The Influence of Pine Volatiles on the Growth of an Ophiostomatoid Fungi Associated with Pine Wilt Disease in Pinus pinaster †

: Phytopathogenic ophiostomatoid fungi play an important role in pine wilt disease (PWD), caused by the pinewood nematode (PWN), since they begin proliferating once the pine hosts decay and can serve as a food source for the PWN. In a recent study, the ophiostomatoid fungi that are associated with naturally infected Pinus pinaster were profiled and cultured. To understand the influence of volatiles that are commonly emitted by pines on fungal growth, the present work aimed at analyzing the influence of α -pinene, β -pinene and trans - β -caryophyllene on a Leptographium isolate. The volatiles promoted fungal growth in the first 24 h, but lost their effect after 48 or 72 h, probably due to compound volatilization. After 5 days, the fungal growth was comparable to that of control cultures, except for α -pinene, which appeared to slightly inhibit fungal growth. Profiling the influence of volatile organic compounds on the PWD complex can contribute to a better understanding of the chemical communication that is occurring between its different intervenients.


Introduction
Ophiostomatoid fungi are a non-taxonomical group of pathogenic fungi, vectored by conifer bark beetles.This interaction between fungi and insect can be considered mutualist, since the fungi benefit from a faster and wider spread, while the insect's survival is improved because the development of some ophiostomatoid fungi damages the host conifer's defenses and provides the insect with essential nutrients [1].
In the pine wilt disease (PWD) complex, these opportunistic fungi are able to take advantage of a pine tree that is weakened after the uncontrollable growth of the pinewood nematode population (PWN, Bursaphelenchus xylophilus) through feeding on its internal structures [2].In this condition, the tree's mycoflora shifts from an abundant and diverse community to a more limited community of opportunistic pathogenic species, where the Ophiostomatales are favored [3].
Even though some works report on the interactions of the very different organisms that form the PWD complex, there is still a lack of information on the specific mechanisms of communication between them.In a preliminary effort to pinpoint the volatile triggers for the interplay between the intervenients in PWD, this work reports on the influence of volatiles that are characteristically emitted by pines, α-pinene, β-pinene and trans-βcaryophyllene on the in vitro growth of a Leptographium isolate, obtained from wood of a naturally infected maritime pine with strong symptoms of PWD.This study aimed to understand if these volatiles are stimulants for fungal growth and if they can play a role in the complex infection cycle of PWD.

Maintance of Leptographium
The mycoflora associated with PWD was previously isolated from wood sections of Pinus pinaster displaying strong symptoms of the disease [3].Briefly, infected wood was collected from 5 maritime pines located in Seia (40 • 15 ′ 57.0 ′′ N, 7 • 42 ′ 47.6 ′′ W).Under aseptic conditions, wood pieces were sectioned, and surface was sterilized (70% ethanol (v/v) for 15 s), washed with sterile distilled water and dried in sterilized filter paper.Sections were then carefully transferred to plates with 2% (w/v) malt extract agar (MEA, Difco, Franklin Lakes, NJ, USA), supplemented with 200 mg/L of cycloheximide and 100 mg/L streptomycin and incubated at 25 • C in the dark.Hyphal tips of emerging colonies were transferred to fresh MEA plates.Representatives from pure cultures were selected for further identification and characterization and deposited in the culture collection of INIAV institute (Micoteca da Estação Agronómica Nacional (MEAN)).Established fungi were transferred to potato dextrose agar (PDA, Difco, Franklin Lakes, NJ, USA) medium and maintained by weekly subculture.

Bioassays with the Volatiles
The fungal isolate selected was identified through morphological observations and DNA sequencing [3] as a Leptographium sp.To analyze the influence of pine volatiles on fungal growth, a Petri dish-based technique was devised using PDA as growth medium (Figure 1).Succinctly, a plug of stock culture of Leptographium (grown for 7 days on PDA medium) was set in the middle of the Petri dish (with 10 mL of PDA), and a disk immersed in a volatile solution was set 5 mm from the side of the dish.The disk was 5 mm in diameter and was obtained from a cellulose Whatman filter paper (Maidstone, UK).Disks were immersed in 2 µL/mL of compound in methanol for 24 h, before being set in the PDA medium as described above.The Petri dishes were then sealed and kept in darkness, at 25 • C, for 5 days, and growth was followed daily by measuring the radius of the fungal culture growing in the section with (section B) and without the disk (A) for comparison (Figure 1).Control experiments were performed with disks immersed for 24 h in pure methanol.

