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.

1. Growing in vitro pinewood nematode
NOTE: Pinewood nematodes are grown by feeding on the fungal mycelium of a non-sporulating strain of Botrytis cinerea (de Bary) Whetzel11.
<ol>
	<li>For routine sub-culture, transfer a culture plug (0.5 cm diameter) from the outermost border of the fungal colony onto a plate of sterile potato dextrose agar (PDA) and keep at 25 &#177; 1 &#176;C for 7 to 10 days, or until the fungal colony ...

Optimizing Pinewood Nematode Infection in &lt;i&gt;Pinus pinaster &lt;/i&gt;

<ol>
	<li>Back, M. A., Bonif&#225;cio, L., In&#225;cio, M. L., Mota, M., Boa, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pine+wilt+disease:+A+global+threat+to+forestry.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+and+indirect+chemical+defence+of+pine+against+folivorous+insects.">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., In&#225;cio, M. L., Gon&#231;alves, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+variation+in+susceptibility+to+pine+wilt+disease+of+maritime+pine+(Pinus+pinaster+Aiton)+half-sib+families.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+the+pinewood+nematode+on+naturally-emitted+volatiles+and+scCO2+extracts+from+Pinus+pinaster+branches:+a+comparison+with+P.+pinea.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=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.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+co-cultures+of+Pinus+pinaster+with+Bursaphelenchus+xylophilus:+a+biotechnological+approach+to+study+pine+wilt+disease.">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., Garc&#237;a-Lara, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+plant+tissue+culture:+means+for+production+of+biological+active+compounds.">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://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> In Vitro Culture of Trees. , (1992).</li><li>Faria, J. M. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&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.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acquisition+of+volatile+compounds+by+gas+chromatography-mass+spectrometry+(GC-MS).">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+and+precise+diagnostic+method+for+detecting+the+pinewood+nematode+Bursaphelenchus+xylophilus+by+loop-mediated+isothermal+amplification.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioactivity+against+Bursaphelenchus+xylophilus:+Nematotoxics+from+essential+oils,+essential+oils+fractions+and+decoction+waters.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+taxonomy+and+morphology+of+the+pine+wood+nematode,+Bursaphelenchus+xylophilus+(Steiner+&amp;Buhrer+1934)+Nickle+1970.">On the taxonomy and morphology of the pine wood nematode, Bursaphelenchus xylophilus (Steiner &amp;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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+methodology+of+nematode+extraction+from+field+samples:+baermann+funnel+modifications.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> DP 10: Bursaphelenchus xylophilus.Diagnostic protocols, International standard for phytosanitary measures.. 27, 1 (2016).</li><li>Rodrigues, A. M., Carrasquinho, I., Ant&#243;nio, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primary+metabolite+adjustments+associated+with+pinewood+nematode+Resistance+in+Pinus+pinaster.">Primary metabolite adjustments associated with pinewood nematode Resistance in Pinus pinaster.</a> Front Plant Sci. 12, (2021).</li><li>Gon&#231;alves, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+Monochamus+galloprovincialis+feeding+on+Pinus+pinaster+and+Pinus+pinea,+oleoresin+and+insect+volatiles.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Medium+and+techniques+for+induction+and+growth+of+monocotyledonous+and+dicotyledonous+plant+cell+cultures.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+axillary+and+adventitious+bud+regeneration+from+Portuguese+genotypes+of+Pinus+pinaster+AIT.">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://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> European Pharmacopoeia. , 241 (2010).</li><li>Likens, S. T., Nickerson, G. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+certain+hop+oil+constituents+in+brewing+products.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+validation+of+an+innovative+headspace+collection+technique:+volatile+organic+compound+patterns+emitted+by+different+developmental+stages+of+Halyomorpha+halys.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+isolation+procedures+for+essential+oils+II.+Ajowan,+Caraway,+Coriander+and+Cumin.">Comparison of isolation procedures for essential oils II. Ajowan, Caraway, Coriander and Cumin.</a> Zeitschrift f&#252;r 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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SPME+fibers+for+the+analysis+of+pesticide+residues+in+fruits+and+vegetables:+A+review.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collection,+identification,+and+statistical+analysis+of+volatile+organic+compound+patterns+emitted+by+phytoplasma+infected+plants.">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>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> ISO 7609:1985 Essential Oils-Analysis by Gas Chromatography on Capillary Columns-General Method. , (1985).</li><li>Rubiolo, P., Sgorbini, B., Liberto, E., Cordero, C., Bicchi, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Essential+oils+and+volatiles:+Sample+preparation+and+analysis.+A+review.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+pine+wood+nematode+Bursaphelenchus+xylophilus+(Steiner+and+Buhrer)+nickle+(=+B.+lignicolus+Mamiya+and+Kiyohara):+An+assessment+of+the+current+position.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=First+report+on+the+synergistic+interaction+between+essential+oils+against+the+pinewood+nematode+Bursaphelenchus+xylophilus.">First report on the synergistic interaction between essential oils against the pinewood nematode Bursaphelenchus xylophilus.</a> Plants. 12 (13), 2438 (2023).</li></ol>

