Strong Induction of Minor Terpenes in Italian Cypress, Cupressus sempervirens, in Response to Infection by the Fungus Seiridium cardinale

Seiridium cardinale, the main fungal pathogen responsible for cypress bark canker, is the largest threat to cypresses worldwide. The terpene response of canker-resistant clones of Italian cypress, Cupressus sempervirens, to two differently aggressive isolates of S. cardinale was studied. Phloem terpene concentrations, foliar terpene concentrations, as well as foliar terpene emission rates were analyzed 1, 10, 30, and 90 days after artificial inoculation with fungal isolates. The phloem surrounding the inoculation point exhibited de novo production of four oxygenated monoterpenes and two unidentified terpenes. The concentrations of several constitutive mono- and diterpenes increased strongly (especially α-thujene, sabinene, terpinolene, terpinen-4-ol, oxygenated monoterpenes, manool, and two unidentified diterpenes) as the infection progressed. The proportion of minor terpenes in the infected cypresses increased markedly from the first day after inoculation (from 10 % in the control to 30–50 % in the infected treatments). Foliar concentrations showed no clear trend, but emission rates peaked at day 10 in infected trees, with higher δ-3-carene (15-fold) and total monoterpene (10-fold) emissions than the control. No substantial differences were found among cypresses infected by the two fungal isolates. These results suggest that cypresses activate several direct and indirect chemical defense mechanisms after infection by S. cardinale.


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Fungal pathogens infect trees by using enzymes, toxins, growth regulators, and by 60 obtaining nourishment from the substances produced by the host. Conifers make use ºC for 10 min, injected into the column with a transfer line at 250 ºC, and submitted to 249 the same chromatographic process described above for the analysis of terpene 250 concentrations.

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No diterpenes were used as standards for the analyses of emission rates 252 because they are not volatile at ambient temperature. The terpene emission rates were 253 expressed in µg g -1 (dry weight (dw)) h -1 . Even though the days of sampling were 254 similar (sunny and warm), the terpene emission rates were standardized at 30 ºC using 255 an algorithm for terpene-storing species (Guenther et al. 1993): 256 257 A u t h o r ' s a c c e p t e d m a n u s c r i p t infected treatments (Mv and Hv) 30 and 90 days after inoculation. These six de novo 287 terpenes were found in all four cypress genotypes. Four of these were oxygenated 288 monoterpenes: oxygenated monoterpene de novo 1 (detected in 15 of 16 samples of 289 Mv and Hv at days 30 and 90, 0.093±0.02 mg g -1 , mean±SE), sabinene hydrate (16/16; 290 0.17±0.03 mg g -1 ), camphor (10/16; 0.16±0.04 mg g -1 ), and α-terpineol (13/16; 0.36±0.1 291 mg g -1 ). The monoterpene de novo 2 (14/16; 0.11±0.04 mg g -1 ) and the diterpene de 292 novo 3 (6/16; 5.4±1.7 mg g -1 ) could not be identified. No differences in concentration 293 were detected between treatment or time for the de novo compounds (REML, 294 fixed=treatment, random=genotype, paired Tukey's post-hoc test, P < 0.05). Thymyl 295 methyl ether (another oxygenated monoterpene) did not appear in the control but was 296 detected in some of the Wounded samples and in all infected treatments from day 10 297 to day 90, reaching a mean concentration of 2.9±1.2 mg g -1 in Hv at day 30 (Table 1).

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Quantitative Differences among Treatments. Total concentrations were lower in the 300 infected treatments than in the control at days 1 and 10 but increased substantially 301 after day 30 (Table 1). Total terpenes were nearly 4-fold higher in the infected 302 treatments compared to control at day 30, and reached a maximum of 140 mg g -1 at 303 day 90 (Table 1). This increase in total terpenes was due partly to increased 304 concentrations of some of the most abundant compounds (α-pinene, diterpene 1) but 305 also to the strong increases in concentrations of several minor compounds. These 306 changes led to a decrease in the proportions of the main compounds. α-Thujene was 307 among the most induced compounds in the infected treatments (up to a 57-fold 308 increase relative to the control), and presented differences from day 10, with 309 concentrations and proportions rising steadily until day 90. Next in order of retention 310 time was sabinene, whose concentrations (60-fold increase) had begun to differentiate 311 by day 10 and whose proportions peaked between days 10-30, and then dropped 312 slightly by day 90 (Fig. 1). Terpinolene concentrations (18-fold increase) had higher statistical differences among treatments. Only cedrol exhibited differences, with Mv 328 higher than Wounded and Hv (REML, fixed=treatment, random=genotype, paired 329 Tukey's post-hoc test, P < 0.05) ( Table 1). δ-3-Carene had a higher proportion in

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Wounded than in all other treatments, and terpinolene, the minor monoterpenes (sum 331 of all monoterpenes except α-pinene and δ-3-carene), and diterpene 2 had higher 332 proportions in the infected treatments than in the control or Wounded (

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proportions also were higher in the infected trees for α-thujene, sabinene, β-myrcene, 367 limonene, terpinolene, terpinen-4-ol, oxygenated, minor and total monoterpenes, β-368 cedrene, manool, and diterpenes 2 and 6. In contrast, longifolene, total sesquiterpenes, opposite trend, having higher proportions in the control or Wounded than in the infected 371 treatments (Table 1). No differences were found among the control trees from days 1 to 372 90, except for total diterpene concentrations at day 90, which were higher than on other 373 sampling days.

