Identification of nematicidal compounds from A. welwitschiae
The A. welwitschiae strain AW2017 was cultured on potato dextrose broth (PDB) medium (50 L) at 25 °C for 5 d [24]. The nematicidal compounds were isolated from AW2017 metabolites as described by Kusano et al. [18] with minor modifications. Briefly, the culture filtrate was extracted with CHCl3/MeOH eluent and evaporated under reduced pressure. The crude material was purified by column chromatography on silica gel, and the active fractions were repeatedly crystallized from Me2CO/EtoAc to produce nematicidal compounds, which were used to analyse its nematicidal activities simultaneously. Final products were identified by comparing the physiochemical properties with those reported previously [4, 18, 34]. All the chemicals used in the present research were purchased from Sigma-Aldrich (China).
Rice culture and propagation of the nematode
Rice seeds (Oryza sativa cv. Nipponbare) were originally obtained from the US Department of Agriculture (GSOR-100) and multiplied in Hunan Province, China. Seeds were soaked in 5.25% sodium hypochlorite for 5 min and germinated at room temperature (25 ± 4 °C) for 4 d. One geminated seed was sown in a polyvinylchloride (PVC) tube containing synthetic absorbent polymer (SAP) [43]. Rice seedlings grew in a greenhouse and were irrigated with 20 ml of Hoagland’s solution twice per week at 28±2 °C with 70-75% relative humidity.
The M. graminicola population was propagated on the susceptible rice variety Nipponbare at 28±2 °C. Nematode eggs were separated from the root galls and hatched in a 75-μm sieve at room temperature for 3-5 d. Hatched second-stage juvenile (J2) suspensions were filtered through a 25-μm sieve and resuspended in distilled water at approximately 200 nematodes mL–1 for the subsequent experiments [27, 43].
Nematicidal activity of αβ-DC against M. graminicola juveniles
To test the larvicidal activity of αβ-DC, approximately 200 hatched J2 individuals were added to each well of 24-well plates containing 1 ml of αβ-DC solution at 480, 240, 120, 60, and 30 μg mL-1. Formulated fosthiazate emulsifiable concentrate (486 mg mL-1 EC) and fluopyram suspension concentrate (417 mg mL-1 SC) were also diluted to 480, 240, 120, 60, 30 μg mL-1 as described above and used as positive controls. A treatment with only l ml of a 1% dimethyl sulfoxide (DMSO) water solution was included as the negative control. Four replicates were set for each treatment. The plates were kept at 25 °C for 48 h. Nematodes were considered dead if their bodies were motionless and straightened after stimulation with 1 M NaOH [5]. The dead and alive juveniles were counted under a stereomicroscope, and the corrected mortality values of juveniles were calculated as described in Liu et al [24]. The median lethal concentration (LC50) of αβ-DC and other chemicals against nematodes were analysed with SAS software (SAS Institute, Cary, NC). Three trials with four replicates were performed for this experiment.
Effect of αβ-DC on the attractiveness of rice roots to M. graminicola
As described in Wang et al. [41], 23 g pluronic F-127 powder was fully dissolved in 100 mL of sterile water at 4 °C while stirring for 24 h. Then, an attraction bioassay was performed at room temperature. Rice roots of 2-week-old plants were drenched with 20 mL αβ-DC (30 μg mL-1) solution or 1% DMSO water solution. One day later, a 1-cm-long root tip was cut and placed into a six-well culture plate containing 1 mL pluronic F-127 gel and approximately 100 hatched J2s. Nematodes attracted to 5 mm around the root tip were counted under a Leica stereomicroscope and were photographed with a DFC400 camera at 6 hpi. The entire experiment was performed thrice, with four replicates.
Morphological observation of nematode giant cells
After J2s enter the vascular cylinder, they inject pharyngeal secretions to induce permanent feeding sites known as giant cells, which are used as food resources throughout their life cycle [9]. To observe the morphological changes of nematode giant cells, each rice plant was drenched with 20 ml αβ-DC (30 μg mL-1). Plants drenched with DMSO solution were used as the control. One day later, each plant was inoculated with 100 J2 s. At 7 dpi, 10 root galls from six plants were fixed in 1×PIPES buffer overnight and then dehydrated in different ethanol dilutions. After being embedded in Technovit 7100 for 2 weeks, gall tissues were sectioned into 10-μm slices with a Leica RM2265 (Leica Microsystems, Beijing, China). Slices on glass slides were stained with 0.05% toluidine blue and sealed with DPX mountant. Microscopic observations were performed under an Olympus SZX 16 at 40× magnification, and images were obtained with an Olympus DP74 (Olympus Optical Company, Tokyo, Japan). The experiment was repeated twice.
Development of nematodes in greenhouse experiments
To determine the direct effect of αβ-DC on the development of nematodes, each rice roots was drenched with 20 mL αβ-DC (240 μg mL-1), fosthiazate EC (48.6 μg mL-1), fluopyram SC (4.17 μg mL-1) or 1% DMSO solution, respectively, at 24 h before nematode inoculation. Each 2-week-old rice plant was inoculated with 200 J2s around the roots and maintained in the greenhouse at 28±2 °C. At 14 dpi, root samples were collected, and nematodes in rice roots were counted under a stereomicroscope after staining with acid fuchsin for 3 min [26]. The total numbers of nematodes in the third or fourth stage (J3/J4) and females were counted. To calculate the ratio of nematodes at various stages, the numbers of nematodes in different life stages (female or J3/J4) were divided by the total numbers of nematodes in roots using Microsoft Excel 6.0 (Redmond, Washington, USA). The entire experiment was performed thrice, each containing 6 individual plants.
Infectivity of nematodes in the field experiment
The field experiment was continuously carried out in a commercial direct-seeding field in Hunan Province, China, from July to September in 2017 and 2018. Rice had been cultivated in this field for at least 40 years and was naturally infested with M. graminicola for 10 years [43]. To ensure the nematodes were evenly distributed in the field, the soil was mixed well with a rotary cultivator. To prevent the spread of nematodes, each plot (4 m×5 m) was constructed with an earthen levee (height of 25 cm and width of 30 cm) as described in Khanam et al. [17]. In total, four treatments were evaluated in a randomized block design, with 4 replicates, as follows: (1) 480 mg mL-1 αβ-DC (15 ml m-2), (2) 486 mg mL-1 fosthiazate EC (2 ml m-2), (3) 417 mg mL-1 fluopyram SC (0.15 ml m-2), and (4) untreated control with only 2 L 1% DMSO solution. Each chemical agent was diluted with 2 L DMSO solution and sprayed evenly on the nursery bed before seeding. Approximately 400 g of seeds were directly seeded on each plot after soaking in water at room temperature for 9 h. At 50 d after direct seeding, 20 seedlings from each plot were uprooted and washed free of soil. Root galling was rated on a scale of 0 to 5, where level 0=no galls, level 1=1~2, level 2=3~10, level 3=11~20, level 4=21~30, and level 5≥30 galls per root system [31]. The gall index was calculated using the formula described in Zhan et al. [43]. The control efficacy was calculated according to the following formula: control efficacy (%) =100× (gall index of control-gall index of treatment) /gall index of control.
Statistical analysis
The mean and standard errors (SE) of the data were subjected to statistical analysis using SAS software version 8.0 (SAS Institute, Cary, NC). Significant differences (P≤0.05) between the treatments were determined according to Duncan’s multiple range test.