Antifungal properties of volatile organic compounds produced by Daldinia eschscholtzii MFLUCC 19-0493 isolated from Barleria prionitis leaves against Colletotrichum acutatum and its post-harvest infections on strawberry fruits

Plant material

Barleria prionitis plants, aged approximately 5 years, were collected from Chiang Rai province, Thailand in October 2019. Healthy B. prionitis leaves from the collected plants were sampled and sealed in plastic bags before being stored at 4 °C and used within 48 h of collection.

Isolation of endophytic fungi

The leaves of B. prionitis were washed with tap water to eliminate soil, dust, and dirt attached to the surface and rinsed with distilled water 3 times. Surface of the leaves was sterilized by immersing them in 70% ethanol for 1 min, 5% sodium hypochlorite for 30 s, and 70% ethanol for 30 s, to remove epiphytic microorganisms. After this, the leaves were washed thoroughly using sterilized distilled water to remove remaining solvents. The surface of the leaves was dried with sterilized tissue paper to provide aseptic conditions. The dried leaves were cut with a sterilized scalpel blade into small pieces (i.e., approximately 0.5 cm ×0.5 cm segments). This surface sterilization procedure was performed following the study by Monggoot et al. (2017).

Each leaf segment was placed on a Petri plate containing potato dextrose agar (PDA) medium mixed with 30 µg/mL of chloramphenicol to prevent bacterial growth. A few drops of sterilized distilled water from the final washing step were placed on PDA plates (n=5) to confirm that no contamination was observed (negative controls). All PDA plates were sealed with Parafilm. The PDA plates were incubated at 27 °C and examined daily for 7 days. Once the mycelia fully appeared, each hypha was further transferred onto a new, freshly prepared PDA plate. This procedure was repeated at least 3 times until a pure culture of each endophytic isolate was achieved. The endophytic isolates were coded as BP(x), where x was a numeric number assigned to each isolate.

Isolation and molecular identification of phytopathogen C. acutatum

Phytopathogenic fungus C. acutatum was isolated from 10 infected strawberry fruits that were collected from a strawberry field located at Angkhang Royal Agricultural Station in Fang district, Chiang Mai province, Thailand in February 2020. Each infected fruit was washed with sterile distilled water before transferring to a Petri plate containing PDA mixed with 30 µg/mL of chloramphenicol. They were incubated at 27 °C for 7 days. All fungi obtained from the infected strawberry fruits were sub-cultured to obtain a pure culture. The isolated phytopathogen was pre-identified based on micro-morphological and macro-morphological characteristics, including morphology of sporulation. The aerial mycelium of C. acutatum isolate was scraped from the PDA surface and pulverized with a mortar and pestle to achieve a mycelium pulp. The genomic DNA was extracted following a protocol described by Tanapichatsakul et al. (2020). The internal transcribed spacer (ITS) region was amplified using ITS5/ITS4 primers (Tanapichatsakul et al., 2020). Polymerase chain reactions (PCR) were performed following a protocol described by Tanapichatsakul et al. (2020). The sequence similarity of isolated fungi from BLAST result was 100% to C. acutatum. In addition, the pathogenicity test of an isolated pathogen was also performed using healthy strawberries to confirm pathogen identification. Ten strawberry fruits were wounded approximately 5 mm diameter and inoculated with a plug of C. acutatum. Fruits were kept in a moist plastic box and incubated at 27 °C for 7 days. Control fruits were inoculated with PDA agar plug. The test was performed in 3 replicates and repeated 3 times (n = 90). The symptoms and morphology of C. acutatum on strawberries were similar with previous studies (Kumvinit & Akarapisan, 2016; Es-Soufi et al., 2018).

In vitro screening of antifungal activity of VOCs produced by isolated endophytic fungi on the radial growth of C. acutatum

The VOCs produced by each isolated endophytic fungus against C. acutatum were screened for antifungal activity using the dual-culture plate method described by Li et al. (2015), with a slight modification on the incubation time. Two sterilized PDA bottom plates (9 cm diameter) were used. An agar plug (5 mm diameter, 7 days old) of C. acutatum was placed at the center of one plate, whereas an agar plug (5 mm diameter, 7 days old) of the individual endophytic isolate was placed at the center of another plate. The two plates were sealed together and wrapped with Parafilm before incubation at 27 °C for 7 days. Plates without the endophytic isolate but with C. acutatum were used as controls. The experiment was performed using 5 replicates for each endophytic isolate and repeated 3 times (n = 15). The mycelial diameter of C. acutatum in each treatment was measured daily using a vernier digital caliper (MITUTOYO-ABS Digimatic Caliper CD-AX, Japan) in a millimeter unit. The obtained results were expressed as inhibition percentage of mycelial growth according to the following formula: ${\displaystyle \text{\%}\text{Inhibition}=\left(\frac{\text{mycelial diameter of untreated fungus}-\text{mycelial diameter of treated fungus}\times 100}{\text{mycelial diameter of untreated fungus}}\right).}$

