LCM fabrication and characterization
Banana fibre from the rachis of Cavendish banana plants was procured from the agro-industrial unit of Earth University, Costa Rica, and was processed in the Forest Biomaterials Department at North Carolina State University. Lignin content and air resistance of the matrix were measured as reported in a previous article21. Briefly, lignin content of the matrix was measured following TAPPI (Technical Association of Pulp and Paper Industry) T236 test protocol. We evaluated porosity of LCM via the Gurley air resistance method following the TAPPI T460 standard protocol. A high air resistance of the LCM is indicative of its low porosity and vice versa.
Experimental sites
Field experiments to evaluate the effectiveness of W&P to protect PCN-susceptible potato cv. ‘Shangi’ against G. rostochiensis (the most prevalent PCN species in Kenya) attack were carried out under rainfed conditions during the main wet season from May to August (2016) and April to July (2017) in Kenya at four sites: Haraka A (00.78537° S, 036.60429° E), Haraka B (00.77588° S, 36.61652° E), Kinangop A (00.58985° S, 036.61740° E) and Kinangop B (00.58854° S, 36.61234° E), Nyandarua County. Farms were selected on the basis of their continuous cultivation of potato over the previous five years; all field sites were situated at elevations >2,500 m above sea level and had clay and sandy loam soils. Physico-chemical and soil texture analyses (Supplementary Tables 2 and 3) were conducted for each site at planting and harvest at the laboratory of the Kenya Agricultural and Livestock Research Organization in Nairobi, using a composite sample from across each site pre-planting and from a composite sample for each treatment per site at each harvest. No remarkable differences in soil composition between treatments were observed (Supplementary Table 4). The accumulated rainfall for the county was 859.9 mm and 458.3 mm for 2016 and 2017, respectively.
Field experiments
Plant parasitic nematode densities were assessed at both Pi and Pf. For both Pi and Pf, nematode infestation was assessed using a 200 ml sub-sample from a composite sample of ~1 kg removed from five randomly selected soil sampling points per plot from ~20 cm depth in each field. PCN cysts were extracted using the Fenwick can method9. As no differences (P < 0.05) were observed between the Pi infestation levels of PCN cysts and other nematodes, a completely randomized design was implemented. The PCN juveniles and other nematodes were identified to the genus level using morphometric traits, while the molecular-based European and Mediterranean Plant Protection Organization diagnostic protocol PM 7/40(3)9 was used to determine the PCN species.
The experiment involved four treatments: ABM–LCM (seeds ‘wrapped’ with LCM impregnated with ∼0.8 ng cm–2 of abamectin (100 ng ABM per sheet), u-LCM (not impregnated with ABM), ABM alone (drenching the soil with ABM Tervigo (8 l ha–1) following the commercial recommendations (0.19 ml active ingredient per potato seed)) and farmer practice (absolute control, which received no additional application of nematicide or wrapping). Quality-declared cv. Shangi seeds were planted in 4 × 4 m plots spaced 30 × 75 cm (65 seeds per plot) with a 1 m distance between plots and four replicate plots per treatment. Plots were prepared individually by hand-hoe. Treatments involving the LCM used a single sheet (10 × 12.5 cm) in which the potato tuber was wrapped loosely. For the non-LCM treatment, two 10 l suspensions of Tervigo were applied per plot onto the unwrapped tubers in the furrow and top drenched after covering with soil; the potato seeds remained unwrapped.
For all treatments, di-ammonium phosphate (NPK 18/46/0) fertilizer was applied at planting (500 kg ha–1); a foliar fertilizer (NPK 20/20/20; Diamond Plant Fertilizer, Kerapros Ltd) (2.5 ml l–1) was applied 6 weeks after planting and the foliar fungicide Mistress 72 wettable powder (1.5 g l–1) (Cymoxanil 8% + Mancozeb 64%; Osho Chemicals Ltd) for early and late blight disease control was sprayed every 2 weeks after sprouting. Weeds were removed manually on three occasions. No additional irrigation was provided.
