. Each treatment was repeated five times. The pathogenicity of Rs-eng-1b RNAi treated nem￾atodes, gfp dsRNA solution treated nematodes, and untreated nematodes were compared. Each treatment was adjusted to a suspension with 100 nematodes per ml. Three small holes of 5 cm depth were made in the soil with a glass rod in a diameter of 0.5 cm from the anthurium stem, and 10 ml of nematode suspension was pippetted in total. The plants were managed as usual except the first 5 days without watering. Five replicates for each treatment were performed. After 56 days, nematodes were extracted from the roots and soil of all treated plants, counted and calculated as above, in addition, above-ground plant weight, root weight, and disease severity were recorded. Disease severity was classified into six levels from low to high according to the discolouration area on the roots: 00 no discolouration on the roots; 10discolouration area less than 5 % on the roots; 20discolouration area less than 25 % and more than 5 % on the roots; 30dis￾colouration area less than 50 % and more than 25 % on the roots; 40discolouration area less than 75 % and more than 50 % on the roots; 50discolouration area more than 75 % on the roots (Fig. 1). Data analysis All data in this study were subjected to analysis of variance (ANOVA) and multiple comparisons of means were conducted by Duncan’s Multiple Range Test at P00.05 using SAS (Release 8.01). Results Alignment and differences of the ITS sequences of two R. similis populations Rs-C and Rs-P The length of the two ITS sequences (Accession num￾ber: JQ619539, JQ619538) of Rs-C and Rs-P were both 625 bp. There were two variant nucleotides in ITS sequence at 5.8 s different between Rs-C and Rs-P, at NO.373 and NO.402 respectively. Isolation and identification of Rs-eng-1b of R. similis Total RNA extracted from Rs-C and Rs-P nematode populations respectively were reverse transcribed into cDNA for SSH. A total of 2,400 clones were randomly selected from secondary round PCR products and validated to have an 89.2 % positive rate by PCR. Positive clones were made through reverse northern blotting and resulted in 124 hybridization spots with significantly different expression (Fig. 2). Finally, 124 positive clones were sequenced and 112 of them were successfully. After removing the vector sequences and the adaptor sequence, 87 unique sequences were obtained and aligned against a non-redundant nucleotide database and the non-redundant protein database in NCBI. The alignment results showed that five of the 87 sequences were homologous to Rs-eng-1b. Of the five sequences, a 467 bp (Accession number: GW395922) and a 742 bp (Accession number: GW395923) could be spliced into a sequence with a length of 1,183 bp. According to the spliced 1,183 bp fragment of Rs￾eng-1b, the nest primers were designed to perform 5′ RACE-PCR and 3′ RACE-PCR. Two fragments in length of 750 bp (5′ RACE) and 1,180 bp (3′ RACE) were obtained. Finally, the two fragments were spliced into a sequence with a length of 1,400 bp. The se￾quence compared with GenBank accessions showed 99 % similarity with Rs-eng-1b (Accession number: EU414839). Therefore, we conferred that the 1,400 bp sequence was Rs-eng-1b sequence of R. similis. Fig. 1 Anthurium andraeanum root infected with Radopholus similis compared with uninfected roots (0). 00no discolouration of the roots; 1075 % discolouration 904 Eur J Plant Pathol (2012) 133:899–910

