Astragalus adsurgens were planted at Heping town in Lanzhou (103.58 E, 36.00 N) with an elevation of 1750m above sea level (m.a.s.l) in Gansu Province in 2010 (Zeng 2016). Lanzhou has a semi-arid continental climate, significant temperature variations and less rainfall, with an average annual precipitation of 300 to 400 mm. The incidence of YSRR in Lanzhou was 100% in 2015 (Zeng 2016). The incidence of YSRR disease occurred on 80% plants of A. adsurgens and twenty symptomatic and asymptomatic A. adsurgens plants were collected at the end of September 2017 and 2018, depending on its symptoms described in 2007 (Li 2007). Plants of Oxytropis ochrocephala used in the study were collected at the end of August 2017 from Haiyuan County, Ningxia Province (105.36° E, 36.29° N), with an elevation of 2173 m.a.s.l.
Pathogenic fungus was the isolate (MHLZU0408) preserved at the Mycological Herbarium of Lanzhou University (MHLZU) (Li and Nan 2007), cultured on wheat hay decoction agar (WHDA) (Li 2007). Endophytes was the strain isolated by Liu (2019) preserved at the Mycological Herbarium of Lanzhou University (MHLZU) (Liu 2019), cultured on potato dextrose agar(PDA) (Li 2007). These fungi had identified by using morphological and molecular characteristics (Li and Nan 2007; Yu et al. 2018). The pure cultures of fungi were collected and then dried in a freeze dryer for 24 h and stored in a refrigerator at -20°C before further experiments.
Extraction Of Dna
DNA from healthy and diseased A. adsurgens stems and leaves (five each), O. ochrocephala plants with and without endophytes (two each) were extracted using Plant DNA Kit (OMEGA Biotech Co.Ltd.) following the manufacturer’s instructions from 10 mg of dried samples previously grounded to powder using sterile pestle. For fungus DNA extraction, the mycelia of pure cultures of A. gansuense and A .oxytropis were scraped from one fresh colony into a 2mL centrifugetube using a sterilized spoon. Genomic DNA was extracted using HP Fungal DNA Kit (OMEGA Biotech Co. Ltd.) following the manufacturer’s instructions.
Polymerase Chain Reaction (PCR) Amplification
The KS gene was amplified using the specific primer pair KS-F(GAGGAAATTGCTATAGTTTCCATGGC) /KS-R (GGCATCCGAAAGACGTTTAAGAAG) (Cook et al. 2017). The PCR amplifications were performed in a 2720 thermal cycler (Applied Biosystems) in a total reaction volume of 25µL mixture containing 1µLof genomic DNA,1µLof forward and reverse primers(10µM), 12.5 µL of 2×Es TaqMasterMix (Dye) and 9.5 µL of sterile water as follows: an initial denaturation step was performed at 94°C for 5 min; followed by 29 cycles of denaturation at 94°C for 30 s, then annealing at 56°C for 30 s and extension at 72°C for 60 s; and a final elongation step at 72°C for 4 min. Amplicons were visualized by gel electrophoresis in a 1% agarose gel stained with BBI 4S GelRed using 1X TAE (40 mM of Tris-acetate and 1 mM of EDTA at pH 8.0, purchased from Sangon Biotech) as a running buffer and photographed (Li 2018).
Assaying The Content Of Swainsonine
One-month-old fungal mycelia were scraped from one fresh colony using a sterile spoon and then dried in a Freeze dryer. About 100 mg of dried and ground stems were then placed into new 2 mL centrifuge tubes. The samples were freeze-dried for 24 h and then wrapped in labeled and sealed filter papers (10 cm × 4 cm). Each sample was continuously extracted with 60 mL methanol at 70°C in a water bath for 12 h using a Soxhlet apparatus extractor. A dry methanol extract was then carried out in a steam spinner to separate the plant material from the solvent layers. The final residue was dissolved in a 10 mL acetic acid solution (2%) in an ultrasonic cleaning machine for 30 min at 60°C. The solution was added to the cation-exchange extraction resin (732) adsorption column (6 cm) in which a small amount of cotton was placed into the tip, and a rubber septum was used to plug the tip end with the dynamic exchange for 2h. The resin and solution were then mixed for 30 min using mechanical rotation to allow the SW cations present in the sample to bind to the resin. The adsorption column was eluted with 50 mL of deionized water, which was then discarded, while for there incolumn, 50 mL ammonia solution (1 mol/L) was used. The ammonia eluate was dried in an evaporating dish and dissolved in 1 mL methanol. The sample solutions were placed into 2 mL centrifuge tubes, centrifuged at 12,000 rpm for 10 min at room temperature, and stored at -20°C until analysis for SW by HPLC-MS [38, 48]. All chemicals or solvents used were of analytical reagent grades. Acetic acid was 2% glacial, while water was deionized. The SW standard was purchased from Sigma USA.
To determine SW concentration by HPLC-MS, a stock solution of SW standards of 15.625, 156.25, 312.5,625, 937.5, and 1250µg/mL using serial dilution was prepared with methanol. After which, the samples and calibration standards were loaded into the autosample rvial holder for analysis (Wang et al. 2012).
The SW peak area was measured from the reconstructed ion chromatogram (m/z = 156) and quantitation based on an external calibration standard. The mobile phase consisted of acetonitrile,40% deionized water, and 60% methanol with 1% formic acid, MH = 174.2 ± 0.75amu, flow rate 0.5 mL/min, chromatographic column: Bestasil C18 (100×2mm, Keystone Scientific), injection volume1uL, capillary temperature 200°C, voltage:16V, peak time: 1.14min, wavelength: 205nm, detection mode: SIM mode, full MSScan = 174.11, using an HPLC-MS to determine the peak area of each concentration as the vertical coordinate and the standard solution concentration served as the horizontal ordinate (Gardner et al. 2001).
Data were analyzed using IBM SPSS Statistics ver. 21.0 (SPSS, Inc., Chicago, USA). Swainsonine concentrations in A. adsurgens and locoweed stems and leaves and fungus were analyzed by one-way ANOVA to test differences. Tukey’s HSD test was used to determine the significant differences between mean values at P < 0.05.