Philippa Kaur

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Mesocosm experiment
To study the effect of earthworms’ bioturbation activities on EPNs recruitment by maize roots, we performed an outdoor mesocosm experiment at the Botanical Garden of Neuchâtel, Switzerland. The mesocosms consisted of 50 × 50 × 30 cm wooden raised garden beds layered with a 1 mm diameter plastic mesh to prevent earthworm escape (Supplementary Fig. S1 in Supplementary material). Each mesocosm was filled with approximately 60 L of the A-layer of an Anthrosol (organo-mineral horizon) enriched with 10% compost and 10% sand. The initial soil was sieved once at 2 cm, and subsequently hand-sieved twice to remove all potential indigenous earthworms present. Before adding compost and sand, natural soil samples were collected, homogenized, dried at 40 °C for 48 h, sieved at 2 mm, and ground using agate mortars for subsequent physicochemical analyses. Specifically, we measured the particle size distribution (modified Robinson pipette method), the organic matter content through loss on ignition by weighing before and after burning 10 g of soil at 450 °C for 2 h, carbon to nitrogen ratio (CN) using an elemental analyser (FLASH2000, Thermo Fisher Scientific, Waltham, Massachusetts, United States); the pH in 1:2.5 soil to water ratio; the cation exchange capacity (CEC) following the cobaltihexamine chloride method; and the total phosphorous (using the Kjeldahl digestion method)25. The initial A-layer of the Anthrosol so was thus characterized as a silty-loamy soil (23% sand, 65% silt, 11% clay), with 7.06% organic matter content, 2.95% organic carbon, with a CN of 11.36, and with 19 ppm of total phosphorus content. The soil pH was 7.65 and the CEC was 5.0 cmolc/kg.
Four experimental treatments were tested (Supplementary Fig. S1): monocultures with and without the endogeic earthworm species Allolobophora icterica; and polycultures with and without the same earthworms. To reduce risks of interspecific variation in earthworms, we used a commercial strain of A. icterica supplied by the Ecotoxicology Department of National Institute for Agricultural Research (INRA Versailles, France).
The simulated monoculture consisted of three maize plants (Zea mays var. Delprim, UFA Delley Semences et Plantes, Delley-Portalban, Switzerland) per mesocosm, whereas one squash plant (Curcubita pepo, var. Rondini, Sativa Rheinau AG, Switzerland) and two bean plants (Phaseolus vulgaris, var. Neckargold, Sativa Rheinau AG) were growing with three maize plants in the simulated polycultures (Supplementary Fig. S1). In total we built 10 mesocosms per treatment (N = 40 mesocosms). All plants were sown early July and grown until mid-October 2018 before the onset of the experiment (Supplementary Fig. S2). Seven weeks after sowing, half of the mesocosms were inoculated with 15 earthworms each. The earthworms were standardized to 6 g of total fresh weight biomass per mesocosm.
In mid-October, the roots of one maize plant per mesocosm were mechanically damaged with a cork borer (punched three times near root area) and watered with 25 ml of a solution containing 500 μg (2.4 µmoles) of jasmonic acid (JA; ( ±)-Jasmonic acid, CAS Number: 77026-92-7, Sigma, St Louis, IL, USA) per plant to induce emission of volatile defence compounds26. JA is a growth phytohormone also called the “wound hormone” as it plays a central role in plant defence and has been shown to induce the release of Eβc in herbivore-attacked maize plants22. Mechanical damage was preferred over direct herbivory on roots to ensure damage reproducibility and to standardize the production of defence volatile compounds. Two days later, four Galleria mellonella (Lepidoptera: Pyralidae) larvae per mesocosm were placed in the soil as sentinel hosts to quantify EPN infection success. Specifically, in each mesocosm, the first two G. mellonella larvae were buried 5 cm deep in the soil and 5 cm away from the stem of a root-broken maize plant (damaged roots), while the second pair of larvae was placed in the same conditions, but close to the roots of an undamaged plant (control roots). Because late-instars G. mellonella larvae are immobile and highly susceptible to EPN infection, they have been extensively used for monitoring EPNs’ presence in soil27,28, as was done here. One day after G. mellonella addition, a solution of less than 2-week old 3000 infective juveniles H. megidis EPNs was inoculated at the centre of all mesocosms. The used H. megidis EPNs (Nematoda: Heterorhabditidae) were supplied by Andermatt Biocontrol AG, Switzerland, and reared on late-instar G. mellonella larvae in accordance with an in vivo rearing protocol described step by step29. Five days after EPN inoculation, all G. mellonella larvae were collected. Dead larvae were directly transferred into White traps to confirm infestation by EPNs, while living larvae were kept in soil-filled 5 × 6 × 4 cm plastic boxes for measuring potential EPN infection. Next, we collected plant traits related to biomass accumulation, including: total aboveground biomass, total vegetative height, and fitness, as the total biomass of all corncobs on each plant. Finally, a fraction of the soil was sampled in each mesocosm for fertility-related analyses; which included CEC and CN measures.