Viability test of the treated C. acutatum

From the in vitro screening above, the treatment that exhibited the highest antifungal activity was selected for a viability test of the treated C. acutatum. An agar plug (5 mm diameter) of C. acutatum treated with the chosen endophytic isolate was placed on a fresh PDA plate and further cultured at 27 °C for 7 days to evaluate its viability. The mycelial growth of C. acutatum was evaluated. The experiment was performed using 5 replicates and conducted 3 times (n = 15).

In vivo antifungal activity of the VOCs produced by the endophytic isolate BP11 against C. acutatum infections on strawberry fruits

The in vivo experiment was performed using healthy strawberry fruits collected in March 2020. All fruits were selected according to their maturity, size, color, and the absence of physical injuries or infections. The fruits were harvested early in the morning and then transported to the laboratory within 3 h. The fruit surfaces were sanitized using 1% sodium hypochlorite for 5 min, then rinsed with distilled water 3 times before being placed on a sterilized tissue paper for aseptic drying.

Inoculation of strawberry fruits with C. acutatum was performed according to the procedure described by Li et al. (2015), with a slight modification. One location on each cleaned strawberry fruit was wounded using a sterile inoculating needle. The wound depth was approximately 5 mm. A plug of C. acutatum (5 mm diameter, 7 days old) was placed into the wound. After inoculation, the strawberry samples were placed inside a plastic box (15.5 cm ×13.5 cm ×7.0 cm). The bottom of the plastic box was covered with sterilized medical gauze. Double layers of autoclaved tissue paper soaked in 100 mL of sterile distilled water were placed underneath the gauze to provide moisture. Then, a 7-day old endophytic isolate BP11 culture plate, without its cover, was placed in the same box, but without direct contact with the strawberry samples. Each box was closed with a fitted plastic lid and air-sealed using Parafilm. All boxes were stored at 27 °C for 7 days. To evaluate the effect of VOCs on the growth of C. acutatum, 4 different treatments were prepared, including pathogen-inoculated fruits in the plastic box (positive control), pathogen-uninoculated fruits in the endophytic isolate BP11-containing plastic box (negative control), pathogen-uninoculated fruits in the plastic box (control), and pathogen-inoculated fruits in the endophytic isolate BP11-containing plastic box (BP11 treatment). There were 15 strawberry samples in each box. Each treatment was performed in 3 replicates and repeated 3 times (n = 135). The mycelial diameter of C. acutatum on strawberries from all treatments was measured using a vernier digital caliper in a millimeter unit. In this experiment, the inhibition percentage was calculated as explained above. Disease incidence and disease severity percentages were calculated using the following formulas: ${\displaystyle \text{\%}\text{Disease incidence}=\left(\frac{\text{number of disease fruits}}{\text{total number of fruits assessed}}\right)\times 100}$ ${\displaystyle \text{\%}\text{Disease severity}=\left(\frac{\text{sum of all disease rating}}{\text{total number of rating}\times \text{maximum disease scale}}\right)\times 100.}$

For the disease severity percentage, the rotten area on the surface of each strawberry fruit was visually evaluated using a scale from 0 to 5. A description for each scale is as follows: 0 = no disease symptom, 1 = a small grey spot covering <1% fruit area, 2 = a grey sunken spot covering 1–10% fruit area, 3 = a grey spot covering 11–25% fruit area, 4 = circular grey sunken spots covering 26–50% fruit area, and 5 = circular to irregular spots covering >51% fruit area.