Eur J Plant Pathol (2012)133:899-910 905 b 回 。。 。。。。。。 。。。。。。。。 Fig.2 cDNA Micr cnR-Cand Rs-P ve clo Expression analysis of Rs-eng-Ib in different of rs-eng-lb in nematodes soaked in rs-eng-lb dsRNA populations and different development stages of R. decreased by 41.8%for the 12 h treatment,61.5%for similis the 24 h treatment,66.2%for the 36 h treatment,and 62.3%for the 48 h treatment (Fig.5),difference were The qPCR results showed that the expression of Rs significant(P=0.05)between the nematodes treated with eng-Ib in Rs-C were about 2.7 times as much as the Rs-eng-76 dsRNA and the untreated nematodes,and expression of Rs-P(Fig.3).According to the expres- nematodes treated with gip dsRNA,respectively.In sion of Rs-eng-Ib in Rs-C eggs,females,juveniles treatments by RNAi silencing of Rs-eng-1b,the silencing and males.Rs-eng-/b had highest expression levels in effect at 36 h and 48 h were significantly higher than the females (Fig.4).The expression in juveniles,males silencing effect at 12 and 24 h.Expression of Rs-eng-/b and eggs accounted for 30.5%.15.5%.and 10.2%of was inhibited by Rs-eng-1b dsRNA and the silencing the expression levels in females. effect was enhanced with increasing exposure time.The non-endogenous gfp dsRNA control had no significant RNAi silence effect on Rs-eng-1b expression influence on the expression of Rs-eng-/6. After Rs-eng-/b dsRNA was synthesized,the RNAi Influence of Rs-eng-/b's silence on reproduction silencing effect of Rs-eng-Ib in Rs-C population was and pathogenicity in R.similis detected by qPCR methods.Compared with relative expression levels of Rs-eng-Ib in corresponding control As the RNAi effect on Rs-eng-1b expression was nematodes soaked ing dsRNA.the relative expression clear,the effect of gene silencing on nematode 1.4 12 08 06 04 02 0 Fig.3 Expression of the endoglucanase Rs-eng-lb in Radopho us similis populations Rs-C an d Rs-P.Bars indicate standar 005) cant differences(P=0.05)between treatments veen treatments Springe

Expression analysis of Rs-eng-1b in different populations and different development stages of R. similis The qPCR results showed that the expression of Rs￾eng-1b in Rs-C were about 2.7 times as much as the expression of Rs-P (Fig. 3). According to the expres￾sion of Rs-eng-1b in Rs-C eggs, females, juveniles, and males, Rs-eng-1b had highest expression levels in females (Fig. 4). The expression in juveniles, males and eggs accounted for 30.5 %, 15.5 %, and 10.2 % of the expression levels in females. RNAi silence effect on Rs-eng-1b expression After Rs-eng-1b dsRNA was synthesized, the RNAi silencing effect of Rs-eng-1b in Rs-C population was detected by qPCR methods. Compared with relative expression levels of Rs-eng-1b in corresponding control nematodes soaked in gfp dsRNA, the relative expression of Rs-eng-1b in nematodes soaked in Rs-eng-1b dsRNA decreased by 41.8 % for the 12 h treatment, 61.5 % for the 24 h treatment, 66.2 % for the 36 h treatment, and 62.3 % for the 48 h treatment (Fig. 5), difference were significant (P00.05) between the nematodes treated with Rs-eng-1b dsRNA and the untreated nematodes, and nematodes treated with gfp dsRNA, respectively. In treatments by RNAi silencing of Rs-eng-1b, the silencing effect at 36 h and 48 h were significantly higher than the silencing effect at 12 and 24 h. Expression of Rs-eng-1b was inhibited by Rs-eng-1b dsRNA and the silencing effect was enhanced with increasing exposure time. The non-endogenous gfp dsRNA control had no significant influence on the expression of Rs-eng-1b. Influence of Rs-eng-1b’s silence on reproduction and pathogenicity in R. similis As the RNAi effect on Rs-eng-1b expression was clear, the effect of gene silencing on nematode Fig. 3 Expression of the endoglucanase Rs-eng-1b in Radopho￾lus similis populations Rs-C and Rs-P. Bars indicate standard errors of mean data (n03) and different letters indicate signifi￾cant differences (P00.05) between treatments Fig. 4 Expression of the endoglucanase Rs-eng-1b in 100 eggs, females, juveniles, and males, respectively of Radopholus similis populations Rs-C. Bars indicate standard errors of mean data (n0 3) and different letters indicate significant differences (P00.05) between treatments a b Fig. 2 cDNA Microarray analysis of differentially expressed genes between Rs-C and Rs-P populations of Radopholus similis. a Hybridization result with random cDNA probe of Rs-C; (b) Hybridization result with random cDNA probe of Rs-P. The red blocks show the positive clones of cDNA fragments differentially expressed between Rs-C and Rs-P population Eur J Plant Pathol (2012) 133:899–910 905