Olfactometer-based bioassays
To dissect the interactive effect of root herbivory and earthworm presence near the roots of maize plants on EPN recruitment, a first (four-arm) olfactometer bioassay was conducted in controlled conditions of temperature, light and humidity (22 ± 2 °C day/16 ± 2 °C night, 55% RH, daytime 08:00 a.m.–06:00 p.m., 230 μmol/m2 s). The belowground olfactometer device (Fig. 2A), modified from Rasmann et al.5, consisted of a central glass chamber filled with white sand (Spielsand classic, Hamann Mercatus GmbH, Germany) extending in side arms connected to terminal glass pots (10 cm high, 15 cm diameter). Pots were filled with 1.2 L of soil (1/4 sand, and 3/4 standard potting soil; Ricoter, Aarberg, Switzerland, 10% relative humidity) as a standard for growing maize in the non-soil substrate when tested in olfactometers30. Three A. icterica earthworms were inoculated in two terminal pots (Fig. 2A). Simultaneously, one maize seedling was sown in each of the four terminal pots, and left to grow for 20 days (two-leaf to three-leaf stage). After 20 days of growth, which is considered as a minimum period for significant bioturbation of the soils31, three second instars of the banded cucumber beetle Diabrotica balteata (Coleoptera: Chrysomelidae) were added to two opposite glass pots containing the plants for the herbivore treatment setup (Fig. 3A). We used D. balteata instead of D. v. virgifera because of quarantine restrictions impeding the use of this species in the climate chambers in Switzerland. D. balteata is a generalist beetle that can feed on maize roots, and has been previously shown to induce maize plants to produce Eβc and attract EPNs32. Therefore, while slight differences might exist in terms of defence induction between the two Diabrotica species, they should be negligible and the results generalizable across Diabroticine beetles. Eggs of D. balteata were supplied by Syngenta Crop Protection (Stein, Switzerland) and larvae reared on a corn-based diet. Overall, the treatments followed a two-by-two factorial design experiment with the presence or absence of earthworms and root herbivores in each four-arm olfactometer (Fig. 2A). After three days, a solution of 2000 H. megidis infective juveniles was inoculated 5 mm below the sand surface in the middle of the central arena. After an additional 24 h, EPNs were retrieved from each side arm using the Baermann decantation funnel method. Next, roots were harvested, carefully washed and flash frozen in liquid nitrogen. Roots were ground in liquid nitrogen and Eβc production from each root system was measured using solid-phase microextraction (SPME) coupled to gas chromatography-mass spectrometry (GCMS) as described in Rasmann et al.5. Finally, for each plant, we scored root and shoot fresh biomass and plant height, measured as the longest leaf length from the ground. The same experiment consisting of 5 olfactometers each time was repeated four times for a total of N = 20 replicates.
A second (two-arm) olfactometer bioassay was performed in order to explore whether earthworm-emitted exudates via the epidermal mucus or faeces would directly interfere with the production of Eβc from maize roots, and the subsequent EPN movement. For this, maize seeds were sown in olfactometer glass pots (10 cm high, 5 cm diameter) with commercial substrate in similar conditions as described above (see Supplementary Fig. S3). Half of the plants were watered with tap water (control) while the other plants were watered with mucus solution. The mucus solution was obtained by caging 10 earthworms (adults and juveniles) in two 1 mm plastic mesh sieves in contact with each other’s open edge. The cage was submerged in 3 mm of tap water and left in darkness at room temperature (25 °C) for one hour, allowing earthworms to move into water and rub their skin against the sieve. The mucus of 20 earthworms (two cages) was collected to water 10 plants (equivalent of two earthworms per plant). The mucus solution (200 mL) was freshly prepared an hour before direct application onto the plants. All plants received the same volume of liquid at the same interval in order to keep substrate between plants as homogenously moist as possible. After 20 days, three second-instar D. balteata larvae were placed in every pot (controls and treatments) and left to feed on maize roots for three days. Connection with the olfactometer system was made 1 day before EPN inoculation, after which, a solution of 2000 infective juvenile EPNs was inoculated in the olfactometer central arena. After 24 h, EPNs were extracted from the two side arms of each olfactometer with the Baermann funnel method and counted under the microscope. Finally, root biomass and Eβc emissions were recorded as described above. The experiment was replicated 10 times.
