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 &lt;i&gt;Pinus pinaster &lt;/i&gt;

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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. 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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. 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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 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

324 Views.  INIAV, I.P., National Institute for Agrarian and Veterinary Research, I.P.. 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 comi...