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Two PCAs (Fig. 4) were conducted with phloem monoterpene concentrations 376 and monoterpene proportions on days 30 and 90 as variables, to provide a general 377 overview of the differences among treatments and infection times. In the concentration 378 PCA, the first two PCs accounted for 69.1% and 11.0% of the total variance, 379 respectively. PC1 distributed the cases by terpene concentration, separating Hv and and PC2 significantly separated the cases of day 30 from those of day 90 (P < 0.05). In 382 the proportion PCA, the first two PCs accounted for the 36.3% and 20.4% of the total 383 variance, respectively. PC1 significantly (P < 0.05) separated the cases with decreased 384 proportion of main terpenes and increased proportion of minor terpenes, and PC2 also 385 separated the cases of day 30 and day 90 (P < 0.05).

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Fungal Isolates. Mv and Hv did not elicit clearly different reactions. Statistically 388 significant differences between terpene concentrations in the infected treatments were 389 observed only for two sesquiterpenes. Cedrol was significantly higher in Mv than in Hv 390 at day 1, and cedrol and β-cedrene were higher in Hv than in Mv at day 90 (Table 1).

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Foliar Terpene Concentration. Leaves also presented abundant terpenes, with high 393 concentrations of monoterpenes, moderate abundances of sesquiterpenes, and traces 394 of diterpenes. No qualitative differences were found among treatments, and few 395 quantitative differences in concentrations were observed ( Table 2).

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No correlation was found between the concentrations (  Fig. 5). No qualitative differences were found, but some 412 quantitative differences appeared. The largest differences were in total monoterpene 413 emissions and δ-3-carene (REML, fixed=treatment, random=genotype, paired Tukey's 414 post-hoc test, P < 0.05), which were higher for the infected trees at day 10 than the 415 control and Wounded. The proportions did not show any clear trend (data not shown).

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At day 1, the emission rates of β-myrcene and limonene were higher in 418 Wounded than in the control (Table 3). At day 10, δ-3-carene had a higher emission 419 rate in Hv than the control and a marginally higher emission rate than in Wounded. α-

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Cedrene also had a marginally higher emission rate in Hv than in the control. Total 421 monoterpenes showed higher emission rates in infected treatments than in the control.

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In contrast, the emission rate of β-pinene was marginally higher in the control than in 423 Wounded. All compounds, except β-myrcene and δ-3-carene, had the highest emission 424 rates in the Hv treatment at day 10. At day 30, differences were observed only in control than in Wounded, and Mv, and β-pinene had a higher emission rate in Mv than 429 in Hv (Table 3). Hv tended to elicit higher emissions and larger differences (sometimes 430 statistically significant) relative to the control and Wounded than did Mv (Table 3,

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The de novo compounds we found could, thus, likely be antifungal phytoalexins 472 because i) sabinene hydrate, camphor, and α-terpineol appeared exclusively in the 473 infected treatments, ii) they are oxygenated monoterpenes, iii) their antifungal activity 474 has been reported in literature (especially α-terpineol), and iv) the report by Madar et al.

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The absence of differences between Mv and Hv suggests that C. sempervirens 552 cannot distinguish between these two S. cardinale isolates. The short time period that 553 this conifer and fungus have coexisted suggests that co-evolution or a capacity to elicit 554 specific responses in their interactions is unlikely. Hv tended to elicit slightly (non-555 significantly) higher reactions compared to Mv, but probably due to the aggressiveness 556 of the isolate and not to a specific reaction of the tree against it. Further study should 557 compare the terpene reaction of C. sempervirens to different canker species or similar 558 fungal pathogens to determine if the tree reaction elicited by S. cardinale is species-559 specific or just a general pathogen defense.

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The main mechanism of reaction to S. cardinale infections in cypresses is 562 based on formation of a necrophylactic periderm, a quantitative (polygenic) trait that in 563 resistant trees is able to compartmentalize and prevent fungal growth in bark tissues. particular fungus but is the same that is activated by cypresses as a consequence of a 568 simple wound (without infection). This mechanism is disturbed by an invading fungus in 569 infected trees. The production of inhibiting terpenes induced by infection in more