Slight induction and strong inhibition of Heterodera glycines hatching by short-chain molecules released by different plant species

The isolate of H. glycines race 3 was multiplied on soybean plants cv. M6410 IPRO (Monsoy^®) in ceramic pots in the greenhouse. Cysts were collected from the roots and soil according to the technique of Shepherd (1970) with modifications. For the extraction of the cysts, the roots were carefully separated from the soil. Then, they were placed on an 850-µm sieve coupled to a 150-µm sieve and subjected to a strong jet of tap water. In this way, the cysts were retained in the 150-µm sieve. For the extraction of cysts present in the soil, 200 cm³ of soil were placed in a cup where water was then added until completing one liter. After 1 min of agitation on the soil shaker, the suspension was poured into an 850-µm sieve coupled to the 150-µm sieve. The cysts were retained in the 150-µm sieve. To obtain the eggs, a set of sieves with an opening of 150, 75, and 25 µm was used. The cysts were deposited on a 150-µm sieve and pressed mechanically with a flat base small beaker and washed with tap water jets. Eggs retained on a 25-μm sieve were transferred to a hatching chamber made from a Petri dish (9 cm diameter) housing a 25-μm sieve (5 cm diameter). Then the hatching chamber was transferred to an incubator at 27 ± 2°C. Second-stage juveniles obtained during the first 24 hr were discarded and those hatched after 48 hr of incubation were used.

H. glycines eggs were exposed to VOCs emitted by soybean, bean, ryegrass, and alfalfa. Such species were selected because the literature reported that they produce SCN hatching factors (Fukusawa et al., 1985; Masamune et al., 1987; Riga et al., 2001) (Glycine max cv. M6410 IPRO), bean (Phaseolus vulgaris cv. Pérola), rye grass (Lolium multiflorum cv. BRS Ponteio) and alfalfa (Medicago sativa cv. P30) were grown in 121-mL wells of a 72-well Styrofoam tray filled with the artificial substrate (60% pine bark, 15% vermiculite, and 25% humus; Tropstrato®, Mogi Mirim, SP, Brazil) and kept in greenhouse. For testing purposes, vegetable materials (leaves and roots) were collected 30 days after germination, with the exception of ryegrass, which was harvested 60 after germination. Soybean and bean leaves and roots, alfalfa leaves and ryegrass roots were superficially disinfected with 1% sodium hypochlorite for 1 min followed by three washes in distilled water. After drying on paper towels, 1 g of each plant material was placed to one of the polystyrene two-compartment Petri dishes (Fernando et al., 2005) and, in the other compartment was poured 5 ml of an aqueous suspension containing approximately 1200 H. glycines eggs. In this assay one individual plant was used for each replicate. The negative control consisted in two-compartment Petri dishes containing only the suspension of eggs in the absence of leaves or roots of the plants. The plates were covered and sealed with polyvinyl chloride (PVC) plastic film and incubated at 25°C in the dark for five days. The evaluation consisted of quantifying the number of J2 hatched five days after the installation of the experiment. For this, the aqueous suspension containing J2 was collected and counted them using a Peters chamber and an inverted light microscope.

Six replicates of treatments were prepared similar to the ones described in the previous item for the characterization of volatile compounds. The samples were added in 20 ml solid phase microextraction (SPME) flasks and kept closed for five days in an incubator at 25°C. The identification of volatile molecules was carried at the Center for Analysis and Chemical Prospecting (Department of Chemistry/UFLA). VOCs were extracted via headspace solid phase microextraction (SPME) (Arthur and Pawliszyn, 1990). The parameters for SPME and gas chromatography-mass spectrometry (GC-MS) were identical to those described in previous work by Gomes et al. (2020).

The effect of four VOCs identified on the gaseous emissions of plants leaves and roots was evaluated on the hatching of H. glycines. These compounds were chosen according to their availability in the market and costs for acquisition, namely: 3-octanol (99%; Sigma-Aldrich, St. Louis, MO, USA), 1-hexanol (99.5%; Chem Service Inc, West Chester, PA, USA), hexanal (99.3%; Chem Service Inc, West Chester, PA, USA), and linalool (96%; TCI America, Portland, OR, USA). Each of these compounds was used in the J2 hatch test. For this, they were individually diluted in 1% aqueous Tween 80 (0.01 g/ml). Then, a 0.5 ml aliquot of the solution plus 0.5 ml aqueous suspension containing 300 H. glycines eggs were placed in 1.5 ml micro tubes. The micro tubes were sealed with PVC plastic film and incubated at 25°C for seven days. Compound solutions were prepared to reach the final concentrations of 200, 600, and 1,000 µg/ml. Water, Tween 80 (1%), and Carbofuran (2,3-dihydro-2,2-dimethyl-1-benzofuran-7-yl N-methyl carbamate; 415 µg/ml) were used as negative control and ZnCl₂ (3 mM) as positive control. After seven days, the tubes were opened and the number of hatched J2 was quantified. The numbers obtained were used to calculate the hatch percentage for each treatment.