906 Eur J Plant Pathol (2012)133:899-910 1.2 18000 1 16000 14000 1200 0.6 CKG-12G-24G-36G-48R-12R-24R-36R-48 Ro-P Rs.CG-12R-12G24R-24G-36R-36 of R callus 56 day and Rs :R.12.R.24 .R-36:Numb 2h336h :G-12G-24 Rs- R-34 and R-48: b in ner odes soakee 61 Rs-C and Rs-P were two populations ep inoculatin inoculated untreated Rs-C wer sRNA being Rs- (P nd Rs- and Rs- Af with b Th respective atode and did vith in 36 gth of. ime.Ner any t gro from Rs-C had the 0.05 rP=0.05 the RNAi effect of Rs eng-16 o on pa 4 h city of r similis wa also studied na odes The nd plant weight an b dsRNA for 12 h and 24 h th、 :li h d 36 h the diffa Rs-P were significantly lower (P=0.05)than for non (P=0.05)bc nd th noculated with untreated rs-c had lower root weight and above-ground plant weight compared to plants weight w significant (P=005)bet the inoculated with untreated Rs-P.The numbers of nem nd the r atodes in the rhizosphere and the disease severity level Ib dsRNA for 12 h and 24 h,and gip dsRNA for 12 Springer

reproduction was studied by respectively inoculating treated and untreated nematodes on carrot callus. The results showed that after being soaked in Rs-eng-1b dsRNA and inoculated onto a carrot callus for 56 days (about two lifecycles of R. similis), Rs-C nematodes treated with Rs-eng-1b dsRNA for 12 h, 24 h, and 36 h had significantly lower (P00.05) reproduction than untreated Rs-C and Rs-P nematodes, and Rs-C nemat￾odes treated with gfp dsRNA, respectively (Fig. 6). The reproduction of nematodes treated with gfp dsRNA was significantly lower (P00.05) than untreat￾ed nematodes and did not decrease with increasing length of exposure time. Nematodes without any treat￾ment from Rs-C had the greatest reproduction. So, with exposure to Rs-eng-1b RNAi treatment increased, nematode reproduction decreased. In addition, the RNAi effect of Rs-eng-1b on path￾ogenicity of R. similis was also studied by respectively inoculating treated and untreated nematodes to anthur￾ium for 56 days. The results showed that above￾ground plant weight and root weight of anthurium inoculated with either untreated R. similis Rs-C or Rs-P were significantly lower (P00.05) than for non￾nematode treated anthurium (Fig. 7a–b). Anthurium inoculated with untreated Rs-C had lower root weight and above-ground plant weight compared to plants inoculated with untreated Rs-P. The numbers of nem￾atodes in the rhizosphere and the disease severity level of anthurium inoculated with untreated Rs-C were significantly higher (P00.05) than for anthurium in￾oculated with untreated Rs-P (Fig. 7c–d). Anthurium plants were inoculated with Rs-C nem￾atodes that had been treated with either Rs-eng-1b dsRNA or gfp dsRNA, or with untreated Rs-C and Rs-P nematodes. After infection for 56 days, fresh anthurium above-ground plants and roots were weighed and compared. The results (Fig. 7a–b) showed that anthurium plants inoculated with the Rs￾C nematodes treated with Rs-eng-1b dsRNA for 36 h had the heaviest above-ground plant and root weights. Its above-ground weight was significantly greater than those in other treatments (P00.05), and its root weight was remarkably heavier (P00.05) than those with gfp dsRNA treatments for 24 h and 36 h, as well as the untreated Rs-C and Rs-P nematodes. For above￾ground plant weight of anthurium inoculated with Rs-C nematodes untreated and treated with Rs-eng- 1b dsRNA for 12 h and 24 h, or with gfp dsRNA for 12 h, 24 h and 36 h, the difference in above-ground plant weight was only significant (P00.05) between the untreated nematodes and the nematodes treated with Rs-eng-1b dsRNA for 24 h. Differences in root weight were significant (P00.05) between the untreat￾ed nematodes and the nematodes treated with Rs-eng- 1b dsRNA for 12 h and 24 h, and gfp dsRNA for 12 h, Fig. 5 Expression of the endoglucanase Rs-eng-1b in Radopho￾lus similis population Rs-C treated with Rs-eng-1b double￾stranded (ds) RNA. CK: expression of Rs-eng-1b in untreated