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.

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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 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><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...

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

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