In vitro antifungal activity of lasiodiplodin, isolated from endophytic fungus Lasiodiplodia pseudotheobromae J-10 associated with Sarcandra glabra and optimization of culture conditions for lasiodiplodin production

A macrolide antibiotic, lasiodiplodin was isolated from the endophytic fungus (EF) Lasiodiplodia pseudotheobromae J-10 associated with the medicinal plant Sarcandra glabra. In vitro antifungal assay demonstrated the inhibitory activity of lasiodiplodin against the growth of six phytopathogenic fungi, with the IC50 values ranging between 15.50 and 52.30 μg/mL. The highest antifungal activities were recorded against Exserohilum turcicum, Colletotrichum capsici, and Pestalotiopsis theae, with IC50 values of 15.50, 15.90, and 17.55 μg/mL, respectively. The underlying mechanism of the antifungal activity of lasiodiplodin against E. turcicum included the alteration of its colony morphology and disturbance of its cell membrane integrity. In addition, the optimization of L. pseudotheobromae J-10 culture conditions increased lasiodiplodin yield to 52.33 mg/L from 0.59 mg/L at pre-optimization. This is the first report on the isolation and identification of antifungal compound from the EF L. pseudotheobromae J-10 associated with S. glabra, as well as on the optimization of L. pseudotheobromae J-10 culture conditions to increase lasiodiplodin yield. The results of this study support that lasiodiplodin is a natural compound with high potential bioactivity against phytopathogens, and provide a basis for further study of the EF associated with S. glabra.


Introduction
Endophytic fungi (EF) reside in the intercellular or intracellular spaces of the plants without causing any negative effects on the host and are known to play an important role in promoting plant health (Chutulo et al. 2018). Furthermore, EF are recognized as natural biocontrol agents to suppress plant pathogens (Aly et al. 2010;Zhang et al. 2010;Que et al. 2022;Zheng et al. 2021). Currently, studies on the isolation of agriculturally important bioactive metabolites from EF (especially those associated with medicinal plants) are gaining popularity.
In our previous study, the L. pseudotheobromae J-10 strain, isolated from the medicinal plant Sarcandra glabra, exhibited a broad-spectrum inhibitory activity against a range of plant pathogenic fungi (Meng et al. 2022). Thus, in this study, we focused on isolation and identification the active component in the L. pseudotheobromae J-10 crude extract and on optimizing the culture conditions to improve the yield and provide a basis for the application of L. pseudotheobromae J-10 metabolites in plant disease control.

Fungal materials
The EF L. pseudotheobromae J-10 was isolated from a healthy S. glabra stem, which was collected in May 2021 from the Yao Autonomous County, Hezhou City, Guangxi Province of China. Six plant pathogenic fungi important in agriculture were selected as test strains for bioassay. Exserohilum turcicum, Colletotrichum capsici, Pestalotiopsis theae, Alternaria oleracea, and Ceratocystis paradoxa were provided by the Laboratory of Phytopathology, College of Agriculture, Guangxi University; Alternaria citri was provided by Guangxi Academy of Specialty Crop.

Fermentation culture of the EF L. pseudotheobromae J-10 and preparation of its crude extract
The strain J-10 was grown in potato dextrose broth (PDB; supplemented with 200 g/L potato extract and 20 g/L dextrose; Xilong Science Co., Ltd, Sichuan, China). Four agar plugs (0.4 × 0.4 cm) were respectively inoculated into each 1000 mL Erlenmeyer flask (20 flasks in total) containing 400 mL PDB medium. Fermentation was carried out for 45 d at 28 ℃ under stilling culture conditions. The mycelium of the fermented culture was extracted thrice with methanol (Xilong Science Co., Ltd) at 28 ℃, and the extracts were combined and evaporated to dryness under reduced pressure to acquire the crude extract (CE). The CE (3.62 g) was further extracted using petroleum ether (Xilong Science Co., Ltd) and acetone (Xilong Science Co., Ltd), respectively, to obtain petroleum ether extract (1.18 g), acetone extract (1.38 g), and methanol residue (0.93 g).

Isolation, purification and identification of the active compound
Based on antifungal activity-directed isolation, the acetone extract was subjected to silica gel open column chromatography and eluted with a linear gradient of petroleum ether/ethyl acetate (2/1 → 1/1 → 1/2 → 1/5 → 0/1) and ethyl acetate/methanol (10/1 → 5/1 → 1/1 → 3/7). Eight fractions (F 1 -F 8 ) were obtained by combining similar fractions using thin-layer chromatography (TLC) detection and analysis. A white needle-like crystal was obtained after evaporation of the solvent in the bioactive fraction (F 2 ), which was then recrystallized to obtain a pure compound (J-10-1A) (Purity ≥ 98%). Identification was based on NMR spectra and ESI-MS (electrospray ionization mass spectrometry) spectrum comparing with the literatures. NMR spectra were recorded on a Bruker ADVANCE III 500 spectrometer (Bruker, Karlsruhe, Germany) at 400 MHz for 1 H and 100 MHz for 13 C, using tetra-methyl-silane (TMS) as an internal standard, and chemical shifts were recorded as δ values. ES-MS spectrum was measured using an Esquire HCT ion trap mass spectrometer (Bruker Daltonics Inc. Billerica, USA) using negative ion mode.

