Bo Fu

The cyst nematode pathogens can cause a yield loss of 30-50% via stunting root growth on grains (Bonfil et al. 2004). Heterodera avenae, H. filipjevi, and H. latipons are identified as the most economically influential and harmful species on wheat (Toumi et al. 2016). H. filipjevi was first detected in Henan Province in 2010, and had subsequently spread to many provinces (Peng et al. 2010; Peng et al. 2016; Cui et al. 2021). In May 2022, wheat plants showing yellowing and premature senescence were observed in different wheat planting areas throughout Shaanxi Province. Nine soil samples were collected from Pucheng County (34°94'32"N, 109°57'23"E) of Shaanxi Province. Among them, six soil samples were detected to have cysts via the sieving-decanting method with an average cyst population density of 10.6 cysts per 100 cubic centimeters of soil. The cysts and second-stage juveniles (J2s) were identified based on both morphological and molecular identification. Morphologically, the cysts are lemon shaped, with a protruding vulval cone. The shell surface had a Z-shaped pattern and covered with a white subgrain layer. The cone of the vulva is bifenestrate in horseshoe-shape. Underbridge exists and is well-developed. The measurements of the cysts (n=16) as follows: body length 670.24 ± 60.67 (593.43 to 778.42) μm; body width 479.03 ± 54.51 (355.91 to 567.52) μm; length/width ratio 1.41 ± 0.16 (1.20 to 1.72); fenestra length 52.30 ± 4.32 (46.82 to 59.13) μm; fenestra width 27.59 ± 3.39 (22.42 to 34.91) μm; vulval slit length 9.17 ± 0.47 (8.69 to 10.37) μm. J2s measurements (n=15) as follow: body length 522.46 ± 24.18 (482.54 to 572.23) μm; body width 26.47 ± 3.08 (22.21 to 30.16) μm; stylet length 23.42 ± 3.24 (16.48 to 29.11) μm. These morphological characteristics and morphometrics are consistent with those of H. filipjevi (Peng et al. 2010). The DNA of single cyst was extracted with four replicates (Shao et al. 2022). The COI fragment sequence was amplified with the specific primers (HfF9/HafR8) according to Niu's research (Niu et al. 2016). The identities of amplification fragments (Genbank PQ650771, PQ650772 and PQ650773) are 98.77%, 99.08% and 98.77% matching the published sequences of H. filipjevi (MG523031.1 and MK093059.1), respectively. We also used the RAPD species-specific primers of H. filipjevi (HfF1/HfR1) to acquire 647bp sequences (Genbank PV165784, PV165785 and PV165786) which have 99.54%, 99.38% and 99.38%identities with published H. filipjevi sequence (KC529338.1) (Peng et al. 2013). Pathogenicity was confirmed by infecting wheat (Triticum aestivum 'Weilong 169'). The eggs of the cyst nematode population hatched at 15℃, and the emerged J2s were used for potted wheat inoculating in greenhouse under 18-24℃. Nine plants in three pots were inoculated and three plants in one pot remained served as the uninoculated control, the inoculum was 800 J2s per pot. After three months, an average of 15.7 cysts were detected from each of the inoculated plants, and the cysts were identified as H. filipjevi via molecular characteristics. To our knowledge, this is the first report of H. filipjevi in shaanxi. As Shaanxi is an important part of the Huang-Huai-Hai wheat region in China, where its occurrence should be attached great importance.

We used Medicago sativa rhizosphere in Shaanxi province of China to isolate and identify hydrogen-oxidizing bacteria that produced ACC (1-aminocyclopropane-1-carboxylate) deaminase, and then studied the mechanism why they can promote the growth of plants. Hydrogen-oxidizing bacteria were isolated by gas-cycle incubation system. We studied the morphological character, physiological characteristics, 16S rDNA sequence analysis and built the phylogenic tree. Thin layer chromatography was used to isolate the strain that produced ACC deaminase. Ninhydrin reaction was used to test the enzyme activity. In total 37 strains were isolated, 8 of which could oxidize H2 strongly and grow chemolithoautotrophically. We initially identified them as hydrogen-oxidizing bacteria. Only strain WMQ-7 produced ACC deaminase among these 8 strains. Morphological and physiological characteristics analysis showed that strain WMQ-7 was essentially consistent with Pseudomonas putida. The 16S rDNA sequence analysis (GenBank accession number EU807744) suggested that strain WMQ-7 was clustered together with Pseudomonas putida in phylogenetic tree, with the sequence identity of 99%. Based on all these results, strain WMQ-7 was identified as Pseudomonas putida. The enzyme activity of strain WMQ-7 was 0.671 U/microg. A strain producing ACC deaminase was identified and tested.