Valeria Reva?Lu?′s Fonseca?Jose′L.Lousada?Isabel Abrantes?Anto′nio R.Figueiredo?Domingos X.Viegas

Basic density,extractive content and moisture sorption properties of Pinus pinaster wood infected with the pinewood nematode,Bursaphelenchus xylophilus

Valeria Reva?Lu?′s Fonseca?Jose′L.Lousada?Isabel Abrantes?Anto′nio R.Figueiredo?Domingos X.Viegas

The pinewood nematode(PWN),Bursaphelenchus xylophilus,has become one of the most severe threats to pine forest worldwide.Nematodes,migrating through resin canals and feeding on the living cells,induce rapid metabolic changes in ray parenchyma cells,create cavitation areas,decrease xylem water content and oleoresin exudation,and cause necrosis of parenchyma and cambial cells.This study focused on the impact of PWN infection on technological parameters of wood and evaluated the impact of anatomic and biochemical incidences of tree defense reactions on basic density,extractive content and moisture sorption properties of Pinus pinaster wood. Samples of infected and uninfected wood were studied. The presence of nematodes reduced wood basic density by 2%and decreased the total content of extractives ininfected wood as compared with uninfected(5.98 and 8.90%of dry wood mass,respectively).Extractives in infected trees had inverse distribution along the trunk as compared with uninfected trees.The adsorption isotherms for infected and uninfected wood had similar positioning. We recorded differences(some statistically significant)in the equilibrium moisture contentofinfected and uninfected wood under varying environmental conditions.Despite the verified differences in wood basic density,extractive content and moisture sorption properties,the overall conclusion is that the PWN had a slight impact on these characteristics of wood.

Pinewood nematodeWood densityExtractivesEquilibrium moisture contentEconomic impact

Introduction

The pinewood nematode(PWN),Bursaphelenchus xylophilus,the causal agent of pine wilt disease(PWD),has become one of the most serious threats to pine forest worldwide.A native of North America,where it is nonpathogenic for native conifer species,the PWN spread to Japan in the early 20th century and later into China,Korea and Taiwan(Zhao etal.2008).In 1999,this nematode was detected for the firsttime in Europe,in continentalPortugal (Mota et al.1999).Later it was detected in Spain and on Madeira Island,Portugal(Abelleira etal.2011;Robertson etal.2011;Fonseca etal.2012).The mostsusceptible host trees belong mainly to the genus Pinus,with affected species varying by geographic location:P.bunjeana,P. densiflora,P.luchuensis,P.massoniana and P.thunbergii, in Asia,and P.nigra,P.sylvestris and P.pinaster inEurope(Evans et al.1996;Vicente et al.2012).In Portugal,the PWN affects maritime pine,P.pinaster(Mota etal. 1999),which occupies 27%of the total forest area(AFN 2010).This is the main coniferous species used in pine timber,pulp and paper and resin production.

Naturaltransmission ofthe nematode from one hosttree to another requires an insect vector.Species of the genus Monochamus are the most important vectors of the nematode(Linit 1988;Sousa et al.2001,2002).Long range dissemination to non-native areas occurs as a result of human activity by tranport of non-manufactured wood and wood products carrying the nematode and/or the insect vector(Schrader and Unger 2003;Gu et al.2006;Jones et al.2008).

The spread of the PWN begins when maturing insectvectors feed on the twigs of healthy pine trees.After entering a new host tree,nematodes rapidly permeate the trunk migrating through resin canals and feeding on parenchyma cells surrounding the canals.Nematode attack induces various biochemical and anatomical changes,such as development of cavitation,plugging of tracheids(water conduits),a decrease in xylem water content and oleoresin exudation,metabolic changes in ray parenchyma cells and denaturation and necrosis ofparenchyma and cambialcells, leading to tree death in a period of a few months(Kuroda et al.1988;Kuroda 1991,2008).

