Nematode collection
In August 2019, five samples of roots and corresponding rhizosphere soils were collected from a tomato (cv. Maohong 801) fields near Zhuma village in Tongxu County of Kaifeng city, Henan Province, China. Each sample consisted of at least five sampling points from patches of poor growth. Samples were placed in plastic bags and stored at 16 to 18 °C [21]. Nematodes were extracted using the modified Baermann funnel method for 2 days [38], by which time 100 g of tomato soil and root samples were extracted from each sample.
Nematode culturing and experiments
Carrot disks were prepared in the following way. The surface of a carrot was sterilized with 95% ethanol. The external tissues were removed with a sterile knife, and then the carrot was cut into disks (approximately 6 mm thick). Each carrot disk was placed in a petri dish, in an incubator maintained at 25 °C approximately 15 days prior to use. One individual female nematode was selected and sterilized with 0.3% streptomycin sulfate, and then transferred to carrot disks at 25 ℃ in the dark for propagation. After that, the purified spiral nematode LQ-1 population cultured on carrot disks were used for morphological and molecular analysis.
Morphological identification
Nematodes were heat-killed, fixed in FG solution (formalin: glycerin: water=10:1:89) [39], and processed to glycerin by the formalin glycerin method [40]. Photomicrographs and morphometric data of the specimen LQ-1 population were obtained using a Nikon Eclipse Ti-S microscope (Japan). Images of key morphological features were processed using Photoshop CS5. The De Man formula was used for measurements. All measurements are expressed in micrometers (μm), unless otherwise stated.
Molecular characterization and phylogenetic relationships
DNA form one individual spiral nematode was extracted using proteinase K-based lysis [41]. The rRNA-internal transcribed spacer (ITS) region and the D2-D3 region of the 28S rRNA gene were amplified with primers TW81/AB28 (5´-GTTTCCGTAGGTGAACCTGC-3´/5´-ATATGCTTAAGTTCAGCGGGT-3´) [42] and primers D2A-D3B (5´-ACAAGTACCGTGAGGGAAAGTTG-3´/5´TCGGAAGGAACCAGCTACTA-3´) [43], respectively. The processes of PCR amplification, cloning and were carried out according to the method of Wang et al [44]. and sequencing was performed by Sangon Biotech Co. Ltd. (Shanghai, PR China). The newly obtained DNA sequences were submitted to the GenBank database.
The obtained ITS region and D2-D3 region of the 28S sequences were subjected to multiple alignment using Clustal W in MEGA 5.0 [45] with other spiral nematode species sequences published in the NCBI GenBank database. Outgroup taxa were selected based on a previous study [20]. Sequence datasets were analysed with Bayesian inference (BI) using MrBayes 3.2.6 [46] under the best-fit model of GTR+G+I, according to Akaike Information Criteria [47]. BI analysis for each gene was run with a random starting tree and four Markov chains for 1×106 generations. The Markov chains were sampled at intervals of 100 generations. After discarding burns in samples, the remaining samples were used to generate a 50% majority rule consensus tree. Posterior probabilities (pp) are given on appropriate clades.
Reproduction tests
Using the carrot disk method, experiments were conducted to determine the reproductive potential of H. microlobus females and the optimum temperature for culturing H. microlobus on carrot disks. In the experiment, 30 females were surface sterilized with 0.3% streptomycin sulfate and then transferred to one carrot disk in a petri dish. These petri dishes were sealed with parafilm and incubated at 25, 27.5, and 30 °C in the dark. The number of nematodes and reproduction rate (Rr = final number of nematodes/initial number of nematodes) on each carrot disk were determined at 90 days after inoculation.
A maceration method was used to collect nematodes [21]: the carrot tissues were placed in sterile water and macerated in a blender, and to isolate nematodes from carrot tissue, the suspension was then passed through 0.250-mm and 0.150-mm-pore sieves. The nematodes were collected through the sieves into a beaker, and carrot tissues were discarded. Then, the nematode suspension in the beaker was left to settle for at least 4 h, and the supernatant was removed. The total nematode number was observed under a stereomicroscope to assess whether H. microlobus reproduction had occurred, and the Rr (final number of nematodes/initial number of nematodes) was determined. There were five replicates for each experiment, and each experiment was conducted twice.