nematodes; G-12, G-24, G-36, and G-48: expression of Rs-eng- 1b in control nematodes soaked by non-endogenous gfp dsRNA solution for 12 h, 24 h, 36 h, and 48 h, respectively; R-12, R-24, R-36, and R-48: expression of Rs-eng-1b in nematodes soaked by Rs-eng-1b dsRNA for 12 h, 24 h, 36 h, and 48 h, respective￾ly. Bars indicate standard errors of mean data (n03) and differ￾ent letters indicate significant differences (P00.05) between treatments Fig. 6 Number of Radopholus similis on carrot callus 56 days after inoculation of 30 females, respectively. Rs-P and Rs-C: untreated population Rs-P and Rs-C; R-12, R-24, R-36: Number of R. similis after inoculating 30 females Rs-C treated by Rs-eng- 1b dsRNA for 12 h, 24 h, and 36 h, respectively; G-12, G-24, and G-36: Number of R. similis after inoculating 30 females of Rs-C treated by non-endogenous gfp dsRNA solution for 12 h, 24 h and 36 h, respectively. Rs-C and Rs-P were two populations of R. similis collected from roots of ornamental plants Calathea makoyana and Philodendron cv Green Emerald, respectively. Bars indicate standard errors of mean data (n05) and different letters indicate significant differences (P00.05) among treatments 906 Eur J Plant Pathol (2012) 133:899–910

Eur J Plant Pathol (2012)133:899-910 907 a b 10 CK Rs-P Rs-C G-12 R-12 G-24 R-24 G-36 R-36 CK Rs-P Rs-C G-12 R-12 G-24 R-24 G-36 R-36 d 1800 10 CK Rs-P Rs-C G-12 R-12 G-24 R-24 G-36 R-36 CK Rs-P Rs-C G-12 R-12 G-24 R-24 G-36 R-36 noatecdRs-Cpopultiom,rated 36 untreated Rs-P and Rs-C populations:R-12.R-24.and R-36 respectively.Comparison of the nematode number in treatments of anthurium inoculated with rs-c nemat the rhizosphere and the disease severity of anthurium odes treated with gfp dsRNA for 12 h,24 h and 36 h, inoculated with nematodes in various treatments respectively,and untreated Rs-C nematodes.Taken showed that anthurium inoculated with rs-c nemato together,these results suggest that the pathogenicity des treated with Rs-eng-/b dsRNA for 24 h and 36 h of r.similis could be reduced markedly after treatmen were significantly lower in the nematode number in with target-specific Rs-eng-16 dsRNA for 12-36 h the rhizosphere and the disease severity than those and that of Rs-eng-Ib dsRNA for 36 h was the lowes inoculated with other nematodes (P=0.05).and tha among them,whereas the pathogenicity of R.similis of Rs-eng-1b dsRNA for 36 h was the lowest (P=0.05) was not impacted upon treatment with non-specific (Fig.7c-d).Nematode number in the rhizosphere and target gio dsRNA for 12-36 h.Nematode number in disease severity in anthurium inoculated with Rs-C the rhizosphere and disease severity of anthurium in nematodes treated with Rs-eng-/b dsRNA for 12 h oculated with untreated Rs-C nematodes were signifi was considerably lower than that in anthurium inocu cantly more than those of anthurium inoculated with lated with untreated Rs-C nematodes (P-0.05).There untreated Rs-P(P=0.05).Therefore,the pathogenicity was no significant difference in the nematode number of Rs-C nematodes was significantly stronger than that in the rhizosphere and disease severity among the of the Rs-P nematodes (P=0.05) Spring

respectively. Comparison of the nematode number in the rhizosphere and the disease severity of anthurium inoculated with nematodes in various treatments showed that anthurium inoculated with Rs-C nemato￾des treated with Rs-eng-1b dsRNA for 24 h and 36 h were significantly lower in the nematode number in the rhizosphere and the disease severity than those inoculated with other nematodes (P00.05), and that of Rs-eng-1b dsRNA for 36 h was the lowest (P00.05) (Fig. 7c–d). Nematode number in the rhizosphere and disease severity in anthurium inoculated with Rs-C nematodes treated with Rs-eng-1b dsRNA for 12 h was considerably lower than that in anthurium inocu￾lated with untreated Rs-C nematodes (P00.05). There was no significant difference in the nematode number in the rhizosphere and disease severity among the treatments of anthurium inoculated with Rs-C