Data Treatment and Statistical Analysis
Data of daily fungal radial growth in section A and B of the Petri dish were collected for each volatile used.To determine preferential fungal growth in response to the presence of the volatile, the following formula for an Interference Index was applied: Interference Index (II) for compound in section A (or B) = Radial growth in experimental section A compound (or B compound )/Radial growth in control section A methanol (or B methanol ).
For IIs above 1 ± standard error of controls, growth was considered stimulated by the compound in the respective section; for IIs below 1 ± standard error of controls, growth was considered inhibited; and for IIs above 1 − standard error of controls and below 1 + standard error of controls, growth was considered not influenced by the compound.
Statistical analysis was performed with SPSS version 29 statistics software.Statistical significance was determined with one-way ANOVA, and individual means were compared using Tukey's post hoc test with p < 0.05; the Shapiro-Wilk test ensured data normality, and the Browns-Forsythe test was used for homoscedasticity.Results are presented as the average and standard error of 6 replicates.

Data Treatment and Statistical Analysis
Data of daily fungal radial growth in section A and B of the Petri dish were collected for each volatile used.To determine preferential fungal growth in response to the presence of the volatile, the following formula for an Interference Index was applied: Interference Index (II) for compound in section A (or B) = Radial growth in experimental section Acompound (or Bcompound)/Radial growth in control section Amethanol (or Bmethanol).
For IIs above 1 ± standard error of controls, growth was considered stimulated by the compound in the respective section; for IIs below 1 ± standard error of controls, growth was considered inhibited; and for IIs above 1 − standard error of controls and below 1 + standard error of controls, growth was considered not influenced by the compound.
Statistical analysis was performed with SPSS version 29 statistics software.Statistical significance was determined with one-way ANOVA, and individual means were compared using Tukey's post hoc test with p < 0.05; the Shapiro-Wilk test ensured data normality, and the Browns-Forsythe test was used for homoscedasticity.Results are presented as the average and standard error of 6 replicates.

Results and Discussion
Fungal growth was analyzed in the section that was in direct contact with the disk that was immersed in the compound to determine its direct influence and in the section opposite the section with the disk to assess an indirect influence of the compound, i.e., the activity of an environment that was saturated in the volatile.The assessment of fungal growth in the presence of methanol revealed that no significant differences were found between these two conditions, except after 5 days of growth, where the fungi growth in the section with the disk was lower (Figure 2).This may be due to the presence of the cellulose disk, which was reached by the fungal colony at this time point, leading to a change in growth rate, probably because of the difference in the contact surface.

Results and Discussion
Fungal growth was analyzed in the section that was in direct contact with the disk that was immersed in the compound to determine its direct influence and in the section opposite the section with the disk to assess an indirect influence of the compound, i.e., the activity of an environment that was saturated in the volatile.The assessment of fungal growth in the presence of methanol revealed that no significant differences were found between these two conditions, except after 5 days of growth, where the fungi growth in the section with the disk was lower (Figure 2).This may be due to the presence of the cellulose disk, which was reached by the fungal colony at this time point, leading to a change in growth rate, probably because of the difference in the contact surface.For the compounds tested, the determined IIs reflected the degree of difference between the result that was obtained for the compound and its respective control.After 24 h of contact with any of the tested volatiles, fungal growth was increased (Figure 2), but in the following time points, it reduced to values that were similar to those of the control.This may be due to the rapid volatilization of the compound at room temperature and its escape through the gaps of the Petri dish.Between the enantiomers of β-pinene, slightly higher values were obtained for the (−) isomer than for the (+) isomer, suggesting a preference of the Leptographium isolate for that compound (a in Figure 2).Additionally, both isomers induced a higher growth in the section with the disk than in the section without in the following 2nd and 3rd days (b in Figure 2).The monoterpene α-pinene showed the highest promotion in fungal growth after 24 h but lost its effect in the following days (c in Figure 2).For the compounds tested, the determined IIs reflected the degree of difference between the result that was obtained for the compound and its respective control.After 24 h of contact with any of the tested volatiles, fungal growth was increased (Figure 2), but in the following time points, it reduced to values that were similar to those of the control.This may be due to the rapid volatilization of the compound at room temperature and its escape through the gaps of the Petri dish.Between the enantiomers of β-pinene, slightly higher values were obtained for the (−) isomer than for the (+) isomer, suggesting a preference of the Leptographium isolate for that compound (a in Figure 2).Additionally, both isomers induced a higher growth in the section with the disk than in the section without in the following 2nd and 3rd days (b in Figure 2).The monoterpene α-pinene showed the highest promotion in fungal growth after 24 h but lost its effect in the following days (c in Figure 2).
On the 5th day, the fungal growth in the section with the disk seemed to be inhibited, suggesting that after reaching the disk that was imbibed with α-pinene (direct contact), the Ophistoma isolate reduced its growth (possible inhibition through contact).This was not observed in the section without the disk.The sesquiterpene trans-β-caryophyllene induced a growth pattern in Leptographium that was similar to that of the other compounds, with a stimulation of growth after 24 h and a reduction to control values in the following days (Figure 3d).
between the result that was obtained for the compound and its respective control.24 h of contact with any of the tested volatiles, fungal growth was increased (Figu but in the following time points, it reduced to values that were similar to those o control.This may be due to the rapid volatilization of the compound at room temper and its escape through the gaps of the Petri dish.Between the enantiomers of β-pin slightly higher values were obtained for the (−) isomer than for the (+) isomer, sugge a preference of the Leptographium isolate for that compound (a in Figure 2).Addition both isomers induced a higher growth in the section with the disk than in the se without in the following 2nd and 3rd days (b in Figure 2).The monoterpene α-pi showed the highest promotion in fungal growth after 24 h but lost its effect in following days (c in Figure 2).
On the 5th day, the fungal growth in the section with the disk seemed to be inhib suggesting that after reaching the disk that was imbibed with α-pinene (direct con the Ophistoma isolate reduced its growth (possible inhibition through contact).This not observed in the section without the disk.The sesquiterpene trans-β-caryophy induced a growth pattern in Leptographium that was similar to that of the compounds, with a stimulation of growth after 24 h and a reduction to control valu the following days (Figure 3d).