The negative impactof the PWN,described in various studies undertaken in Japan,Canada,China and USA,is usually related to annualloss of timber and restrictions on the transport of susceptible wood and wood products, leading to signifi cant economic losses(Bergdahl 1988; Webster and Mota 2008;Mota and Vieira 2008;Vicente etal.2012).The presentstudy focuses on other important aspect such as the impact of PWN infection on the chemical and physical characteristics of wood.The understanding of the nematode impact on the wood properties is of greatimportance for the evaluation of the potential industrial resource.The impact of the PWN on the mechanical properties,gross calorific value(GCV) and chemical composition of PWN-infected P.pinaster wood were recently studied.A decrease of 4–13%in the main mechanical parameters of infected wood(basic density,axial compressive stress,modulus of elasticity and work to maximum bending load)was detected(Rodrigues et al.2010).The GCV of PWN-infected wood was lower than in uninfected wood(19.79 and 20.25 MJ kg-1,respectively)(Reva et al.2012).The differences in GCV,hydrogen and nitrogen contents were statistically significant.Carbon,oxygen,sulfur and ash contents did not differ statistically.

The objective ofthis research was to evaluate the impact of biochemicaland anatomicalchanges due to tree defense reactions on basic density,extractive contentand moisture sorption properties of PWN-infected P.pinaster wood by comparative analysis of these parameters in infected and uninfected wood.

The study of hygroscopicity of P.pinaster biomass is relevant to forest fire propagation.Fuel moisture content affects various fire behavior factors,such as the preheating and ignition of unburned fuels,rate of fire spread(or fire growth),rate ofenergy release,and production ofsmoke by burning and smoldering fuel(Nelson 2001).Residualsofpine forestexploitation,when they have no economic value(i.e., potentialuse in bioenergy production)due to high transport costs,are usually left behind.According to the Legislative Act95/2011(2011)imposed by the Portuguese authoritiesas regulatory measures to preventthe propagation of the nematode,residuals ofpine forestexploitation should be burned ormay be leftin the forestwhen chipped to dimensions less than 3 cm.In this context,presence of less-hygroscopic surface dead fuels mightinfluence surface fire behavior.

It is generally recognized and confirmed by various studies that extractives affect the sorption properties of wood(Nearn 1955;Higgins 1957;Spalt 1958;Wangaard and Granados 1967;Jankowsky and Galva?o 1979;Hernandez 2007).We analyzed the relationship between extractive contentand equilibrium moisture content(EMC) for infected and uninfected wood.

Materials and methods

Plant material

Pinus pinaster trees of the same age(approximately 40 years)showing PWD symptoms(browning/reddening of the needles)and without symptoms were cut and sampled in a planted pine forest in central Portugal (Oliveira de Hospital,Coimbra District).Cross-sections of the stem at 1.5,6 m and top and crown branches were collected.Nematode screening and identification was performed for all sampled trees since pine tree wilting can result from causes other than nematodes,and trees with no visible symptoms can either be healthy or infected by nematodes.Nematodes were extracted from wood samples of cross-sections of the stem and crown branches (100 g)using the tray method(Whitehead and Hemming 1965).B.xylophilus was identified on the basis of species-specific diagnostic morphological characters(Fonseca et al.2008;EPPO 2009)and quantified.As a result of the test of nematode screening and identification,wood samples of 14 trees,7 infected and 7 uninfected,were selected for analysis of wood basic density,extractive content and EMC.The numbers of PWN/100 g of wood for collected sections of infected trees are shown in Table 1.

Table 1 Number of pinewood nematodes(PWN)/100 g wood for wood samples collected from Bursaphelenchus xylophilus infected Pinus pinaster trees

Wood basic density

The test of wood basic density was performed for two diametrically opposed circular sectors that were cut from each stem cross-section.The mean density of the two sectors was taken as the final value of the cross-sectional density.The green volume of each of these samples was measured by the water displacement or immersion method (Haygreen and Bowyer 1982).

The samples were submerged for 24 h(hot and cold water was used to ensure full saturation).Samples were then removed from water,and the excess water was removed by absorbent paper.Green volume was determined by weighing a recipientvessel with water and with water plus the sample using an electronic balance with precision of 0.1 g.The mass of water displaced was converted to the volume of the sample(1 g of water is equalto 1 cm3).Then,the samples were oven dried at(100±3)C to constant mass,and the oven-dry mass was measured.