Quality of strawberry fruits after treating with VOCs produced by the chosen endophytic isolate (BP11)

The quality of strawberry fruits was evaluated after exposure to VOCs produced by the endophytic isolate BP11 for 7 days. In this experiment, fruit firmness, total soluble solids, and pH were measured following the method of Cai et al. (2015). Fruit firmness was measured by a TA-XT2i texture analyzer (Stable Micro Systems Ltd., UK) with a P50 cylinder plunger probe at the equator of the equidistant region. Fruit firmness was recorded from the maximum force. The texture analyzer was adjusted following this condition: 5.0 mm/s pre-test speed, 1.0 mm/s test speed, and 5.0 mm/s post-test speed, all with a penetration distance of 5 mm. Strawberry juice was extracted and filtered through two layers of cheesecloth. Total soluble solids of the strawberry fruit juice were measured using a hand-held refractometer (WYT-4) (Top Instrument Co., Ltd., China). The pH value was also determined by a pH meter (DELTA 320) (Top Instrument Co., Ltd., China). In this experiment, all treated strawberry samples from above were used.

Identification and analysis of VOCs produced by the endophytic isolate BP11

The VOCs produced by the endophytic isolate BP11 were extracted using solid phase micro-extraction (SPME). Prior to extraction, the endophytic isolate BP11 was cultured in a glass container (9 cm diameter) containing PDA medium at 27 °C for 7 days and covered with an aluminum cap with a PTFE-coated silicone septum. The SPME sampling apparatus with a SPME holder and a 1.0 cm fused silica fiber was purchased from Supelco (PA, USA). A 50/30 µm divinylbenzene-carboxen-polydimethylsiloxane (DVB-CAR-PDMS) SPME fiber was selected to extract the VOCs. Before extraction, the SPME fiber was preconditioned at 230 °C inside the injection port of a gas chromatograph (HP 6890) (Agilent Technologies, CA, USA) for 30 min. The preconditioned SPME fiber was then inserted into the glass container containing the endophytic isolate BP11 to trap the VOCs for 30 min prior to immersion in the injection port of the HP model 6890 gas chromatograph coupled to an HP model 5973 mass-selective detection instrument (Agilent Technologies) for 10 min in splitless mode. An HP-5ms (30 m ×0.25 mm i.d., 0.25 µm film thickness) column (Agilent Technologies) was used for compound separation. The oven temperature was initially set to 40 °C and then increased at a rate of 2 °C/min to the final temperature of 200 °C. The injector temperature was set to 250 °C. Helium was used as a carrier gas and was set to a flow rate of 1 mL/min. The mass spectrometer was operated using electron impact ionization (70 eV electron ionization voltages). A scan mode (cover the range of m/z 30-300) was used. The ion chamber and the mass transfer temperatures were set to 250 °C and 230 °C, respectively.

The C₉-C₁₇ n-alkanes were used to calculate the retention time index. Mass spectra stored in NIST05 Library, Wiley7N Mass Spectral Library, and Adams Library (2017) were used for compound identification. A gas chromatograph (HP 6890, Agilent Technologies) equipped with a flame ionization detector was used for quantitative analysis of the VOCs. The identified volatile constituents were reported as the percentage of relative peak areas calculated using the normalization method without correction factors.

In vitro antifungal activity of synthetic VOCs on C. acutatum

Synthetic volatile standards of 3,5-dimethyl-4-heptanone (95%), ethyl sorbate (95%), benzaldehyde dimethyl acetal (95%), trans-sabinene hydrate (95%), methyl geranate (90%), and elemicin (95%) were used in this experiment. Ethyl sorbate, benzaldehyde dimethyl acetal, methyl geranate, and elemicin were purchased from Santa Cruz Biotechnology (TX, USA) while trans-sabinene hydrate was purchased from Sigma-Aldrich (China). These volatile standards were in a liquid form. The antifungal activity of each synthetic volatile compound was investigated according to the method of Gotor-Vila et al. (2017), with a slight modification. In this experiment, 2 sterilized PDA bottom plates (9 cm diameter) were used. An agar plug (5 mm diameter, 7 days old) of C. acutatum was placed at the center of one plate, whereas a paper filter (90 mm diameter) spiked with an aliquot of the volatile standard was placed at the center of another plate. Both plates were sealed together and wrapped with Parafilm before incubation at 27 °C for 7 days. Each synthetic VOC was tested separately using 3 different aliquots (12.5, 25.0, and 50.0 µL) of its liquid solution. Because the Petri plates have a remaining headspace volume of 44 mL, the corresponding concentrations of the synthetic compound were 0.28, 0.56, and 1.12 µL/mL headspace, respectively. Petri plates without synthetic VOCs but with C. acutatum were used as controls. For each synthetic volatile compound, 5 replicates for each spiking volume were used and repeated 3 times (n = 45 for all 3 levels). The mycelial diameter of C. acutatum in each treatment was measured using a vernier digital caliper in a millimeter unit. The obtained results were expressed as inhibition percentage of mycelial growth as explained above.