The experiment was terminated at 110 days post-planting. Data were collected on percentage germination per plot, plant height, stems per plant, root mass, yield and number of tubers per plot (kg plot–1) measured from all harvested tubers. The final juvenile (J2-Pf) and cyst (Cy-Pf) densities per plot and of Meloidogyne species were determined as described.
Laboratory-based experiments
The experiments were conducted using potato root exudates from cv. Shangi planted in 2 l plastic pots in sterilized sand (autoclaved at 121 °C for 40 min, Astell Scientific autoclave) in the screenhouse at 23 ± 2° C and 60–70% relative humidity at the International Centre of Insect Physiology and Ecology (icipe), Nairobi, Kenya (1.2219° S, 36.8967° E). Plants were watered three times weekly with a nutrient solution prepared according to ref. 36. Potato root exudates were collected from 3- to 6-week-old plants using the dipping method37. Batches of five potato plants per treatment were gently uprooted, rinsed in water, placed into 500 ml of distilled water and their exudates collected over 24 h. The exudates were filtered through Whatman no. 1 filter paper and stored at –80° C for use over a period of 8 weeks for the hatching assay.
For the chemotaxis assays, freshly collected exudates within 24 h after collection were used for each assay. Cysts used in the assays were obtained from a farmer’s field shortly following harvest of potatoes in Nyandarua County, Kenya (00.78537° S, 036.60429° E). The soil was air dried, and cysts were extracted as described previously. The collected cysts were dried on a milk filter paper before handpicking with the aid of a stereomicroscope (LEICA M125).
Chemical reagents
LC-MS grade methanol (LC-MS LiChrosolvR, Merck (99.97%)), formic acid (98–100%), water (LC-MS Chromasolv), α-solanine (95%), solanidine (95%) were sourced from Sigma-Aldrich and α-chaconine (95%) from PhytoLab.
Potato cyst nematode hatching bioassays
The PCN hatching assay was conducted with and without the LCM as illustrated in Supplementary Fig. 1a. Two test solutions, distilled water and potato root exudates retrieved from plants grouped into four ages (from plants aged 3, 4, 5 and 6 weeks old), were used to evaluate the egg hatching response. Exudates were collected from different plants for each age group and each group replicated four times. Six treatment combinations were then assessed against each exudate group: exudates alone (no LCM; positive control), ABM–LCM + exudates, u-LCM + exudates, distilled water alone (no LCM; negative control), ABM–LCM + water and u-LCM + water. For each replicate, five cysts were placed in container A and were held in place by a nylon mesh that permitted hatching juveniles to pass through. The experiment was arranged in a randomized design with four replicates per treatment. For treatments involving the LCM, the base of container B was lined with the matrix; for the treatments without matrix, a nylon mesh was used. The experiment was arranged in a six-well culture plate labelled as container C (Supplementary Fig. 1a) into which the test solution was added and maintained in the dark for 8 weeks. The number of juveniles emerging from cysts were counted weekly and the test solution replenished. At 8 weeks, eggs remaining in the cysts were assessed for viability38.
Potato cyst nematode chemotaxis bioassays
PCN eggs were stimulated to hatch using freshly collected potato root exudates, and freshly hatched juveniles were collected daily on a 20 µm sieve, rinsed and held in distilled water for use. The responses of hatched infective juveniles to potato root exudates in the presence and absence of LCM were tested in a dual-choice sand bioassay (Supplementary Fig. 1b), according to ref. 37, with some modifications. Root exudates from 5-week-old plants were used. Each chamber was filled with 5 g of sterile sand. Sand in chambers A and B was mixed with 1 ml of freshly collected exudate (<24 h) and distilled water, respectively. The effects of u-LCM or ABM–LCM were compared with a nylon mesh partition between the chambers. Chamber C contained 3 mg of moist autoclaved sand and 500 µg of water containing 200 J2s. Each chamber was suspended in water and juveniles collected on 20 µm sieves. The juveniles recovered after 48 h were counted and scored under a stereomicroscope as either positive responders to stimuli or negative responders/non-responders depending on the chamber in which they were recovered. The experiment was arranged in a randomized design with three replicates per treatment. The experiment using root exudates from 5-week-old plants was conducted six times.