Conclusions
Analyzing the influence of volatiles that are characteristic of susceptible pines o growth of fungi that are associated with PWD is an important contributio understanding the relationships and communication between the different interven in PWD.The tested Leptographium isolate appeared to be more influenced by α-pi than by the other compounds.This monoterpene is a very important volatile, emitte the pine, and is an attractant for many pine bark beetles.Further research will asses combinations of volatiles simulating the emissions of the pine in its natural environm

Conclusions
Analyzing the influence of volatiles that are characteristic of susceptible pines on the growth of fungi that are associated with PWD is an important contribution to understanding the relationships and communication between the different intervenients in PWD.The tested Leptographium isolate appeared to be more influenced by α-pinene than by the other compounds.This monoterpene is a very important volatile, emitted by the pine, and is an attractant for many pine bark beetles.Further research will assess the combinations of volatiles simulating the emissions of the pine in its natural environment.

Figure 1 .
Figure 1.Scheme of the experimental setup used for bioassays with the Leptographium isolate.The fungal plug (dark circle) is set between the sections without (A) and with (B) the cellulose disk (blank circle).

Figure 1 .
Figure 1.Scheme of the experimental setup used for bioassays with the Leptographium isolate.The fungal plug (dark circle) is set between the sections without (A) and with (B) the cellulose disk (blank circle).

6 Figure 2 .
Figure2.The radial growth of the Leptographium isolate on potato dextrose agar (25 °C) in the section without (white columns) and with (gray columns) a cellulose disk imbibed in pure methanol (control).Data are presented as the mean ± standard error of six independent biological replicates.Different letters indicate statistically significant differences (p < 0.05).

Figure 2 .
Figure 2.The radial growth of the Leptographium isolate on potato dextrose agar (25 • C) in the section without (white columns) and with (gray columns) a cellulose disk imbibed in pure methanol (control).Data are presented as the mean ± standard error of six independent biological replicates.Different letters indicate statistically significant differences (p < 0.05).

Figure 3 .
Figure 3. Interference indexes (ratio of radial growth of the Leptographium isolate in contact wi compound over that of the control) for fungi growing in the section without (white line) and (gray line) a cellulose disk imbibed in (−)-β-pinene (a), (+)-β-pinene (b), α-pinene (c), or tra caryophyllene (d).Data are presented as mean ± standard error of six independent biolo replicates.Different letters indicate statistically significant differences (p < 0.05).The red represents a condition of no influence (experimental = control), with respective standard error b on variation of control values (interrupted red lines).

Author Contributions:Figure 3 .
Figure 3. Interference indexes (ratio of radial growth of the Leptographium isolate in contact with the compound over that of the control) for fungi growing in the section without (white line) and with (gray line) a cellulose disk imbibed in (−)-β-pinene (a), (+)-β-pinene (b), α-pinene (c), or transβ-caryophyllene (d).Data are presented as mean ± standard error of six independent biological replicates.Different letters indicate statistically significant differences (p 0.05).The red line represents a condition of no influence (experimental = control), with respective standard error based on variation of control values (interrupted red lines).