Constitutive Chemical Compounds in Different Tissues of Seven Pine Species and Their Relationship with Susceptibility to Pine Wood Nematode ( Bursaphelenchus xylophilus ) †

: Pine wilt disease was detected in Galicia (North West of Spain) in 2010. Two-year-old seedlings of seven pine species were inoculated with B. xylophilus , and three different groups were stablished: non-susceptible ( P. canariensis , P. taeda , P. halepensis , and P. pinea ); susceptible ( P. pinaster , P. radiata ), and highly-susceptible ( P. sylvestris ). We aimed to determine the interspecific variation of constitutive compounds levels, groups and species, and their relationships with nematode multiplication and mortality. Needles of the non-susceptible group had significantly less water and more nitrogen, potassium, iron, and starch than the others groups; the cortex and phloem of the non-susceptible group had more nitrogen, phosphorus, manganese, and starch, and less potassium, calcium, iron, total polyphenols, condensed tannins and liposoluble substances than the highly susceptible group. The xylem of the non-susceptible group had more N, P, Mg, Mn, total polyphenols, and starch than the other groups. Higher levels of constitutive N and/or starch in any tissue was related with less mortality and nematode multiplication; higher P in the three tissues was also correlated with less nematode multiplication. Moreover, liposoluble substances, soluble carbohydrates and condensed tannin concentration in the needles were negatively correlated with nematode multiplication. On the contrary, in the needles, water content and K were positively correlated with mortality and nematode invasion.


Data analysis
Differences among susceptibility groups for the studied constitutive chemical compounds on three different tissues (needles, stem (bark and phloem), and stem xylem) were analyzed using a nested ANOVA following the model: X = µ i + R j + SPP k(j) + ξ l(ijk) , where R (group of susceptibility), with three levels (NS=non-to slightly susceptible (P.canariensis, P pinea, P. halepensis and P. taeda), S= susceptibility (P.pinaster and P. radiata), and HS=highly susceptible (P.sylvestris),;SPP (species, nested to R, with seven species, mentioned before.Duncan test was performed to determine differences among groups of susceptibility to every chemical compound analyzed in different tissues.Non-parametric Spearman correlation between average values of constitutive chemical compounds by species (n=7) and later wilt, mortality and nematode invasion were performed on every tissue.Principal components analysis of constitutive chemical compounds on different seedling tissues were performed.

Cortex + phloem Needles Xylem
Principal components analysis of constitutive chemical compounds on different seedling tissues.
Needles of non-susceptible group had significantly less water and more Nitrogen, Potassium, Iron, and starch than the others groups.
Cortex + phloem of non-susceptible group had more Nitrogen, Phosphorus, Manganese, and starch and less Potassium, Calcium, Iron, total polyphenols, condensed tannins and liposoluble substances than the highly susceptible group.
Xylem of non-susceptible group had more Nitrogen, Phosphorus, Magnesium, Manganese, total polyphenols, and starch than the other groups.
Higher levels of constitutive N and/or starch in any tissue was related to less mortality and nematode multiplication; Higher P on the three tissues was also correlated with less nematode multiplication.Moreover, liposoluble substances, soluble carbohydrates and condensed tannins concentrations on the needles were negatively correlated with nematode multiplication.On the contrary, needles water and K were positively correlated with mortality and nematode invasion.
Two-year-old seedlings of seven pine species were inoculated with B. xylophilus, and three different groups were stablished: non-susceptible (P.canariensis, P. taeda, P. halepensis, and P. pinea); susceptible (P.pinaster, P. radiata), and highly-susceptible (P.sylvestris) Chemical compound data were obtained for needles, branches, stem xylem and stem bark tissues from trees harvested prior to inoculation.Water content, condensed tannins, total polyphenols, lipid-soluble substances, macronutrients (N, P, K, Ca, and Mg) and some micronutrient (Fe and Mn) levels were determined as described Menéndez-Gutiérrez et al. (2018).
Soluble carbohydrate and starch analysis, non-described before, were determined as Chow and Landhansser ( 2004) and Dubois ( 1956), with some modification.Sugars were extracted in ultraturrax with (EtOH: H 2 O) (80:20) (v : v), centrifuged, and soluble carbohydrates were analyzed in extract by Dubois method as glucose, after ethanol was eliminated in rotary evaporator.Residue contained starch was hydrolyzed with H 2 SO 4 5N, and then, colorimetrically analyzed in the same manner as soluble carbohydrates.Results were expressed in mg glucose.g -1 lyophilized tissue.