A third (two-arm) olfactometer bioassay was performed to the test whether earthworm bioturbation activity in bulk soil affects nematode mobility alone, independently of earthworms being in contact with the maize root system. For this, soil-filled side arms and central arena and sand-filled terminal pots (10 cm high, 5 cm diameter) were assembled into a two-arm olfactometer (Fig. 4A). The same natural soil that was used for the mesocosm experiment at the Botanical Garden was used and sieved to 2 mm to ensure effective bioturbation and burrowing by earthworms. Ten soil samples were randomly taken from the soil stock, put on filter paper, emerged in water and left for decantation for 48 h to test the presence of any indigenous nematodes. None were observed and the soil was consequently not sterilized. Based on the study of Chiriboga et al.20 on diffusion of Eβc in different soil textures, soil and sand moisture were set respectively at 20% and 10% to maximise diffusion of volatiles. On the first day, three A. icterica earthworms were added to one half of olfactometer, and none in the other half. Earthworm bioturbation was restricted to half of the central arena by a 0.5 mm-mesh screen dividing it and by an anti-EPN mesh screen at the end of the side arms. Earthworms were left to work the soil for 4 days in climatic chamber (18 ± 2 °C, continuous darkness). Four days were considered enough time for three earthworms to properly burrow 0.5 L of soil. On day 5, the terminal pots were connected to olfactometer central system, and custom-made dispensers containing CO2 generating material (300 mg and sodium hydrogencarbonate and citric acid 3:1) and synthetic Eβc (300 μL, β-Caryophyllene, CAS Number 87-44-5, Sigma, St Louis, IL, USA) were prepared as described in Turlings et al.22, and inserted into the terminal pots filled with sand to ensure that EPN attraction was equally stimulated by both sides of the olfactometer (Fig. 4A). Five hours after inserting the dispensers, a suspension of 2000 H. megidis infective juveniles was inoculated in the central arena and left for 24 h, after which EPN presence in each arm was retrieved using Baermann funnels. The experiment was replicated eight times.
Statistical analysis
All statistical analyses were performed on R33.
Mesocosm outdoor experiment
We scored the probability of infection by dividing the number of larvae infected by EPNs around each plant by two. We then assessed the full interactive effect of culture type (two levels), earthworms (two levels), and root induction (two levels) on the probability of infection with generalized linear model analysis (GLM) with quasi-binomial distribution. We next performed the same GLM, but by comparing the interactive effect of earthworms and root induction, by splitting the data into monoculture and polyculture systems. Probabilities of infection scores were visualized using the library popbio34. The interactive effect of culture type and earthworms on CEC, CN, plant biomass and corn earcobs biomass was assessed using two-ways ANOVAs, followed by TukeyHSD post-hoc tests. For plant traits, we included mesocosm as a blocking effect in the model.
Four-arm olfactometer bioassay
Analysis of variation in nematode recruitment across earthworms by root herbivory treatments was performed using generalized linear model analysis (GLM) with quasi-Poisson distribution to take data overdispersion into consideration, and by including the experiment date as a blocking factor in the model. Differences among treatments were assessed using analyses of deviance and F statistics. The analysis of the effect of herbivores, earthworms and their interactions on log + 1-transformed Eβc emissions were performed using two-way ANOVA, and by including experiment as blocking factor, and root biomass as covariate in the model. Differences among treatments were assessed using Tukey HSD tests. The effect of root herbivory and earthworms on plant biomass accumulation was assessed on the first principal component analysis (PCA) axis that included plant height, root and shoot fresh biomass (Supplementary Fig. S4).
Mucus experiment
Analysis of variation in nematode recruitment across treatments was performed using GLM with quasi-Poisson distribution. Analysis of the effect of earthworm mucus on log + 1-transformed Eβc emissions and root biomass were performed using one-way ANOVAs.
Bioturbation and synthetic Eβc olfactometer bioassay
The analysis of variation in nematode recruitment across earthworm presence/absence treatment was performed using GLM with quasi-Poisson distribution, and followed by analysis of deviance. More

Specimens examined
Seven Australian genetic strains (clonal genotypes) of phylloxera were assessed in this project: G1, G4, G7, G19, G20, G30 and G38; genotype numbers follow14. These strains were obtained from live insect colonies maintained by Agriculture Victoria Rutherglen, Victoria. Root aphids, mealybugs and other non-target invertebrates were collected from vineyards for assay specificity testing. Additional aphids, from grass and vegetable host plants, as well as Phylloxeroidea (Adelges spp. and Pineus sp.) from pine trees, were also collected to create a comprehensive Aphidomorpha (Phylloxeroidea/Aphidoidea) species panel to test the specificity of the new LAMP assay.