Potato cyst nematode development experiments
The effect of the LCM on PCN development was assessed using wrapped and unwrapped seed potatoes (cv. Shangi) inoculated with PCN in 2 l pots filled with autoclaved sand. Treatments included potatoes wrapped in ABM–LCM, potatoes wrapped in u-LCM and unwrapped potatoes (control). The experiment was arranged in randomized design with six replicates per treatment. The pots were inoculated with 20 cysts at planting and maintained for 8 weeks in the screenhouse at 23 ± 2° C and 60–70% relative humidity at icipe. Plants were watered three times weekly with a nutrient solution prepared according to ref. 36. At 2-week intervals for 8 weeks, six replicates per treatment were randomly selected and the number of each development stage of PCN on the roots recorded. The experiment was conducted twice.
The different nematode developmental stages present in the roots were assessed after staining with acid fuchsin39. Briefly, the uplifted roots were gently rinsed in distilled water to remove soil debris. The roots were chopped into 1–2 cm segments, placed in 1.5% sodium hypochlorite solution for 4 min, agitating occasionally, then rinsed in tap water followed by distilled water. The clean roots were placed in 30 ml of water containing 1 ml of acid fuchsin (3.5 g in 25% acetic acid) (BDH), which was heated to boiling, cooled and rinsed under running water before placing in heated glycerine acidified with a few drops of 5 N hydrochloric acid. After cooling, root segments were pressed between two microscope slides, and the different nematode development stages were counted under a stereomicroscope at ×40 magnification.
Root exudate chemical composition after exposure to LCM
The chemical composition of root exudates from 5-week-old plants was assessed following exposure to the LCM in vitro. A 0.05 g of LCM (u-LCM and ABM–LCM) was immersed in 1 mg ml–1 of freeze-dried root exudate for 24 h. The LCM was removed and the remaining solution centrifuged to remove any remaining particles. This experiment was replicated three times. Chromatographic separation was then performed using an ACQUITY ultra-performance liquid chromatography (UPLC) I-class system (Waters Corp.) fitted with an ACQUITY UPLC BEH C18 column (2.1 × 150 mm, 1.7 μm particle size; Waters Corp.) according to the method by ref. 25. Briefly, the mobile phase comprised water acidified with 0.01% formic acid (solvent A) and methanol (solvent B) and followed a gradient system. The gradient system used was 0–2 min: 5% B; 2–4 min: 40% B; 4–7 min: 40% B; 7.0–8.5 min: 60% B; 8.5–10.0 min: 60% B; 10–15 min: 80% B; 15–19 min: 80% B; 19.0–20.5 min: 100% B; 20.5–23.0 min: 100% B; 23–24 min: 95% B; 24–26 min: 95% B. The flow rate was held constant at 0.2 ml min–1. The UPLC was interfaced with an electrospray ionization Waters Xevo TQ-S operated in full-scan MS in positive ionization modes, and data were acquired over the m/z range 100–2,000.
Identification of the compounds present in root exudates pre- and post-exposure to the LCM was performed using a Thermo Scientific Q Exactive Mass Spectrometer coupled to a Vanquish UHPLC System by gradient elution using an ACE Excel 2 C18-PFP column (2.1 µm, 100 mm). The mobile phase used was composed of water acidified with 0.1% formic acid (solvent A) and 0.1% formic acid in acetonitrile (solvent B) and followed a gradient system. The gradient system used was 0–3 min: 100% A; 3–23 min: 80% B; 23.0–26.5 min: 80% B; 26.5–30.0 min: 100% A. Solvent flow rate was at a constant 0.35 ml min–1 and 2 μl of the sample injected. Full-scan MS analysis was performed in positive ion mode at mass resolution 35,000 scanning from m/z 70–1,000.