nemat￾odes treated with gfp dsRNA for 12 h, 24 h and 36 h, respectively, and untreated Rs-C nematodes. Taken together, these results suggest that the pathogenicity of R. similis could be reduced markedly after treatment with target-specific Rs-eng-1b dsRNA for 12–36 h, and that of Rs-eng-1b dsRNA for 36 h was the lowest among them, whereas the pathogenicity of R. similis was not impacted upon treatment with non-specific target gfp dsRNA for 12–36 h. Nematode number in the rhizosphere and disease severity of anthurium in￾oculated with untreated Rs-C nematodes were signifi￾cantly more than those of anthurium inoculated with untreated Rs-P (P00.05). Therefore, the pathogenicity of Rs-C nematodes was significantly stronger than that of the Rs-P nematodes (P00.05). Fig. 7 Above-ground plant weight (a), root weight (b), number of nematodes in the rhizosphere (c), and disease severity (d) of Anthurium andraeanum at 56 days after being inoculated with Radopholus similis. CK: uninoculated control; Rs-P and Rs-C: untreated Rs-P and Rs-C populations; R-12, R-24, and R-36: inoculated Rs-C population treated with dsRNA of R. similis Rs￾eng-1b for 12 h, 24 h, and 36 h, respectively; G-12, G-24, G-36: inoculated Rs-C population treated with non-endogenous gfp dsRNA solution for 12 h, 24 h and 36 h, respectively. Bars Eur J Plant Pathol (2012) 133:899–910 907

908 Eur J Plant Pathol (2012)133:899-910 Discussion and juvenile of R.similis may secrete abundantly EGases for infection.In addition to infection,the During feeding and migration in the plant,parasitic female is also responsible for breeding,and therefore nematodes inject enzymes through the stylet into plant requires additional nutrients (Gowen et al.2005).So, tissue to degrade the cell wall.The most extensively females may secrete more EGases than juveniles due studied nematode cell wall-degrading enzyme is B-1, to their higher activity in gaining nutrents.In contrast, 4-endoglucanase (EGase)(Haegeman et al.2009).Rs males of R.similis have a degraded stylet and pharyn eng-/b is known as EGases which functions in geal glands are non-parasitical (Williams and Siddiqi degrading cellulose(Goellner et al.2001),a key com 1973;Luc 1987;Gowen et al.2005).Eggs need the ponent in forming cell walls.In this study,Rs-eng-1b least nutntion due to their immobility.Smant et al was isolated and obtained according to our constructed (1998)reported that the EGases gene was only SSH library built from mixed-stage nematodes of Rs expressd in the mobile stages of G C and Rs-P populations with different pathogenic abil namely in pre-parasitic and parasitic J2 and in adul ity on Musa paradisiaca.The sequence of Rs-eng-1b males.but not in sedentary females.Our present find in here was 1,427 bp that had a 99%similarity with ings and the results reported by Smant ct al.(1998 rst repor ,OS-g-1b(E414859)( nd nay on of Rs-eng-/b n out hig nif 11g1 a DNA T and in th vell de glands (willia and Siddiai 1973 t al 2005)EGas tto be 公 de cell walls of hosts 16hto361 hel odes to gh the tissue and absorb nutrition from there e.So the fen (2009 ile Springer

Discussion During feeding and migration in the plant, parasitic nematodes inject enzymes through the stylet into plant tissue to degrade the cell wall. The most extensively studied nematode cell wall-degrading enzyme is β-1, 4-endoglucanase (EGase) (Haegeman et al. 2009). Rs￾eng-1b is known as EGases which functions in degrading cellulose (Goellner et al. 2001), a key com￾ponent in forming cell walls. In this study, Rs-eng-1b was isolated and obtained according to our constructed SSH library built from mixed-stage nematodes of Rs￾C and Rs-P populations with different pathogenic abil￾ity on Musa paradisiaca. The sequence of Rs-eng-1b in here was 1,427 bp that had a 99 % similarity with the first report of Rs-eng-1b (EU414839) (Haegeman et al. 2008). The expression of Rs-eng-1b in Rs-C was about 2.7 times higher than the expression in Rs-P, and the pathogenicity of Rs-C was significantly greater than