Phylloxera DNA extractions and molecular variation
DNA was extracted from 100% ethanol preserved whole phylloxera adult, crawler, and eggs (destructive extraction method) using a DNeasy Blood and Tissue extraction kit (Qiagen, Australia), following the manufacturers protocol. The DNA concentrations were quantified using a Qubit 2.0 Flourometer (Invitrogen, Life Technologies, Australia) following the manufacturers protocol and stored at − 20 °C. These samples provided “clean” DNA preparations to use for phylloxera DNA barcoding, to assess Cytochrome Oxidase I (COI) genetic variation between strains, and for development of the LAMP assay.
All specimens, both phylloxera and the non-target invertebrates, were identified morphologically, prior to DNA extraction, followed by molecular identification using standard DNA barcoding methods7,17. DNA sequences from the 5′ region of the COI locus were obtained using LCO1490 / HCO2198 primers19. The thermal cycling conditions consisted of a PCR amplification profile of: 2 min at 94 °C, 40 cycles of at 94 °C for 30 s, 50 °C for 45 s and 72 °C for 45 s, and a final extension of 2 min at 72 °C.
PCR amplicons were sanger sequenced by Macrogen Inc. (Seoul, Korea) and sequences were compared to public databases (NCBI and BOLD), to determine similarity (i.e.  > 99% matches) with previously identified reference species. DNA sequences from the laboratory strains of phylloxera were used to develop phylloxera specific LAMP primers in this research.
An additional in-field compatible non-destructive DNA extraction method for “crude” DNA extractions was tested. Intact specimens of phylloxera (egg, crawler, and adult) were processed using a modified HotSHOT protocol “HS6” from20. There are several variations of HotSHOT method which have been used previously to extract DNA from mice (HS4)16 and Zebrafish (HS3)21. The HotSHOT (HS6) protocol has been refined to further improve the efficiency of extracted DNA by combining NaOH and Tris–EDTA buffer in a single step20. In our study twenty microlitres of HotSHOT extraction buffer consisting of 25 mM NaOH + TE buffer, pH 8.0 (Invitrogen , Australia) (1:1 volume) was pipetted into each 8 well strip of LAMP PCR tubes (OptiGene, UK). Phylloxera samples were removed from ethanol and air dried on a paper towel for approx.1 min. Single whole adult, crawler, single and/or multiple eggs were transferred with a toothpick (single use to prevent cross contamination), into each well. Each sample was immersed in HotSHOT buffer with up to six samples processed simultaneously in the portable real-time fluorometer (Genie III, OptiGene, UK). The protocol used the Genie III as an incubator at 95 °C for 5 min followed by  > 1 min incubation on ice. The DNA was quantified using a NanoDrop ND-1000 Spectrophotometer (Thermo Fisher, Australia) and stored at − 20 °C.
Development of the LAMP assay
LAMP primer design
Phylloxera specific primers were designed from an alignment of publicly available COI (5′-region) DNA sequences (from BOLD, http://www.boldsystems.org), which included (i) target (phylloxera) and (ii) non-target species that were either closely related to phylloxera (i.e. Phylloxeroidea), or aphids (Aphididae) known to utilise grapevines or plant roots as hosts (Supplementary Table 1).
For all primers, the GC content (%), predicted melting temperature (Tm), and potential secondary structure (hairpins or dimers) were analysed using the Integrated DNA Technologies (IDT) online OligoAnalyzer tool (https://sg.idtdna.com/calc/analyzer), using the qPCR parameter sets. Complete sets of LAMP primers were analysed together to detect potential primer dimer interactions using the Thermo Fisher Multiple Primer Analyzer tool (www.thermofisher.com). Potential within-species genetic variation in phylloxera LAMP primers was examined using combined DNA sequences of Australian phylloxera and reference sequences available on BOLD (i.e. the seven laboratory strains, plus 230 × 5′-COI reference sequences on BOLD, accessed May 2018).
A synthetic 221 bp DNA fragment, designed from the complete fragment used for the assay (Fig. 1), was synthesized as a gBlock (Integrated DNA Technologies, Iowa, USA) to provide a reliable LAMP positive control. This fragment consisted of all eight LAMP primer regions (Fig. 1) concatenated together, with the non-primer DNA removed from between primers and replaced a run of “ccc” DNA bases between the primer regions and at the ends of the fragment, to increase the overall GC content of the synthetic fragment.