This adsorption experiment was repeated using individual standards of PCN hatching factors α-solanine, α-chaconine and solanidine. A 10 µg ml–1 concentration of the standards was prepared separately in a 1.5 ml Eppendorf tube, and 0.05 g of the ABM–LCM and u-LCM were exposed to the standards for 24 h. Both the LCM-exposed and unexposed standards were analysed to compare differences. This experiment was conducted only once.
For comparison with the LCM, the chemical composition of root exudates following exposure to cellulose was analysed in an in vitro experiment. Exudates from 5-week-old plants were used, with three replicates per treatment. After freeze-drying (see the preceding), 1 mg of exudate was dissolved in 1 ml of methanol/water (3/7 v/v), and 0.05 g of cellulose was immersed in the solution. After 24 h, the solution was centrifuged and the supernatant collected and analysed, as described. This experiment was conducted only once.
Identification of root exudate chemicals adsorbed by LCM
Following exposure to 30 ml of 1 mg ml–1 root exudates for 24 h, 2.0 g of the LCM (u-LCM and ABM–LCM) was removed, dried and placed in 5% methanol in a sonicator bath at room temperature for 30 min. The extract was then freeze-dried, and 1 mg ml–1 of the extract was reconstituted in 30% methanol, centrifuged (Spectrafuge 16M) at 14,000g for 10 min and then analysed for chemical composition as described using three replicates.
The bioactivity of the LCM extract was also assessed using a hatching bioassay to determine whether the extracts remained active. Five pre-soaked cysts were placed in 200 µl of the u-LCM and ABM–LCM extracts per well, using a Linbro 96 multi-well sterile plate, with three replicates per treatment. Emerging juveniles were counted on a weekly basis over 4 weeks and compared with a negative water control. This experiment was conducted only once.
An assessment of the LCM longevity and ability to adsorb PCN hatching compounds under field conditions was undertaken in the field at icipe. Potatoes were planted following the procedure for the field evaluation of the LCM described in the preceding using ABM–LCM and u-LCM with three replications per treatment. Plants were uprooted on a 2-week cycle for 8 weeks, and the physical state of the matrix was observed and photographed. Matrix fragments were also analysed to detect and determine the presence of hatching factors using liquid chromatography triple quadrupole mass spectrometry analysis.
Statistical analysis
Data from field experiments were first checked for normality and equality of variances before analysis using the Kruskal–Wallis test to determine differences among the treatments and a pairwise Wilcox test used for a pairwise comparison between treatments. The numbers of juveniles hatching in the bioassays were fitted in a quasi-Poisson with negative binomial models to determine the differences between the treatments. The number of weekly hatched juveniles was log transformed and subjected to analysis of deviance and multiple comparisons of the means performed using Tukey’s honestly significant difference test. Comparisons between the treatments were also performed after fitting a linear mixed model, and the mean interactions of the treatments were analysed using least squares means. The number of responding juveniles from the dual-choice assays was analysed using the proportionality test to check the effect of the treatments on the attraction potential of the stimulus. The differences across the treatments were determined for each of the three chambers separately, including stimulus, control and the release chamber. The numbers of juveniles recovered from the stimulus chamber and the release chamber were fitted in a generalized linear model (GLM) with a negative binomial distribution, and those recovered from the control chamber were fitted in a zero-inflated model with a negative binomial distribution. A pairwise comparison of the means was performed when there were significant differences in the means.
The different stages of nematode development were represented as a mean of six replicates and subjected to ANOVA after fitting in a GLM with a negative binomial distribution. The means were adjusted using least-square means and separated using Tukey’s honestly significant test. Data from the repeated experiments were pooled, as no differences were observed between them, and analysed to assess treatment effect on the development of PCNs. The analyses were conducted separately for each week and for each developmental stage. The data were fitted in a GLM assuming a negative binomial distribution with the exception of J2 counts at week 2, for which a zero-inflated model with negative binomial distribution was used and the female count at week 4 was fitted with the zero hurdle model to address both dispersion and zeros in the data. All the models took experiment and treatment as covariate.
Reporting Summary
Further information on research design is available in the Nature Research Reporting Summary linked to this article.
