Potential of Trichoderma spp. and Pinus sylvestris Bark Extracts as Biocontrol Agents against Fungal Pathogens Residing in the Botryosphaeriales

: Botryosphaeriales represent a diverse order of fungal pathogens of various woody plant species. In Serbia, these fungi are important pathogens of forest, ornamental, and fruit trees causing die-back, cankers, leaf blights, fruit


Introduction
Sawmill industries represent a huge generator of bark waste estimated to more than three hundred million tons [1].The disposal and storage of bark waste is a global problem since it is mostly used as an energy source through direct combustion or transported to landfills [1,2].However, removed bark represents a raw material with a whole scale of valuable purposes.Substrate formulations, soil conditioners, plant protection, health and industrial products, and bioremediation agents are some of the potential applications of this material considered to be a waste [1,[3][4][5][6].Coniferous bark, as a component of commercial substrates, increases water and nutrient availability and exhibits antifungal, antibacterial, and insecticidal effects [3][4].Antimicrobial and antifungal activity of extracted bark constit-Citation: Karličić, V.; Jovičić-Petrović, J.; Marojević, V.; uents has been well documented [5][6][7], suggesting that they are environmentally sound alternatives to plant protection chemicals.In addition, different microorganisms with such roles inhabit above-ground and below-ground plant parts [8,9].Microbial communities inhabiting plant parts mainly originate from the rhizosphere and some of them are known as plant growth-promoting microorganisms, for example, Bacillus spp., Pseudomonas spp., Azospirillum spp., Arthrobacter spp., Aspergillus spp., Penicillium spp., Piriformospora spp., Phoma spp., and Trichoderma spp.These microorganisms increase disease resistance by triggering plant defensive mechanisms [8,10].Members of the fungal genus Trichoderma (Ascomycota, Hypocreales, Hypocreaceae) are well-known biocontrol agents with plant growthpromoting characteristics [10].These fungi are most diverse in the above-ground plant parts such as bark, but they are also widely distributed in soils [11].Nowadays, more than 250 Trichoderma-based biofungicides are commercially available worldwide [12].
Species of the Botryosphaeriales (Ascomycota) are distributed worldwide and occur on various tree species.These fungi are known as endophytes, latent pathogens, pathogens, and saprophytes and they usually cause disease when their hosts are under stress [9].In Serbia, Botryosphaeriales are important pathogens of woody and vascular plants causing die-back, cankers, leaf blights, shot hole disease, fruit, and root rot [13][14][15][16][17][18].The pathogenicity of Botryosphaeriales is of a particular importance in urban environments such as city parks which are mostly comprised of plants belonging to diverse taxonomic groups and these fungi can infect multiple hosts [19].Botryosphaeriales species are difficult to control because these pathogens reside in vascular woody tissues and cause cankers [20].Additionally, there is increasing concern about the environmental effects and safety of conventional fungicide-based control measures against diseases caused by these fungi [21].
The aims of the present study were the following: (1) to isolate Trichoderma spp.from Pinus sylvestris bark and (2) to evaluate the antifungal activity of P. sylvestris bark extracts and Trichoderma spp.against Botryosphaeria dothidea, Neofusicoccum parvum, and Dothiorella sarmentorum in vitro.

Isolation of Trichoderma spp.
Trichoderma spp.were isolated from P. sylvestris bark obtained from the Eko Farm Kovačević (Gornji Milanovac, RS), using a serial dilution method.One milliliter of 10 −3 dilution was spread evenly on potato dextrose agar plates (PDA, HiMedia, India) that were kept at 25 ± 1 °C for 7 days.The colonies with morphological and cultural characteristics of Trichoderma spp.(yellow-green colonies with a characteristic conidiophore branching pattern) were selected, purified, and kept in 20% glycerol at −80 °C, until use.

Extract Preparation
Pinus sylvestris bark was ground with a mill, sifted through 0.5 mm sieve, dried at 50 °C and maintained at room temperature.Two different water extracts were prepared using distilled water as a solvent.Extraction was carried out in 0.5 L Erlenmeyer flasks by immersing the bark powder (40 g dry weight) in 400 mL of the solvent.The Erlenmeyer flasks were placed in a water bath (Memmert WNB 14, Germany) for 2 h at 80 °C and at 120 °C in an autoclave (Panasonic MLS-37812) for 20 min.The extraction mixtures were filtered with a Whatman ® Grade 52 filter paper (Merck, Germany) and the residues were dried in a convection oven at 105 °C for 24 h (Binder, Germany).The water extract yields were calculated according to the following formula [22]: Yield of extract (%) = (W1 × 100)/W2 where W1 is the weight of the dry extract residue after solvent removal and W2 is the initial weight of bark powder (40 g).Prepared WEs were kept in a refrigerator (4 °C) until used.