LAMP primer ratio optimisation
Primer master mix was prepared following published protocols11. Optimisation of the six primer (Table 1) ratio and concentration for the phylloxera LAMP assay was also conducted according to11.
LAMP assay conditions
The phylloxera LAMP assay was performed following the same method as published11. All LAMP assays were run in the Genie III at 65 °C for 25 min followed by an annealing curve analysis from 98 °C to 73 °C with ramping at 0.05 °C/s. The total run time being approximately 35 min.
Comparison of molecular methods for phylloxera identification
Analytical sensitivity of the LAMP assay compared with qPCR
Clean DNA was extracted from phylloxera (3 adult samples were pooled for one DNA extraction) using the destructive method. A fourfold serial dilution of two biological replicates was prepared using ultrapure water. Starting DNA concentrations were quantified using a Qubit 2.0 Flourometer (Invitrogen, Life Technologies, Australia) following the manufacturers protocol. Phylloxera DNA was serially diluted from 1.0 ng/µL to 0.000061 ng/µL (1:1 to 1:16,384). Sensitivity of the LAMP assay was tested using the serially diluted DNA in the Genie III, following the same assay conditions as mentioned above. The time of amplification and anneal derivative temperature was recorded for all samples.
The same serial dilution of DNA extracts was also used in real-time qPCR assay. The primers and probe set (manufactured by Sigma, Australia) and cycling conditions used were as published9. Real-time qPCR was performed in QuantStudio 3 Real time PCR system (Thermo Fisher Scientific, Australia) in a total volume of 25 µL with technical replicates for each dilution. Each reaction mixture included 12.5 µL Platinum Quantitative PCR SuperMix-UDG (Invitrogen, Australia), 0.5 µM of each forward and reverse primers, 0.2 µM Taqman probe, 4 µL of template DNA and made up to 25 µL with RNA-free water. An NTC with 4 µl of water instead of DNA was included in each run to check for reagent contamination. The thermal cycling conditions consisted of a two-step denaturation: 2 min at 50 °C and 10 min at 95 °C, followed by 40 cycles of amplification in a two-step procedure: 95 °C for 15 s and 60 °C for 1 min. The mean Cq value (cycling quantification value) of the 8 dilutions were recorded for comparison with the time of amplification obtained from the LAMP assay.
Performance of non-destructive DNA samples (HotSHOT HS6)
DNA was non-destructively extracted from whole phylloxera egg, crawler, and adult (n = 3) using the “crude” HotSHOT HS6 protocol, as mentioned above. The extracted DNA was tested across the full range of molecular techniques available for detection of phylloxera: real-time qPCR (using 5 µL and 2 µL DNA per reaction)9, microsatellite genotyping14, DNA barcoding7, as well as the new LAMP assay. Real-time qPCR and DNA barcoding were performed as described above. Microsatellite genotyping was performed following a laboratory protocol (Blacket et al. unpublished) that screens the six phylloxera microsatellite loci used to define Australian genotypes5,14, modified to utilise fluorescently labelled primer tails following22 and a multiplex PCR kit (Qiagen, Australia). Capillary separation of fluorescently labelled microsatellites was performed commercially by AGRF (Melbourne), using LIZ-500 size standards. Genotyping of fsa files was performed using the microsatellite plugin in Geneious R11 (https://www.geneious.com).
Phylloxera specimens (adults and crawlers) were visually examined pre-DNA extraction, with slides prepared from specimens’ post-DNA extraction to confirm retention of key morphological characters. Microscopic slides were prepared following the protocols as published22 and were deposited as reference specimens in the Victorian Agricultural Insect Collection (VAIC) (https://collections.ala.org.au).
Evaluation of gBlock DNA fragment for use as synthetic DNA positive in LAMP assay
To evaluate detection sensitivity a tenfold serial dilution of the gBlock DNA fragment was prepared using ultrapure water (Invitrogen, Australia). Synthetic DNA was serially diluted from ~ 100 million copies down to ~ 10 copies (108 copies to 10 copies). Sensitivity of the LAMP assay was tested using the serially diluted synthetic DNA in the Genie III, following the same assay conditions as mentioned above (run time increased from 25 to 35 min). Following this another LAMP run was conducted to determine the best dilution to be used as synthetic DNA for positive template in LAMP assay. The same fourfold serial dilution (1.0 ng/µL to 0.00098 ng/µL) of clean phylloxera DNA prepared previously was used as template to compare with one hundred thousand copies (105) of synthetic DNA. The amount of phylloxera DNA was then calculated from the amplification time of 105 copies of synthetic DNA. More

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