title: Evaluation of endobacteria (Stenotrophomonas maltopholia) on pathogenesis-related gene expression of pine wood nematode and the role in pine wilt disease
design: Pine wood nematode (AmA3) was isolated from the infected Pinus massoniana (Anhui, China).The endobacteria S.maltopholia was initially isolated from pine nematode that separated from infected P. massoniana (Zhejiang, China). This bacterium had high frequency of isolated from PWN strains in our previous study.The strains of PWN and endobacteria were provided by Jiangsu Key Laboratory for Prevention and Management of Invasive Species.The suspension of 6000 PWN was used to inoculate to 6 years old P. massoniana in the wild (Nanjing Forestry University).The PWN were isolated from the dead P. massoniana using a Baermann funnel. Then, these PWN were pipetted into the Potato Dextrose Agar (PDA) plate (6 cm diam.) containing Botrytis cinerea. After grown at 25℃ for 7 days, the nematodes were collected in 10 mL centrifuge tubes and treated as wild-type PWN (Bx_w).The above-mentioned suspension (1mL) of PWN was poured onto a sterilized cover slip in a petri dish and incubated at 25℃ for lay eggs in 4～6 h, after which the nematodes were discarded .The nematode eggs were collected and soaked in 15% H2O2 for 60 min at 25℃. And the eggs were rinsed 3 times with sterile water and placed at 25℃ in the dark on the mycelia of B.cinerea grown on the PDA medium. The propagated nematodes were collected in 10 mL centrifuge tubes under aseptic conditions and treaded as aseptic PWN (Bx_a). The endobacteria strain of S. maltopholia was that maintained on slant Nutrient Agar medium(NA) in tubes transferred to cultured in 50mL NB and cultured on shaker for 24h.The concentration of S.maltopholia was adjusted to 8.0×108 CFU and 200μL suspension was sprayed to PDA plates containing B. cinerea. Each plate was inoculated with 1000 bacteria-free nematodes (AmA3) and incubation for 7 days at 25℃. Then we collected PWN in 10 mL centrifuge tubes with Baermann funnel under aseptic condition. The collected nematodes (Bx_b) were washed 3 times with sterile water.Total RNA was respectively extracted from three frozen nematode samples (Bx_a, Bx_b, and Bx_w)( All the samples were immediately frozen in liquid nitrogen and then stored at -75℃ until use.) using RNAprep Kit (Tiangen, China) and purified using RNAclean Kit (Tiangen, China), according to the manufacturer’s instructions. The RNA integrity and quantity were determined with an Agilent 2100 Bioanalyzer (Agilent, Inc.) before cDNA synthesis. The isolated mRNA strands were broken into short fragments used as templates to synthesized cDNA, using cDNA Synthesis SystemKit (Roche, Inc.).Then the three cDNA samples were respectively used to construct cDNA libraries with TruSeq RNA Sample Prep Kit (Illumina) following the manufacturer's instructions. The cDNA libraries quality was determined with Agilent 2100 Bioanalyzer and ABI StepOnePlus Real-Time PCR System (Counterpane, Inc.). The three cDNA libraries were sequenced using Illumina HiSeqTM 2000 system.