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 perf...

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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>n-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 &#38; Hildebrandt Basal salt medium</td>
			<td>Duchefa Biochemie</td>
			<td>S0225</td>
			<td></td>
		</tr>
		<tr>
			<td>Schenk &#38; 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

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.

Author Spotlight: In Vitro Co-Culture System of Pine Shoots and Pinewood Nematode for Studying Host Volatile Response

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

We have established an In Vitro co-culture system of Pine shoots with the Pinewood nematodes to market and study changes in host volatile response to these phytoparasites. Greenhouse biohouses allow control of environmental variability. However, the genetic diversity of Pine seedlings still influences the resulting volatile profiles.In Vitro Pine shoots infected with the Pinewood nematodes allow sampling of easily obtainable plant clones, which reduces the influence of variability coming from Pine genetic diversity. Plant volatiles are isolated by hydrodistillation in distillation extraction, but headspace techniques allow the profiling of volatiles emitted by the plant. Resin-packed columns will be used as a non-invasive technique to profile volatiles emitted by In Vitro ground Pine infected with Pine nematode for direct analysis of the chemical interplay between the host and the phytoparasite.

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...

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

In Vitro Cultivation and Maintenance of Pinewood Nematodes Using Botrytis cinerea

To begin, transfer a culture plug of botrytis cinerea from the outermost border of the colony onto a sterile potato dextrose auger plate. Incubate the plate at 25 degrees Celsius for seven to 10 days, or until the fungal colony reaches the edge of the plate. Culture B cinerea in steam-sterilized, hydrated, certified organic barley grains for seven to 10 days.Or until the surface of the cereal is fully colonized. Next, inoculate the B cinerea culture with a Pinewood nematode suspension. Incubate the flask in the dark for seven to 10 days, or until the fungal colony is completely consumed and the nematode climbs the flask walls.To isolate Pinewood nematodes, rinse the walls of the flask with tap water. Then pour the contents onto a paper towel, placed in a Baermann funnel or tray, and immerse the nematodes in water. After 24 to 48 hours, recover the nematodes using a 38 micrometer mesh sieve.Wash the accumulated nematodes into a container with tap water. Using this methodology, the Pinewood nematode population increased 100 fold within an eight day growth period.