The VOCs that caused hatching inhibition in the previous trial were selected to assess their toxicity to the J2. For this purpose, was determined the lethal concentration values required to kill 50% of the nematode population (LC₅₀) of the compounds 1-hexanol and 3-octanol. The compounds were prepared similarly to that described in the previous item; however, the final concentrations were adjusted to 10, 50, 100, 150, 200, 250, and 300 µg/ml. Then, 0.5 ml of the aqueous solution of each compound plus 0.5 ml of the J2 suspension (300 J2) were placed in 2 ml micro tubes. The micro tubes were sealed with plastic PVC film and incubated at 25°C for 48 hr. After this period, the micro tubes were opened and an aliquot of 100 µl of each replicate was added in holes of 96-well polystyrene plates to determine J2 mortality. The determination of dead nematodes was performed by adding 20 µl of 1 mol/l sodium hydroxide solution (NaOH) to each hole in the plate (Chen and Dickson, 2000). Nematodes that remained immobile after 3 min were considered dead. The J2 mortality percentages obtained in each treatment were used to calculate the LC₅₀ of each of the compounds, which represents the concentration capable of killing 50% of the J2 population of H. glycines.

The experiments were carried out twice in completely randomized designs with five replicates per treatment in hatching tests, and with six replicates for the lethal concentration experiment. Combined analysis of the experiments (Experiment 1× Experiment 2) was performed and in all tests, it was significant (p < 0.05), therefore, the results of each experiment were analyzed separately. Previously, the results of the experiments were subjected to tests of normality (Shapiro–Wilk’s test) and homogeneity of the variance (Bartlett’s test). Thus, the data for each experiment were subjected to the F test and when significant (p < 0.05) they were subjected to the Scott–Knott’s means test (p < 0.05). Mortality percentages were subjected to regression analysis and the determination of the LC₅₀ of the compounds was performed. The Sisvar (version 5.6) and SigmaPlot® (12.0) programs were used for statistical and graphical analysis, respectively.

In two experiments, the VOCs emitted by the different plant species induced a significant increase (p = 0.0000) in the percentage of H. glycines J2 hatched, compared to the negative control. In the first experiment, VOCs released by leaves and roots of soybean and bean provided the largest hatching increases, reaching values up to 71.4% higher than the control. In the same experiment, the volatiles of alfalfa leaves and ryegrass roots also caused significant increases in J2 hatching. In the second experiment, the highest hatching percentage was observed from eggs exposed to the volatiles of soybean roots, being 42.8% higher than the control. The VOCs emitted by other plant species also induced a significant increase in the number of hatched J2 compared to the control (Table 1).

GC-MS analysis of the VOCs released by the roots of soybean, ryegrass, and bean revealed the presence of 22, 19, and 17 compounds, respectively. In the emissions of soybean, bean, and alfalfa leaves, 14, 5, and 7 compounds were found, respectively. More compounds were found in soybean and bean roots emissions than in leaves (Table 2). In general, most of the compounds found belong to the classes of alcohols, esters, and ketones, but with incidence of some compounds in the furans, aldehydes, terpenes, pyrazines, and other classes. The compounds 1-hexanol and 2-octanone were detected in all analyzed root emissions (ryegrass, soybean, and bean), but were not identified in the leaf samples. The compounds 2-octanol, 3-methyl-1-butanol, 2-methyl-1-butanol, 6-methyl-5-heptan-2-ol, 2-ethyl-1-hexanol, 2-octanone, 3-octanone, 6-octen-2-one, pentan-3-one, and ethylene benzene were detected only in soybean and bean roots. Some compounds were identified exclusively in certain plant material, such as: heptyl acetate, octyl acetate, isopulegol acetate, n-nonyl acetate, p-ethylanisole, and 4-ethyl-1,2-dimethoxybenzene in ryegrass roots; 3-octanol, lavandulol, 2-butanone, ionone and linalool in soybean leaves; hexanal, b-felandrene, 2-isopropyl-3-methoxypyrazine and 2-sec-butyl-3-methoxypyrazine in soybean roots; and, 1-octen-3-ol, ethyl 2-methylbutyrate, and ethyl butyrate on alfalfa leaves. Three compounds were not identified including two sesquiterpenes. All compounds detected showed low intensity peaks in the samples and, therefore, were categorized only as minor peaks (+).

In two experiments, none of the compounds tested at concentrations of 200, 600, and 1000 µg/ml, namely 3-octanol, 1-hexanol, hexanal and linalool, induced J2 hatching at the same level as the positive control (ZnCl₂). Furthermore, the solubilizing agent used to dissolve the compounds – 1% Tween 80 – when tested alone was more effective in inducing hatching than the solutions containing the compounds. In the second experiment, the solubilizing agent Tween 80 at 1% showed similar hatching percentage (p = 0.0000) to the positive control ZnCl₂.

If, on the one hand, none of the compounds acted as hatching factors, the compounds 3-octanol and 1-hexanol caused, in the three concentrations tested in both assays, a reduction in hatching (p < 0.05) similar to that observed with carbofuran (415 µg/ml), which caused the lower percentage of hatching (Table 3).

In two assays, the increase of 1-hexanol and 3-octanol concentrations caused increase in H. glycines J2 mortality. Through regression analysis, the LC₅₀ values of compounds 1-hexanol and 3-octanol were 210 and 228 µg/ml, respectively, in the first experiment and, 230 and 124 µg/ml in the second one (Fig. 1).

Figure 1: