Name: Infection of In Vivo and In Vitro Pines with the Pinewood Nematode Bursaphelenchus xylophilus and Isolation of Induced Volatiles (Video) | JoVE Journal
Uploaded: 2024-09-27T14:10:00.000+00:00
Duration: 8 min 42 s
Description: Learn to establish in vitro co-culture systems of pine shoots with Bursaphelenchus xylophilus for analyzing volatile organic compounds. This method enables controlled study of host-pathogen interactions.

ai_agent_IP

ai_agent

maintenance

instantTrialDurationMinutes

isBiopharma

enableNewMyJoveSectionDesign

educationFeatures

seoMetaApi

ai_copilot_institution_blacklist

ai_chat_institution_blacklist

freeAccessWindow

ssoRoleCapture

scheduledMaintenanceBanner

joveLabsFeatures

disable_autoplay_for_bots

extended_trial_duration

5 minutes

This research was partly funded by the EU under the PurPest project through grant agreement 101060634, and by Funda&#231;&#227;o para a Ci&#234;ncia e a Tecnologia (FCT), through projects NemACT, DOI 10.54499/2022.00359.CEECIND/CP1737/CT0002; NemaWAARS, DOI 10.54499/PTDC/ASP-PLA/1108/2021; CESAM UIDP/50017/2020+UIDB/50017/2020+ LA/P/0094/2020; CE3C, DOI 10.54499/UIDB/00329/2020; GREEN-IT, DOI 10.54499/UIDB/04551/2020 and 10.54499/UIDP/04551/2020.

Optimizing Pinewood Nematode Infection in Pinus pinaster

<ol><li>Back, M. A., Bonifácio, L., Inácio, M. L., Mota, M., Boa, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Pine+wilt+disease%3A+A+global+threat+to+forestry%5BTitle%5D)+AND+%22Plant+Pathol%22%5BJournal%5D)">Pine wilt disease: A global threat to forestry.</a> Plant Pathol. 73 (5), 1026-1041 (2024).</li>
<li>Mumm, R., Hilker, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Direct+and+indirect+chemical+defence+of+pine+against+folivorous+insects%5BTitle%5D)+AND+%22Trend+Plant+Sci%22%5BJournal%5D)">Direct and indirect chemical defence of pine against folivorous insects.</a> Trend Plant Sci. 11 (7), 351-358 (2006).</li>
<li>Carrasquinho, I., Lisboa, A., Inácio, M. L., Gonçalves, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Genetic+variation+in+susceptibility+to+pine+wilt+disease+of+maritime+pine+%28Pinus+pinaster+Aiton%29+half-sib+families%5BTitle%5D)+AND+%22Annals+Forest+Sci%22%5BJournal%5D)">Genetic variation in susceptibility to pine wilt disease of maritime pine (Pinus pinaster Aiton) half-sib families.</a> Annals Forest Sci. 75 (3), 85(2018).</li>
<li>Gaspar, M. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Impact+of+the+pinewood+nematode+on+naturally-emitted+volatiles+and+scCO2+extracts+from+Pinus+pinaster+branches%3A+a+comparison+with+P.+pinea%5BTitle%5D)+AND+%22J+Supercritical+Fluids%22%5BJournal%5D)">Impact of the pinewood nematode on naturally-emitted volatiles and scCO2 extracts from Pinus pinaster branches: a comparison with P. pinea.</a> J Supercritical Fluids. 159, 104784(2020).</li>
<li>Rodrigues, A. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Pinus+halepensis%2C+Pinus+pinaster%2C+Pinus+pinea+and+Pinus+sylvestris+essential+oils+chemotypes+and+monoterpene+hydrocarbon+enantiomers%2C+before+and+after+inoculation+with+the+pinewood+nematode+Bursaphelenchus+xylophilus%5BTitle%5D)+AND+%22Chem+Biodiver%22%5BJournal%5D)">Pinus halepensis, Pinus pinaster, Pinus pinea and Pinus sylvestris essential oils chemotypes and monoterpene hydrocarbon enantiomers, before and after inoculation with the pinewood nematode Bursaphelenchus xylophilus.</a> Chem Biodiver. 14 (1), e1600153(2017).</li>
<li>Faria, J. M. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+vitro+co-cultures+of+Pinus+pinaster+with+Bursaphelenchus+xylophilus%3A+a+biotechnological+approach+to+study+pine+wilt+disease%5BTitle%5D)+AND+%22Planta%22%5BJournal%5D)">In vitro co-cultures of Pinus pinaster with Bursaphelenchus xylophilus: a biotechnological approach to study pine wilt disease.</a> Planta. 241 (6), 1325-1336 (2015).</li>
<li>Espinosa-Leal, C. A., Puente-Garza, C. A., García-Lara, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+vitro+plant+tissue+culture%3A+means+for+production+of+biological+active+compounds%5BTitle%5D)+AND+%22Planta%22%5BJournal%5D)">In vitro plant tissue culture: means for production of biological active compounds.</a> Planta. 248 (1), 1-18 (2018).</li>
<li>Bonga, J. M., von Aderkas, P. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aBonga%2C+JM+%22In+Vitro+Culture+of+Trees%22">In Vitro Culture of Trees</a>. , Springer. Netherlands, Dordrecht. (1992).</li>
<li>Faria, J. M. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Nematotoxic+and+phytotoxic+activity+of+Satureja+montana+and+Ruta+graveolens+essential+oils+on+Pinus+pinaster+shoot+cultures+and+P.+pinaster+with+Bursaphelenchus+xylophilus+in+vitro+co-cultures%5BTitle%5D)+AND+%22Indus+Crops+Prod%22%5BJournal%5D)">Nematotoxic and phytotoxic activity of Satureja montana and Ruta graveolens essential oils on Pinus pinaster shoot cultures and P. pinaster with Bursaphelenchus xylophilus in vitro co-cultures.</a> Indus Crops Prod. 77, 59-65 (2015).</li>
<li>Vallarino, J. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Acquisition+of+volatile+compounds+by+gas+chromatography-mass+spectrometry+%28GC-MS%29%5BTitle%5D)+AND+%22Plant+Metabol.+Meth+Mol+Biol%22%5BJournal%5D)">Acquisition of volatile compounds by gas chromatography-mass spectrometry (GC-MS).</a> Plant Metabol. Meth Mol Biol. , 225-239 (2018).</li>
<li>Kikuchi, T., Aikawa, T., Oeda, Y., Karim, N., Kanzaki, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+rapid+and+precise+diagnostic+method+for+detecting+the+pinewood+nematode+Bursaphelenchus+xylophilus+by+loop-mediated+isothermal+amplification%5BTitle%5D)+AND+%22Phytopathology%22%5BJournal%5D)">A rapid and precise diagnostic method for detecting the pinewood nematode Bursaphelenchus xylophilus by loop-mediated isothermal amplification.</a> Phytopathology. 99 (12), 1365-1369 (2009).</li>
<li>Faria, J. M. S., Barbosa, P., Bennett, R. N., Mota, M., Figueiredo, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Bioactivity+against+Bursaphelenchus+xylophilus%3A+Nematotoxics+from+essential+oils%2C+essential+oils+fractions+and+decoction+waters%5BTitle%5D)+AND+%22Phytochemistry%22%5BJournal%5D)">Bioactivity against Bursaphelenchus xylophilus: Nematotoxics from essential oils, essential oils fractions and decoction waters.</a> Phytochemistry. 94, 220-228 (2013).</li>
<li>Nickle, W. R., Golden, A. M., Mamiya, Y., Wergin, W. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((On+the+taxonomy+and+morphology+of+the+pine+wood+nematode%2C+Bursaphelenchus+xylophilus+%28Steiner+%26Buhrer+1934%29+Nickle+1970%5BTitle%5D)+AND+%22J+Nematol%22%5BJournal%5D)">On the taxonomy and morphology of the pine wood nematode, Bursaphelenchus xylophilus (Steiner &Buhrer 1934) Nickle 1970.</a> J Nematol. 13 (3), 385-392 (1981).</li>
<li>Viglierchio, D. R., Schmitt, R. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((On+the+methodology+of+nematode+extraction+from+field+samples%3A+baermann+funnel+modifications%5BTitle%5D)+AND+%22J+Nematol%22%5BJournal%5D)">On the methodology of nematode extraction from field samples: baermann funnel modifications.</a> J Nematol. 15 (3), 438-444 (1983).</li>
<li>IPPC. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((IPPC+DP+10%3A+Bursaphelenchus+xylophilus%5BTitle%5D)+AND+%22Diagnostic+protocols%2C+International+standard+for+phytosanitary+measures%22%5BJournal%5D)">IPPC DP 10: Bursaphelenchus xylophilus.</a> Diagnostic protocols, International standard for phytosanitary measures. 27, 1-40 (2016).</li>
<li>Rodrigues, A. M., Carrasquinho, I., António, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Primary+metabolite+adjustments+associated+with+pinewood+nematode+Resistance+in+Pinus+pinaster%5BTitle%5D)+AND+%22Front+Plant+Sci%22%5BJournal%5D)">Primary metabolite adjustments associated with pinewood nematode Resistance in Pinus pinaster.</a> Front Plant Sci. 12, 10(2021).</li>
<li>Gonçalves, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Effect+of+Monochamus+galloprovincialis+feeding+on+Pinus+pinaster+and+Pinus+pinea%2C+oleoresin+and+insect+volatiles%5BTitle%5D)+AND+%22Phytochemistry%22%5BJournal%5D)">Effect of Monochamus galloprovincialis feeding on Pinus pinaster and Pinus pinea, oleoresin and insect volatiles.</a> Phytochemistry. 169, September 2019 112159(2020).</li>
<li>Schenk, R. U., Hildebrandt, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Medium+and+techniques+for+induction+and+growth+of+monocotyledonous+and+dicotyledonous+plant+cell+cultures%5BTitle%5D)+AND+%22Canadian+J+Botany%22%5BJournal%5D)">Medium and techniques for induction and growth of monocotyledonous and dicotyledonous plant cell cultures.</a> Canadian J Botany. 50 (1), 199-204 (1972).</li>
<li>Tereso, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Improved+axillary+and+adventitious+bud+regeneration+from+Portuguese+genotypes+of+Pinus+pinaster+AIT%5BTitle%5D)+AND+%22Propag+Ornam+Plants%22%5BJournal%5D)">Improved axillary and adventitious bud regeneration from Portuguese genotypes of Pinus pinaster AIT.</a> Propag Ornam Plants. 6 (1), 24-33 (2006).</li>
<li>Council of Europe European Directorate for the Quality of Medicines. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=%22European+Pharmacopoeia%22">European Pharmacopoeia</a>. , 241(2010).</li>
<li>Likens, S. T., Nickerson, G. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Detection+of+certain+hop+oil+constituents+in+brewing+products%5BTitle%5D)+AND+%22Proc+Ann+Meet+-+Am+Soc+Brewing+Chem%22%5BJournal%5D)">Detection of certain hop oil constituents in brewing products.</a> Proc Ann Meet - Am Soc Brewing Chem. 22 (1), 5-13 (1964).</li>
<li>Karimi, A., Gross, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Development+and+validation+of+an+innovative+headspace+collection+technique%3A+volatile+organic+compound+patterns+emitted+by+different+developmental+stages+of+Halyomorpha+halys%5BTitle%5D)+AND+%22Front+Horti%22%5BJournal%5D)">Development and validation of an innovative headspace collection technique: volatile organic compound patterns emitted by different developmental stages of Halyomorpha halys.</a> Front Horti. 3, (2024).</li>
<li>Koedam, A., Scheffer, J. J. C., Baerheim Svendsen, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Comparison+of+isolation+procedures+for+essential+oils+-+II.+Ajowan%2C+Caraway%2C+Coriander+and+Cumin%5BTitle%5D)+AND+%22Zeitschrift+f%C3%BCr+Lebensmittel-Untersuchung+und+-Forschung%22%5BJournal%5D)">Comparison of isolation procedures for essential oils - II. Ajowan, Caraway, Coriander and Cumin.</a> Zeitschrift für Lebensmittel-Untersuchung und -Forschung. 168 (2), 106-111 (1979).</li>
<li>Abdulra'uf, L. B., Hammed, W. A., Tan, G. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((SPME+fibers+for+the+analysis+of+pesticide+residues+in+fruits+and+vegetables%3A+A+review%5BTitle%5D)+AND+%22Crit+Rev+Anal+Chem%22%5BJournal%5D)">SPME fibers for the analysis of pesticide residues in fruits and vegetables: A review.</a> Crit Rev Anal Chem. 42 (2), 152-161 (2012).</li>
<li>Gross, J., Gallinger, J., Rid, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Collection%2C+identification%2C+and+statistical+analysis+of+volatile+organic+compound+patterns+emitted+by+phytoplasma+infected+plants%5BTitle%5D)+AND+%22Methods+Mol+Biol%22%5BJournal%5D)">Collection, identification, and statistical analysis of volatile organic compound patterns emitted by phytoplasma infected plants.</a> Methods Mol Biol. 1875, 333-343 (2019).</li>
<li>ISO. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((ISO+7609%3A1985+Essential+Oils-Analysis+by+Gas+Chromatography+on+Capillary+Columns-General+Method%5BTitle%5D)+AND+%22ISO%3A+Geneva%2C+Switzerland%22%5BJournal%5D)">ISO 7609:1985 Essential Oils-Analysis by Gas Chromatography on Capillary Columns-General Method.</a> ISO: Geneva, Switzerland. , (1985).</li>
<li>Rubiolo, P., Sgorbini, B., Liberto, E., Cordero, C., Bicchi, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Essential+oils+and+volatiles%3A+Sample+preparation+and+analysis.+A+review%5BTitle%5D)+AND+%22Flav+Fragr+J%22%5BJournal%5D)">Essential oils and volatiles: Sample preparation and analysis. A review.</a> Flav Fragr J. 25 (5), 282-290 (2010).</li>
<li>Fielding, N. J., Evans, H. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+pine+wood+nematode+Bursaphelenchus+xylophilus+%28Steiner+and+Buhrer%29+nickle+%28%3D+B.+lignicolus+Mamiya+and+Kiyohara%29%3A+An+assessment+of+the+current+position%5BTitle%5D)+AND+%22Forestry%22%5BJournal%5D)">The pine wood nematode Bursaphelenchus xylophilus (Steiner and Buhrer) nickle (= B. lignicolus Mamiya and Kiyohara): An assessment of the current position.</a> Forestry. 69 (1), 34-46 (1996).</li>
<li>Faria, J. M. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((First+report+on+the+synergistic+interaction+between+essential+oils+against+the+pinewood+nematode+Bursaphelenchus+xylophilus%5BTitle%5D)+AND+%22Plants%22%5BJournal%5D)">First report on the synergistic interaction between essential oils against the pinewood nematode Bursaphelenchus xylophilus.</a> Plants. 12 (13), 2438(2023).</li>
</ol>

We have nothing to disclose.

The protocol presented here outlines an enhanced methodology to analyze volatile compounds in maritime pine infected by the PWN, where environmental and genetic variability is reduced and does not influence the outcomes. Using pure lines of in vitro maritime pine genotypes, extracted and emitted volatiles can be analyzed as a host response to one of the most damaging biotic threats to pine forests.
Maintenance of reference cultures or the growth of large amounts of PWNs is easily performed in the lab by growing in vitro B. cinerea (non-sporulating strain) and using it as a food source for the nematode. This procedure is commonly very accessible, given the right conditions; however, when pipetting the PWN suspension, be sure to break the fungal colony surface. Otherwise, the PWNs will be retained in the water droplets. When successful, the fungal mycelium will be consumed, and PWNs will greatly multiply. Nematode sterilization is a critical step, but once sterile PWNs are obtained, the culture can be either maintained in B. cinerea, being then a monoxenic culture only contaminated with this fungus, or on the in vitro pine shoots6,29.
The use of greenhouse-grown 2-year-old P. pinaster seedlings has the advantage of simulating a closer condition to natural PWD infections; however, variability is harder to control3,5. In vitro co-cultures of P. pinaster shoots with the PWN are a refined working system that is more adequate for studies that require a finer control of environmental and biological variation6,9. However, their use is limited to studies on the tissue level, e.g., ultrastructural morphology or on biochemical regulation mechanisms under biotic or abiotic stress. In other fields, these systems offer a great potential for following minute changes in host metabolome, proteome, or transcriptome induced by the PWN in the first stages of infection.
In the establishment of in vitro pine shoots, the surface sterilization of pine seeds resorts to a strong biocide; however, optimizations can be performed with a variation on exposure time or biocidal agent. Pine seed coats are generally strong and can withstand pure ethanol for a longer period than weaker seeds. However, liquid diffuses inside the shell, so care must be taken that ethanol does not reach the embryo. In vitro, pine shoots can also be used for the maintenance or micropropagation of specific pine genotypes. Although harder to establish in an uncontaminated environment, non-seminal material can also be used to establish the in vitro shoot cultures. However, multiplication and growth may be slower.
Hydrodistillation and distillation-extraction procedures are volatile extraction techniques. The first will yield a pure extract of pine volatiles, called an essential oil; the second has the advantage of simultaneously using a solvent, so lighter molecules that are prone to escape due to high temperatures of boiling water can potentially be retained. These are extracts and do not always represent the volatile mixtures that plants emit into the environment. These&#160;headspace volatiles are more accurately sampled through solid-phase microextraction procedures, e.g., using coated fibers or packed tubes. Despite using boiling water as an extractive agent, which can induce the formation of artifacts, the hydrodistillation procedure has several advantages, e.g., a) the non-use of organic solvents, thus providing a pure extract, b) allowing yield determination, c) affording a chlorophyll-free extract, and d) providing, in addition to the essential oil, two other extracts, an hydrolate and decoction water, each of which with its own composition and characteristic bioactivity. The Likens-Nickerson apparatus is a special distillation-extraction unit that allows the simultaneous condensation of the hydrodistillate coming from the flask with plant material and of the organic solvent coming from the second flask. As the organic solvent is immiscible with, and lighter than, water, it is visible as the supernatant floating over the distilled water in the U-shaped central separatory trap above the apparatus stopcock. The fact that the distillate return-arms in the separatory trap are at different levels allows the distilled water to return to the flask with plant material and the extracting organic solvent to return to the second flask. n-Pentane changes from being transparent to translucent, indicating the distillate&#39;s presence. Solid phase microextraction is one of the several ways to collect headspace volatiles and can be adjusted to different types of applications and volumes of plant material in nature, in vials, in glass desiccators, and in PTFE bags, among others17. All these extraction procedures provide complementary information on the volatile profile. However, individual in vitro pine shoots are small and have a lower weight, and consequently lower amounts of volatiles when compared to pine seedlings. For in vitro pine shoots, distillation-extraction is more suitable to obtain extracted volatiles, while resin packed tubes are more suitable to trap emitted volatiles.

The pinewood nematode (PWN), Bursaphelenchus xylophilus (Steiner &#38; B&#252;hrer 1934)&#160;Nickle 1970, is a plant parasitic nematode that mainly parasitizes Pinus species. This phytoparasite is vectored by insects of the genus Monochamus into trees of susceptible pine species during the insect&#39;s maturation feeding. The PWN kills the tree by attacking its resin canals and reducing resin flow, and by damaging its vascular tissue, causing interruptions in the water column. Lack of water at the tree canopy induces the first visible symptoms of pine wilt disease (PWD), i.e., the pine needles become chlorotic after the cessation of photosynthesis and drooping due to desiccation. Pine generally responds to biotic and abiotic stress through the production of resin and volatile compounds1. Thus, understanding the mechanisms of pine defense is important to determine the specific effects of PWN attacks and to find alternative methods for pest control2.
Currently, experimentation under field conditions is dependent on the availability of infected pines, the confirmation of PWN infection, and variable environmental conditions. Under greenhouse conditions, these parameters can be more easily controlled; however, the genetic diversity of the host becomes a strong source of variability3. For example, in a study on the resistance response of Pinus pinaster, the production of the terpene limonene and resin acids was associated with PWN infection4. However, due to the small number of samples and the variability of natural conditions, detectable changes were only registered for half the samples. In another study using greenhouse-grown pine seedlings, even though environmental conditions were more easily controlled, natural pine genetic diversity induced a great variability in the volatiles extracted5. Since disease-induced pine volatiles can be greatly influenced by environmental and genetic variation, resorting to in vitro shoot cultures is a good alternative for studies on the chemical and biochemical response of pine tissue to PWN infection5,6. By propagating a plant genotype in vitro, its genetic makeup can be maintained and cloned indefinitely, leading to the establishment of a higher amount of genetically identical individuals in less space and under less time than in in vivo conditions. These cultures are a simple working system under easily manipulated nutritional and environmental conditions, so they offer additional advantages to conventional systems in the evaluation of the production and emission of volatiles7,8. These systems are particularly advantageous for research in woody species, which most of the time require substantial resources, i.e., target trees are sometimes located in hard-to-reach sites, require expensive equipment, a dedicated workforce, and longer time periods of analysis8. In vitro, co-cultures of pines with the PWN enable the assessment of metabolic interactions between the nematode and the plant at different stages9. For the analysis of volatiles, this is very important since profiling techniques have become highly accurate, and minute variations in sampling can result in substantial changes in volatile profiles. Gas chromatography coupled with mass spectrometry (GC-MS) is a powerful technique for the analysis of volatiles and allows a quick and streamlined profiling of volatiles10. The protocol presented here describes techniques for infecting in vivo pine seedlings under greenhouse conditions and in vitro shoot cultures of genetically identical pines optimized for the extraction and profiling of induced volatiles.

<table><tbody><tr><td>38 mesh test sieve</td><td>Retsch</td><td>60.131.000038</td><td></td></tr><tr><td>6-Benzylaminopurine (6-BAP)</td><td>Duchefa Biochemie</td><td>B0904</td><td></td></tr><tr><td>Charcoal activated</td><td>Duchefa Biochemie</td><td>C1302</td><td></td></tr><tr><td>Clevenger apparatus</td><td>WINZER Laborglastechnik</td><td>25-000-02</td><td></td></tr><tr><td>Hydrogen peroxide solution</td><td>Sigma-Aldrich</td><td>H1009-500ML</td><td></td></tr><tr><td>Indole-3-butyric acid (IBA)</td><td>Duchefa Biochemie</td><td>I0902</td><td></td></tr><tr><td>Likens-Nickerson apparatus</td><td>VitriLab LDA.</td><td>c/IN29/32</td><td></td></tr><tr><td>Microbox round containers</td><td>Sac O2</td><td>O118/80+OD118</td><td></td></tr><tr><td>&lt;em&gt;n&lt;/em&gt;-Pentane</td><td>Sigma-Aldrich</td><td>1.00882</td><td></td></tr><tr><td>PARAFILM M sealing film</td><td>BRAND</td><td>HS234526B-1EA</td><td></td></tr><tr><td>Phyto agar</td><td>Duchefa Biochemie</td><td>P1003</td><td></td></tr><tr><td>Potato Dextrose Agar</td><td>BD DIFCO</td><td>213400</td><td></td></tr><tr><td>Scalpel blade no. 24</td><td>Romed Holland</td><td>BLADE24</td><td></td></tr><tr><td>Schenk &amp;amp; Hildebrandt Basal salt medium</td><td>Duchefa Biochemie</td><td>S0225</td><td></td></tr><tr><td>Schenk &amp;amp; Hildebrandt vitamin mixture</td><td>Duchefa Biochemie</td><td>S0411</td><td></td></tr><tr><td>SPME fiber assembly Polydimethylsiloxane (PDMS)</td><td>Supelco</td><td>57300-U</td><td></td></tr><tr><td>SPME Fiber Holder</td><td>Supelco</td><td>57330-U</td><td></td></tr><tr><td>Sucrose</td><td>Duchefa Biochemie</td><td>S0809</td><td></td></tr><tr><td>Tenax TA- stainless steel tubes- conditioned + capped</td><td>Markes International</td><td>C1-AAXX-5003</td><td></td></tr></tbody></table>

infection of in vivo and in vitro pines with the pinewood nematode bursaphelenchus xylophilus and isolation of induced volatiles

Infection of In Vivo and In Vitro Pines with the Pinewood Nematode Bursaphelenchus xylophilus and Isolation of Induced Volatiles

The pinewood nematode (PWN) is a phytoparasite that causes pine wilt disease (PWD) in conifer species. This plant parasitic nematode has heavily contributed to pine deforestation in Asian countries, e.g., Japan, China, and Korea. Over the last two decades, in Europe, Portugal and Spain have been greatly affected. Research on the mechanisms of PWN infection and/or PWD progression in susceptible host species relies on the controlled infection of pine seedlings under greenhouse conditions. This technique is laborious and mobilizes substantial economic and human resources. Additionally, it can be prone to variability that results from the genetic diversity associated with some pine species but also from the interference of external factors. As an alternative, in vitro co-cultures of pine with PWNs offer a more advantageous system for studying biochemical changes since they a) allow controlling single environmental or nutritional variables, b) occupy less space, c) require less time to obtain, and d) are&#160;free from contamination or from host genetic variation. The following protocol details the standard in vivo PWN infection of Pinus pinaster, the maritime pine, and the establishment of the novel in vitro co-cultures of pine shoots with the PWN as an improved methodology to study this phytoparasite influence on pine volatiles. PWN-induced volatiles are extracted from in vivo and in vitro infected pines by hydrodistillation and distillation-extraction, and the emitted volatiles are captured by solid phase microextraction (SPME), using fiber or packed column techniques.

The PWN reproduces quickly under optimal conditions, and generation times can be as low as 4 days, with each female lying about 80 eggs during her life28. Using the methodology described above, large amounts of PWNs can be obtained depending on fungal growth. Within an 8-day growth period, PWNs can have a 100-fold increase in population numbers (Figure 1). To increase the consistency in the amounts of PWNs, use sterilized PWNs since contamination with unknown bacteria or fungi will negatively impact the PWN population.
The successful infection of pine seedlings with the PWN will result in symptoms of PWD at around 20 days after infection (Figure 2). However, P. pinaster is known to possess a high genetic variability on susceptibility to PWD, and variations on the time of symptoms onset are very common3.
In vitro pine shoots do not have this genetic variability, and environmental and nutritional conditions are controlled and set according to the user's needs. In vitro maritime pine shoot cultures grow well in SH elongation medium, showing a growth rate of about 0.9 mm per week6. Ideally, sterile PWNs can be added to shoots with at least 5 cm (from the base to the highest needle). Because in vitro pine shoots are smaller and the PWN infection area is reduced, nematode population growth is less expressive, so doubling times can be between 6 and 7 days6. Nevertheless, chlorotic needles can be observed within a month of co-culture (Figure 3). In comparison to greenhouse-grown pines, in vitro pine shoot cultures allow synchronization of PWD symptoms, control of temperature and photoperiod, control of nutritional status, and avoidance of contamination.
Two-year-old P. pinaster seedlings have an essential oil yield of around 0.2% ± 0.1% (v/fresh weight) and a volatiles profile rich in α-pinene, β-pinene or δ-3-carene, with regular chemical variability. Currently, two main chemotypes have been identified, one where α-pinene and β-pinene are dominant and another where α-pinene and β-pinene share dominance with δ-3-carene5. Maritime pine in vitro shoots can be cultured according to the desired chemotype, whose volatiles are fully profiled through GC-MS. While volatile profiles from volatiles obtained by distillation-extraction can be different from those of essential oils, in P. pinaster, they are similar (Figure 5A). Volatile extraction techniques are adequate for assessing induced changes in pine volatile metabolite profiles. Volatile trapping techniques give information on the volatiles emitted by pines. Chemical profiles from SPME techniques can be different, but differences are usually dependent on the amount of plant material and the volatiles trapped (Figure 5B,C).
<img alt="Fungal growth in beakers A and B, experimental setup for mycology research, mold colony formation." class="xfigimg" src="/files/ftp_upload/67149/67149fig01.jpg" data-altupdatedat="1788304762000" data-alt="Figure 1"> 
Figure 1: In vitro growth of pinewood nematode. (A) Non-sporulating Botrytis cinerea strain grown in sterilized hydrated barley seeds and (B) in vitro pinewood nematode culture after feeding on fungal mycelium. Scale bar = 1 cm. <a href="https://www.jove.com/files/ftp_upload/67149/67149fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Healthy vs wilted tree comparison; plant growth experiment results, showing effects of conditions." class="xfigimg" src="/files/ftp_upload/67149/67149fig02.jpg" data-altupdatedat="1788304762000" data-alt="Figure 2"> 
Figure 2: Greenhouse trials with pine seedlings. (A) A 2-year-old Pinus pinaster seedlings without and (B) with symptoms of Pine Wilt Disease. Scale bar = 10 cm. <a href="https://www.jove.com/files/ftp_upload/67149/67149fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Plant growth comparison; seedling health diagram; experiment with growth inhibitor effect observed." class="xfigimg" src="/files/ftp_upload/67149/67149fig03.jpg" data-altupdatedat="1788304762000" data-alt="Figure 3"> 
Figure 3: In vitro experiments with pine microshoots. Pinus pinaster in vitro shoot cultures (A) before and (B) after pinewood nematode infection. Chlorosis as a result of nematode infection can be seen in microshoot needles (arrows). Scale bar = 1.5 cm. <a href="https://www.jove.com/files/ftp_upload/67149/67149fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Fractional distillation setups A-C with condensers; D shows gas analysis experiment station." class="xfigimg" src="/files/ftp_upload/67149/67149fig04.jpg" data-altupdatedat="1788304762000" data-alt="Figure 4"> 
Figure 4: Setup for the different volatiles isolation systems. Isolation of (A) essential oils by hydrodistillation (Clevenger apparatus), (B) volatiles by distillation-extraction (Likens-Nickerson apparatus), and headspace volatiles by solid phase microextraction (SPME) with (C) fibers or (D) packed tubes. Scale bar = 10 cm. <a href="https://www.jove.com/files/ftp_upload/67149/67149fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Chromatography graph for terpene analysis showing α-Pinene, β-Pinene, Limonene; experimental results." class="xfigimg" src="/files/ftp_upload/67149/67149fig05.jpg" data-altupdatedat="1788304762000" data-alt="Figure 5"> 
Figure 5: Representative chromatograms of Pinus pinaster shoot volatiles. (A) essential oil, (B) volatiles collected by solid-phase microextraction fiber, and (C) emitted volatiles using packed tubes. Identified using a laboratory-built reference library. <a href="https://www.jove.com/files/ftp_upload/67149/67149fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

The protocol describes the in vivo and in vitro pinewood nematode infection of Pinus pinaster and their volatilome analysis through Gas Chromatography (GC) and GC coupled to Mass Spectrometry (GC-MS).