In vivo antifungal activity of elemicin against C. acutatum infections on strawberry fruits

Strawberry fruits were prepared and wounded as explained above. They were placed at the bottom of the plastic box (15.5 cm ×13.5 cm ×7.0 cm) prepared in a similar manner as explained above. Then, a Petri plate containing 3 paper filters (90 mm diameter), each moistened with 50 µL of the elemicin standard, was placed in the same box, but without direct contact with the strawberry samples. Each box was closed with a fitted plastic lid and air-sealed using Parafilm. All boxes were stored at 27 °C for 7 days. Treatment that included only the pathogen-inoculated fruits in the plastic box was used as a positive control. In each box, there were 15 strawberry samples. Each treatment was performed in 3 replicates and repeated 3 times (n = 135). The mycelial diameter of C. acutatum on strawberry samples from all boxes was measured using a vernier digital caliper in a millimeter unit. The inhibition, disease incidence, and disease severity percentages were calculated as described above.

Quality of strawberry fruits after treating with elemicin

The quality of strawberry fruits was evaluated after treating with elemicin. This evaluation was done similarly to the procedure outlined above.

Molecular identification of the endophytic isolate BP11

Genomic DNA of endophytic isolate BP11 was extracted using a Genomic DNA Extraction Mini-Kit following the method of Tanapichatsakul et al. (2020). DNA loci including ITS, LSU, and RPB2 regions were amplified by PCR following the protocol of Samarakoon et al. (2019). The obtained sequences in this study were subjected to BLAST search in GenBank (https://blast.ncbi.nlm.nih.gov/Blast.cgi). BLAST search indicated that the isolate BP11 belongs to Hypoxylaceae taxa. The sequences of representative Hypoxylaceae taxa used in the phylogenetic analyses were chosen from GenBank based on the BLASTn searches and recently published data (Samarakoon et al., 2019). Sequence datasets from all fungi obtained from ITS, LSU, and RPB2 rDNA primers were used to construct the phylogenetic tree. The accession numbers of all strains are shown in Table S1. The combined sequence data were first aligned using MAFFT version 7 (http://mafft.cbrc.jp/alignment/server/). The alignment was further improved using BioEdit v. 7.0.5.3. The last alignment of the combined sequence datasets was investigated and the phylogenetic tree inferred based on maximum likelihood (ML) and Bayesian inference analyses (BI). ML and BI analyses were achieved using the RAxML-HPC2 on XSEDE (v. 8.2.12) via the CIPRES Science Gateway platform and MrBayes on XSEDE, MrBayes 3.2.6 via the CIPRES Science Gateway platform, respectively. Bayesian posterior probabilities (PP) were investigated using Markov Chain Monte Carlo sampling (BMCMC). The phylogenetic tree was constructed in FigTree v.1.4.3. The endophytic isolate BP11 was deposited in the Mae Fah Luang University Culture Collection (MFLUCC), Chiang Rai, Thailand under the accession number MFLUCC 19-0493.

Analysis of data

Data from each experiment were presented as the mean ± standard deviation. When applicable, data were subjected to Analysis of Variance (ANOVA), followed by post-hoc multiple pairwise comparisons using Duncan’s multiple range tests (α = 0.05) or Dunnett comparisons with a reference treatment (α = 0.05). Linear regression analysis was conducted to evaluate the relationship between the observed inhibition percentage and the concentrations of synthetic VOCs tested. The statistical analyses were conducted using the SPSS 20.0 software (IBM Corp; 2011, NY, USA).

In vitro antifungal activity of VOCs produced by endophytic isolates and viability of the treated C. acutatum

A total of 34 endophytic isolates were obtained from the B. prionitis leaves. After 7-day incubation, only 26 of them (Fig. S1) were able to produce VOCs that inhibit the mycelial growth of C. acutatum, but to varying degrees (Fig. 1). For these endophytic isolates, the average inhibition percentages ranged between 3.3 and 80.8%. The endophytic isolate BP11 was found to produce the VOCs with the highest inhibition percentage (80.8, p < 0.05). Moreover, the in vitro antifungal activity of the VOCs produced by the endophytic isolate BP11 on C. acutatum across 7 days of the incubation period was depicted in Fig. S2. The highest antifungal activity was observed from day 4 to day 7. During which time, the average inhibition rate was about 80%. From the viability test, C. acutatum treated with the VOCs produced from the endophytic isolate BP11 could not reproduce its mycelia on the PDA plates over a 5-day incubation period (data not shown).

In vivo antifungal activity of VOCs produced by the endophytic isolate BP11 against C. acutatum infections on strawberry fruits

Strawberry samples after undergoing various treatments are shown in Fig. 2. Physical damage on the strawberry fruits was neither detected in the pathogen-uninoculated fruits containing the endophytic isolate BP11 (negative control) nor the pathogen-uninoculated fruits (control). In comparison to pathogen-inoculated fruits (positive control) (Fig. 2C), less physical damage was observed on fruits inoculated with C. acutatum and treated with the VOCs produced by the endophytic isolate BP11 (Fig. 2D).

VOCs produced by the endophytic isolate BP11

Gas chromatographic-mass spectrometric (GC-MS) analysis of the VOCs produced by the endophytic isolate BP11 led to the identification of 60 compounds. These VOCs are listed in Table 1. The volatile profile of the endophytic isolate BP11 was dominated by the presence of hydrocarbons (35.0%), followed by monoterpene hydrocarbons (39.4%), and sesquiterpene hydrocarbons (37.3%). The major volatile compounds produced by the endophytic isolate BP11 were elemicin (23.8%), benzaldehyde dimethyl acetal (8.5%), ethyl sorbate (6.8%), methyl geranate (6.5%), trans-sabinene hydrate (5.4%), and 3,5-dimethyl-4-heptanone (5.1%). In addition, the VOCs produced from the endophytic isolate BP11 possessed sweet and fruity odor.

In vitro antifungal activity of synthetic VOCs against C. acutatum

The in vitro antifungal activities of the 6 most abundant VOCs against C. acutatum are summarized in Fig. 3. The results show that these chosen synthetic VOCs had varying degrees of antifungal activity. For every volatile compound tested, the highest inhibition percentage was detected on day 1 and continued to decrease in the later days (p < 0.05). Elemicin demonstrated the highest inhibition percentages across the observation period, covering a range of 15.3% to 76.6% (p < 0.05) (Fig. 3). Ethyl sorbate showed poor antifungal activity against C. acutatum.

Positive associations were found between the amounts of synthetic VOCs used (per mL headspace) and the inhibition percentages. In the case of elemicin, the resulting linear regression slope on day 1 of the incubation period was 22.1 (Fig. S3). Across the observation period, the slopes of elemicin were in a range of 22.1 to -24.7. Moreover, the linear regression slopes of other compounds (on day 1) was also positive with a value of 22.5, 18.0, 13.4, 22.3, and 8.9 for 3,5-dimethyl-4-heptanone, ethyl sorbate, benzaldehyde dimethyl acetal, trans-sabinene hydrate, and methyl geranate, respectively (data not shown).

In vivo antifungal activity of elemicin against C. acutatum infections on strawberry fruits

Strawberry fruits after treatment with elemicin are shown in Fig. 2E. Less physical damage was observed on the fruits inoculated with C. acutatum and treated with elemicin than the positive control samples. The inhibition, disease incidence, and disease severity percentages of this treatment were 70.9%, 21.5%, and 15.9%, respectively (Table 2). These results were similar to those obtained from the endophytic isolate BP11 treatment.

Strawberry quality after treating with VOCs produced by the endophytic isolate BP11 or elemicin

The quality of strawberry fruits obtained from various treatments, including the synthetic compound elemicin, in terms of firmness, total soluble solids, and pH are demonstrated in Table 3. After 7 days of treatment, the average firmness of pathogen-inoculated fruits (positive control) was 1.2 N. It was significantly lower than other treatments. The average firmness of pathogen-inoculated fruits (positive control), pathogen-uninoculated fruits containing the endophytic isolate BP11 (negative control), pathogen-uninoculated fruits (control), pathogen-inoculated fruits containing the endophytic isolate BP11 (BP11 treatment), and pathogen-inoculated fruits containing elemicin (elemicin treatment) ranged from 3.8 to 3.9 N. These values were not significantly different. No significant difference was observed for the total soluble solids content (7.7–7.9) and the pH (3.3–3.4) of the strawberry fruits from all treatments.

Phylogenetic tree and molecular identification

The phylogenetic tree of the endophytic isolate BP11 is shown in Fig. 4. Based on the results, the endophytic isolate BP11 was identified as Daldinia eschscholtzii MFLUCC 19-0493. Its genetic sequence was submitted to the GenBank database under the accession numbers MN704648, MW485486, and MW495050 from ITS, LSU, and RPB2 regions, respectively.