Wood basic density(BD,q,kg m-3)was calculated as the ratio of dry mass and green volume.The mean basic density for each tree(qtree)was calculated by Eq.(1):

where,q1.5mand q6-8mare the basic density ata tree height of 1.5 m and 6–8 m above the ground,respectively (Fonseca and Lousada 2000;Lousada et al.2008).

Extractive content

The content of extractives was determined for two diametrically opposed circular sectors thatwere cutfrom each stem cross-section.The mean value of extractive contentof two sectors was taken as a finalvalue ofextractives for the studied cross-section.

Wood samples were broken into chips and ground in a laboratory mill(Retsch-Mu¨hle)with a 2-mm mesh screen size.The moisture content of the samples was determined before extraction by the oven-drying technique.Sawdust samples of 5 g(humid basis)were used to perform the extractive content analysis carried out with a Soxhlet extraction apparatus and based on the norm Tappi204 cm-07 and Tappi207 cm-08 norms(TAPPI 2007,2008).Two organic(dichloromethane and ethanol)and a non-organic (distilled water)reagents were used(200 mL of each solvent).The extraction started with dichloromethane,followed by distilled water and ethanol,with period of extraction 10,12 and 10 h,respectively.To determine the quantity of extractives removed by dichloromethane and ethanol,a small amount of solvent with extractives was evaporated to dryness and the resulting residues were weighed.The quantity ofextractivesremoved by waterwas established by weighing a dry sample before and after extraction.The totalextractive contentwas calculated as a sum ofthe extractives obtained foreach reagentand related to the mass of dry sample.

Equilibrium moisture content

The EMC was determined using a climatic chamber Fitoclima 300 EDTU.Crown branches were broken into chips of 2–2.5 cm lengths.Samples of 10 g were prepared and tested until constant mass to determine,for each sample, the EMC.Tests were performed at15,20,25,30 and 35C (T),and 30,50,70,75 and 80%ofrelative humidity ofair (RH).The mean values of EMCs obtained were taken as final EMC values of infected and uninfected wood for studied parameters of T and RH.

Statisticalanalysis

Regression analysis was performed to evaluate the impact of the PWN on the basic density and extractive contentof P.pinaster wood,and to study the relationship between EMC and extractive content.The correlation coefficient matrix was determined for EMC and extractives for infected and uninfected wood.The Student’s t-test was applied to compare values of wood density,extractive content and EMC for infected and uninfected wood.The null hypothesis was that studied parameters for infected and uninfected wood are identical(H0:linf=luninfvs.H1:).The normality ofdistribution(Shapiro–Wilktest)and homogeneity of variances(Levene’s test)of data samples were verified before the application of the Student’s t-test.Statistical data analysis was performed with STATISTICA StatSoft Inc.software.

Results

Wood basic density

The basic density of infected and uninfected wood ranged from 394 kg m-3(stem,top)to 507 kg m-3(stem,1.5 m) and from 413 kg m-3(stem,top)to 507 kg m-3(stem, 1.5 m),respectively(Table 2).Wood basic density decreased along the tree stem(from base to top)for both infected and uninfected wood,however,infected trees had lower density values at 6-8 m and on the top of the stem. Nematode attack induced 2%variation in mean basic density of trees(466 kg m-3for infected and 475 kg m-3for uninfected).Number of nematodes(PWN/100 g wood) was not significantly correlated with wood basic density (r=-0.31,p=0.22).

Extractive content

Extractives include a wide range of chemical compounds which can be extracted from wood by organic(dichloromethane and ethanol-benzene)or non-organic solvents (cold or hot distilled water)(TAPPI 2007,2008).Organic solvents remove waxes,fats,resins,sterols,non-volatile hydrocarbons,low-molecular-mass carbohydrates,salts, and polyphenols.Non-organic solvent removes tannins, gums,sugars and colouring matter.

Substances extracted by dichloromethane and water represent the main part of the total extractive content for both infected and uninfected wood(Table 2).For uninfected trees,the total extractive content decreased continuously along the tree(base-to-top direction)from 9.59 to 8.42%,while for infected trees,the extractives had an inverse distribution,increasing from 5.47 to 6.64%.In general,the total extractive content in infected trees (5.98%)was less than that of uninfected trees(8.90%). The differences in the total extractive content and in the content of water-and ethanol-soluble compounds of infected and uninfected wood were statistically significant at p