The pinewood nematode (PWN) is a phytoparasite that causes pine wilt disease (PWD) in conifer species. This plant parasitic nematode has heavily ...

infection-vivo-vitro-pines-with-pinewood-nematode-bursaphelenchus

Research

JoVE Journal

Biology

A protocol for investigating the mating behavior of the pinewood nematode, Bursaphelenchus xylophilus, is presented. Behavioral features of B. xylophilus are described in the mating process.

Observation and Quantification of Mating Behavior in the Pinewood Nematode, Bursaphelenchus xylophilus

Here, we introduce a detailed soaking method of RNA interference in Bursaphelenchus xylophilus to facilitate the study of gene functions.

Application of RNA Interference in the Pinewood Nematode, Bursaphelenchus xylophilus

Here we present a protocol for obtaining entomopathogenic fungi from a forest wood borer and a substitutive way to evaluate their entomopathogenic activities using a Coleopteran model insect. This method is efficient and convenient for exploring entomopathogenic fungal resources from wood-boring insect pests in natural forests.

Isolation, Behavioral Identification, and Pathogenicity Assessment of Entomopathogenic Fungi from a Forest Wood Borer

The present protocol describes a method for inducing the dispersal of third-stage juveniles (pre-dauer juveniles, JⅢ) of Bursaphelenchus xylophilus into cryptobiosis through osmotic regulation of potassium chloride (KCl) ions. This method can provide technical support for research on the stress resistance mechanisms of B. xylophilus.

Inducing the Entry of Third Stage Dispersal Juveniles of Bursaphelenchus xylophilus into Cryptobiosis Through Osmotic Regulation

The protocol describes infection of Solanum tuberosum roots with plant parasitic nematodes under in vivo greenhouse conditions and potato in vitro transgenic roots for histochemical analysis of root structure through optical microscopy.

In vivo and In vitro Infection of Potato Roots with Plant Parasitic Nematodes for the Assessment of Induced Structural Changes

Clathrin-mediated endocytosis depends on adaptor proteins that coordinate cargo selection and clathrin coat assembly. Here we describe procedures to study adaptor-clathrin physical interaction and live cell imaging approaches using as a model the yeast endocytic adaptor protein Sla1p.

In vivo and in vitro Studies of Adaptor-clathrin Interaction

A push-pull method for collecting plant volatiles is described.  The method allows for a comparison of volatiles induced by herbivore feeding, exogenous methyl jasmonate, and mechanical damage.  This technique is also used to investigate the volatile response of undamaged branches to exposure to volatiles from herbivore-damaged branches within blueberry plants.

Herbivore-induced Blueberry Volatiles and Intra-plant Signaling

A method to analyse the distribution of bone marrow hematopoietic progenitors in flow cytometry as well as to efficiently isolate highly purified hematopoietic stem cells (HSCs) is described. The isolation procedure is essentially based on magnetic enrichment of c-Kit+ cells and cell sorting to purify HSCs for cellular and molecular studies.

Phenotypic Analysis and Isolation of Murine Hematopoietic Stem Cells and Lineage-committed Progenitors

Primary hepatocytes provide a valuable tool to evaluate biochemical, molecular, and metabolic functions in a physiologically relevant experimental system.  We describe a reliable protocol for rat in situ liver perfusion, which consistently generates viable hepatocytes up to 1.0 × 108 cells per preparation with cell viability between 88 ~ 96%.

Isolation and Primary Culture of Rat Hepatic Cells

A technique to identify translational pause sites on mRNA is described. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA, followed by the size analysis of the nascent chains using a denaturing gel electrophoresis.

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

Learn to establish in vitro co-culture systems of pine shoots with Bursaphelenchus xylophilus for analyzing volatile organic compounds. This method enables controlled study of host-pathogen interactions.

Infection of In Vivo and In Vitro Pines with the Pinewood Nematode Bursaphelenchus xylophilus and Isolation of Induced Volatiles (Video) | JoVE Journal

Infection of In Vivo and In Vitro Pines with the Pinewood Nematode Bursaphelenchus xylophilus and Isolation of Induced Volatiles

Explore More Videos