In Vivo Infection Process of Pinus pinaster Seedlings with Pinewood Nematodes

To begin, set suspensions of mixed life-stage pinewood nematodes to 1000 nematodes per milliliter. Count the nematodes on a concave slide under the binocular microscope with up to 40x magnification. Before inoculating nematodes, manually remove needles from a section below the upper five centimeters of the pine stem, and make a superficial longitudinal incision using a sterilized scalpel.At the wounding zone, place a piece of sterilized cotton to hold 0.5 milliliters of pinewood nematode suspension, and fix it with a transparent strip to maintain humidity. Maintain the greenhouse in humid conditions and water frequently. Follow pine wilt disease progression regularly, and score symptomatology by quantifying the percentage of discolored and wilted needles.At 20 days after inoculation, cut the seedlings at the base of the stem for volatiles extraction. Place the seedlings in brown paper bags and freeze at minus 20 degrees Celsius. The successful infection of pine seedlings with the pinewood nematode resulted in symptoms of pine wilt disease at around 20 days after infection.

Establishment and Infection of In Vitro Pinus pinaster Shoot Cultures with Pinewood Nematodes

To begin, wash certified Pinus pinaster seeds in running tap water to remove larger debris. Then wash the finer debris using a common detergent solution with vigorous agitation. In a flow hood, add a commercial bleach solution until the seeds are covered and mix vigorously for 15 minutes.After discarding the bleach solution, rinse the seeds three times with sterilized tap water. Now immerse the seeds in 80%ethanol for 15 minutes with vigorous agitation. After disposing of ethanol, wash the seeds three times with sterilized ultrapure water.For pine germination, break the sterilized seed coats with a sterile mechanical lathe. Isolate the pine nuts and set them in a closed container with sterilized wet filter paper to hydrate overnight. The next day, stratify hydrated pine nuts at four degrees Celsius for seven days to break dormancy and synchronize germination.Then maintain the pine nuts in the dark at 25 degrees Celsius until germination. Transfer the aseptic germinated pines to controlled environmental conditions for two weeks, or until the main stem begins developing. After two weeks, cut the aerial portion of the seedling above the root with a scalpel and transfer it to a sterilized Schenk Hildebrandt culture medium.Maintain the culture in controlled environmental conditions as described previously. Subculture periodically by cutting the callous tissue at the base of the shoot with a sterile scalpel. Transfer the shoot cluster using sterile tweezers to a fresh multiplication culture medium.Next, inoculate the microshoots into the Schenk Hildebrandt culture medium containing three grams per liter of activated charcoal for micro stem elongation. Now place the elongated shoots in the Schenk Hildebrandt culture medium without phytohormones or activated charcoal. Add 100 to 150 mixed life stage sterilized pinewood nematodes at the bottom of the microshoot.Maintain the culture in controlled environmental conditions as described previously. The establishment and infection of in-vitro pine shoots showed the appearance of chlorotic needles within a month of co-culture with pine wood nematodes.

Isolation of Volatile Compounds from Pinus pinaster Seedlings Using Headspace Trapping with Packed Tubes

To begin, prepare a setup for trapping headspace volatiles with packed stainless steel traps. Place pine material set in covered glassware or inside PET bags to accumulate headspace volatiles. For a closed-loop system, connect the pump outlet to the PTFE tubing.Then attach the PTFE tubing to activated charcoal air filters. Connect the air filters to additional tubing leading to the glassware or PET bags with the pine material. Next, attach the packed tube to the glassware or PET bag outlet and connect additional PTFE tubing from the stainless steel tube outlet to the mass flow pump inlet.Using a mass flow pump, pump 100 liters of filtered air through the pine system at 0.6 liters per minute to collect volatiles. After collecting volatiles, close the packed tubes at both ends with airtight storage caps. Finally, rinse all PTFE tubing in glassware with 70%ethanol and heat in an oven at 230 degrees Celsius for two hours.Volatile profiles of maritime pine in vitro shoots obtained through hydro distillation or distillation extraction were similar within the chemotype analyzed. Chemical profiles obtained through solid phase micro extraction techniques showed variations depending on the amount of plant material and volatiles trapped.

242 Views.  INIAV, I.P., National Institute for Agrarian and Veterinary Research, I.P..  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).

Optimizing Pinewood Nematode Infection in Pinus pinaster

Summary