that of Rs-P. Consequently, the expression of Rs-eng-1b was positively correlated with the pathoge￾nicity of R. similis. The expression of Rs-eng-1b in the nematodes treated by Rs-eng-1b dsRNA for more than 12 hr decreased significantly, and the Rs-eng-1b dsRNA treatment caused the nematodes to have a lower reproduction than gfp dsRNA treated and un￾treated Rs-C. Furthermore, the growth of anthurium inoculated by the nematodes treated by Rs-eng-1b dsRNA was better than that of untreated nematodes and nematodes treated with gfp dsRNA, and the effect of Rs-eng-1b RNAi treatment was enhanced with extending time of Rs-eng-1b RNAi treatment. In view of these results, it is assumed that a higher expression of Rs-eng-1b results in more degradation of cellulose, and therefore contributes to nematode invasion of the hosts resulting in more serious damage to the plant. Analysis of the expression of Rs-eng-1b performed at different nematode stages of Rs-C showed that Rs￾eng-1b expression was highest in females and lowest in eggs. Interestingly, the expression difference of Rs￾eng-1b in females, males, juveniles, and eggs, con￾forms to their individual developmental features. The female and juvenile of R. similis are the infective forms which have a powerful stylet and well devel￾oped pharyngeal glands (Williams and Siddiqi 1973; Gowen et al. 2005). EGases is thought to be secreted in order to degrade cell walls of hosts during infection, and may help nematodes to migrate through the host tissue and absorb nutrition from there. So the female and juvenile of R. similis may secrete abundantly EGases for infection. In addition to infection, the female is also responsible for breeding, and therefore requires additional nutrients (Gowen et al. 2005). So, females may secrete more EGases than juveniles due to their higher activity in gaining nutrients. In contrast, males of R. similis have a degraded stylet and pharyn￾geal glands are non-parasitical (Williams and Siddiqi 1973; Luc 1987; Gowen et al. 2005). Eggs need the least nutrition due to their immobility. Smant et al. (1998) reported that the EGases gene was only expressd in the mobile stages of G. rostochiensis, namely in pre-parasitic and parasitic J2 and in adult males, but not in sedentary females. Our present find￾ings and the results reported by Smant et al. (1998) indicate that EGases may be related to parasitism and nematode infection. Haegeman et al. (2008) reported the expression of EGases in different life stages of R. similis by a semi-quantitative RT-PCR. In their study Rs-eng-1b was expressed at a very low level in eggs, and higher levels in males and females, but not in juveniles. They concluded that other EGases not iden￾tified in juveniles might complement the role of Rs￾eng-1b. Employing RNAi to study gene function of nem￾atodes, there are in principle three approaches to input dsRNA: microinjection, feeding approaches using bacteria with expression of dsRNA, and soak￾ing nematodes in dsRNA (Urwin et al. 2002). However, the first two approaches are unrealistic: due to the small size of nematodes it is difficult to microinject dsRNA, nor do PPNs feed upon bacte￾ria. Therefore RNAi input on PPNs is generally performed by soaking the nematodes for more than 4 h for effective gene silencing (Rosso et al. 2009). Chen et al. (2005) soaked G. rostochiensis in dsRNA of Gr-eng-1 for 24 h, Bakhetia et al. (2007) soaked H. glycines in dsRNA of Hg-eng-1 for 16 h and Cheng et al. (2010) soaked Bursaphelenchus xylophilus in dsRNA of Bx-eng-1 for 24 h for the best RNAi effect. However as shown in this study, overlong periods can also cause decreased expression. In this study, R. similis soaked in dsRNA of Rs-eng-1b for 36 h, expression of Rs-eng-1b of R. similis decreased significantly. All these results indicate that RNAi treatment for eng-1 in different nematodes requires 16 h to 36 h exposure to display an effective silencing. Haegeman et al. (2009) silenced the putative 908 Eur J Plant Pathol (2012) 133:899–910