Assay of Water Bark Extracts' Antifungal Activity In Vitro
The antifungal activity assay was performed against the growth of B. dothidea (CMW39314), D. sarmentorum (CMW39365), and N. parvum (BOT275).The B. dothidea, D. sarmentorum, and N. parvum were isolated from Picea abies, Thuja occidentalis, and Prunus laurocerasus, respectively, and have been identified in previous studies [14,19,23].These isolates are maintained at −80 °C in the culture collection of the Institute of Lowland Forestry and Environment (ILFE), University of Novi Sad.Before use, the isolates were subcultured onto freshly prepared PDA and allowed to grow for one week at 25 °C, in the dark.
The essay was conducted using PDA plates containing 20% and 30% of bark extract.Agar plugs (5 × 5 mm) of the 7-day-old cultures of B. dothidea, D. sarmentorum, and N. parvum were placed in the centre of 85 mm Petri dish with P. sylvestris bark extracts.The control plates contained only PDA.Each treatment was replicated three times.Inoculated Petri dishes were incubated at 25 °C, in the dark.Radial growth of the mycelium was measured in two directions until the control fungus had reached the edge of the plate.The mycelial growth inhibition percentage was calculated according to the formula [7]: Mycelial growth inhibition (%) = ((DC − DT)/DC)) × 100, where DC is the average diameter of a fungal colony of the control group, and DT is the average diameter of a fungal colony of the treatment group.

Assay of Trichoderma spp. Antifungal Activity In Vitro
The ability of Trichoderma spp. to antagonize B. dothidea, D. sarmentorum, and N. parvum was assessed using a confrontation assay on PDA plates.The plates were double inoculated with agar plugs (5 × 5 mm) of a 7-day-old culture of B. dothidea, D. sarmentorum, N. parvum, and Trichoderma spp. at a 3 cm distance.Each treatment was replicated three times.Inoculated Petri dishes were incubated at 25 ± 2 °C, in the dark, until the control fungus had reached the edge of the plate.The mycelial growth inhibition percentage was calculated using the same formula as above.The degree of antagonistic activity was estimated as: very high (% > 75), high (% = 61-75), moderate (% = 51-60), and low (% < 51) [25].

Statistical Analyses
The statistical analyses were performed using the analyses of variance (ANOVA) and software Statistica (StatSoft, Tulsa, OK, USA).Mean values of data were compared using the Tukey test at a significance level of p = 0.05.

Results and Discussion
After extraction, the water-soluble fraction from the non-soluble particulate material was separated by filtering.Weight losses detected after bark drying revealed different levels of extraction depending on the temperature regime.Extraction yields of P. sylvestris bark at 80 °C were 4.10%, while yields were 5.14% at 120 °C.A temperature increase favors the solubility of phenolic compounds in water resulting in an increased rate of extraction and decreased extraction time [26].
Generally, on the one hand, water is not considered to be the best solvent since it shows no selectivity in the extraction and, consequently, both phenolic and non-phenolic compounds are dissolved.On the other hand, industrial usage gives preference to water over other solvents [2].The highest extraction yields of P. sylvestris bark were obtained with hot water (4.80-13.70%)as compared with benzene, ether, and ethanol as a solvent [2].In addition, several solvents (water, ethanol, and acetone) were used for P. radiata bark extraction and the highest yields were achieved with water (11.99%) at 120 °C [27].
The antifungal properties of the extracts were tested using two concentrations (20 and 30%).Extract concentration of 20% inhibited mycelial growth of B. dothidea by 35-39% (Table 1), while 30% concentration increased the level of inhibition to 39-44%.The case of D. sarmentorum water extract prepared at 80 °C showed the highest inhibition rate (60%).
According to [24], the effects of extracts on B. dothidea mycelium growth are considered to be slightly toxic, while the same treatments showed a moderately toxic effect on D. sarmentorum growth.Similarly, hydrophilic extracts of P. sylvestris applied in 1% concentration caused slight toxicity towards Schizophyllum commune (48.00%),Gloeophyllum trabeum (49.14%), and Fibroporia vaillantii (47.03%), while 5% concentration was classified as moderately toxic in all three cases with an inhibition percentage over 55% [28].Pinus sylvestris bark extracts showed no inhibition of radial growth of N. parvum, but the sparse aerial mycelium was noted.The water extracts increased asexual sporulation of Trichoderma spp., but did not cause growth inhibition.Fungal isolations from P. sylvestris bark yielded two isolates of Trichoderma spp.(BKG 3 and 4) which were subjected to a confrontation test with B. dothidea, D. sarmentorum, and N. parvum (Table 2).The test showed that Trichoderma spp. was able to inhibit the growth of the pathogens five days after the confrontation.Trichoderma sp.BKG 4 showed very high antagonism against B. dothidea and D. sarmentorum.In the test with N. parvum, the activity qualified as high.Trichoderma sp.BKG 3 showed high antagonism against B. dothidea and D. sarmentorum, while the effects on N. parvum mycelial growth were declared as moderate.The interaction established between the two Trichoderma isolates, B. dothidea and D. sarmentorum, induced morphological changes characterized as overgrown with replacement which indicates mycoparasitism as the mode of the action [29,30].The confrontation test of N. parvum with Trichoderma isolates resulted in a deadlock at a distance with enhanced production of dark pigmentation in N. parvum.This is expected as pigments indicate the meeting of the two genetically different mycelia while an inhibition zone indicates the diffusion of antifungal metabolites [30,31].
Preliminary results of the study suggested, for the first time, in vitro potential of P. sylvestris bark extracts and Trichoderma spp.for suppressing the growth of B. dothidea, N. parvum, and D. sarmentorum isolated from ornamental trees with the die-back symptoms, in Serbia.It is also the first study to indicate the in vitro biocontrol potential of P. sylvestris bark extracts against diseases caused by Botryosphaeriales.However, more research is needed to further examine different antagonistic mechanisms using biochemical and microscopical examinations and to identify Trichoderma spp.Moreover, future work should also focus on the extraction of active compounds and on optimization of the extraction procedure.