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