Determination of antifungal activity
The antifungal activity of J-10-1A was determined using a previously described method (Luo et al. 2017;. J-10-1A was dissolved in acetone: H 2 O (1:1, v/v) to obtain the desired concentration. The solution was evenly mixed with molten PDA medium (1:9, v/v) and poured into a Petri dish. The solvent of a broad-spectrum fungicide carbendazim instead of samples solution was used to prepare a positive control, and the acetone:water (1:1 by volume) was also used in the same way to prepare a negative control. The pathogenic fungi were inoculated on the agar medium (diameter 0.4 cm) and incubated at 28 ℃ for 72 h. The diameters of the fungal colonies were measured and the inhibitory rates were calculated. IC 50 values at 95% confidence intervals (95% CI) were calculated using the least-squares method.

Morphological characterization of Exserohilum turcicum
The fungal pathogen, E. turcicum was cultured on the PDA plates containing lasiodiplodin (test) or acetone: H 2 O solution (1:1, v/v) (control) and incubated for 72 h at 28 ℃. Thereafter, the mycelium morphology was observed by macroscopic and microscopic methods. A DM3000 microscope (Leica Microsystems Instrument Incorporated Company, Germany) was used for the microscopic observations.

Determination of the cell membrane permeability
Cell membrane permeability of E. turcicum was determined according to a previously described method with slight modifications Luo et al. 2019). The mycelia (1 g) were incubated in a rotary shaker at 120 rpm and 28 ℃ for 7 d and suspended in 10 mL of lasiodiplodin solution at 15.50 μg/ mL (the IC 50 of lasiodiplodin against E. turcicum). Acetone: H 2 O (1:1, v/v) solution was used as the control. Thereafter, the electrical conductivity (J 0 ) of each sample was measured. Subsequently, the samples were incubated at 28 ℃ and 120 rpm for 30, 60, 90, 120, 180, and 360 min, and the electrical conductivities (J 1 ) of the supernatants were measured for each time point. Lastly, the 360 min culture samples were boiled and the final electrical conductivity (J 2 ) was measured. Each treatment was replicated thrice. The relative permeability of the cell membranes was calculated using the following equation: where J 0 is the initial electrical conductivity, J 1 is the electrical conductivity of lasiodiplodin-treated samples at each time point, and J 2 is the final electrical conductivity of the 360 min lasiodiplodin-treated sample after being boiled.

Orthogonal designed experiment
On the basis of a previous orthogonal designed experiment using five factors, four levels were adopted to optimize the culture conditions to improve the yield of lasiodiplodin in L. pseudotheobromae J-10. L 16 (4 5 ) orthogonal table design was adopted for each factor level designed, and the settings are shown in Table 1.

Analytical procedures
An equal amount of methanol was added into the fermentation flask (containing broth and mycelia) standing for 24 h, and the solid and liquid components were separated by filtration. The solid component was extracted twice using methanol and filtered. Subsequently, all the filtrates were combined, evaporated to dryness, and then dissolved in 1 mL of methanol. The lasiodiplodin content in the solution was analyzed by high-performance liquid chromatography (HPLC) system (Shimadzu, Kyoto, Japan). A reversedphase Inertsil ODS-HL C 18 column (250 mm × 4.6 mm, 5 μm, Shimadzu, Inc., Kyoto, Japan) was used for separation using methanol: H 2 O (60:40, v/v) as a mobile phase at 1 mL/min flow rate. The temperature was maintained at 40 °C, and the UV detection wavelength was set at 230 nm. The sample injection volume was 5 μL. The LC-solution multi-PDA workstation was employed to acquire and process chromatographic data. Lasiodiplodin was detected and quantified using the HPLC standards (Purity ≥ 98%; Yuanye Biotechnology Co., Ltd, Shanghai, China). The linear equation of lasiodiplodin analysis was as follows: where Y was the peak area, X was the quality (μg) of the sample injected each time, and R was the correlation coefficient.

Statistical analysis
All the tests were replicated thrice, and the results were represented as mean ± standard deviation (SD). Analysis of variance (one-way ANOVA; PROC ANOVA; SAS version 8.2) was used to detect significant differences in the data. p-value ≤ 0.05 was considered statistically significant.

Lasiodiplodin toxicity toward phytopathogenic fungi
The results of lasiodiplodin toxicity analysis toward six plant pathogenic fungi are shown in

Effects of lasiodiplodin on Exserohilum turcicum colony and mycelial morphologies
E. turcicum was inoculated on plates containing lasiodiplodin (test) or acetone: H 2 O solution (1:1, v/v; control), and after incubating for 72 h at 28 ℃, the colony and mycelial morphologies were determined. There were significant differences in the colony and mycelial morphologies between the lasiodiplodin-treated (test) group and the control group. The lasiodiplodin-treated colonies (Fig. 2b) were shriveled, and their diameters were smaller than those of the control group (Fig. 2a). Light microscopic analysis revealed that   compared with the control (Fig. 2c), lasiodiplodin-treated mycelia had several skinny, shriveled, and distorted aberrations; moreover, non-septate mycelia was observed (Fig. 2  d).

Effects of lasiodiplodin on Exserohilum turcicum cell membrane permeability
The changes in the relative permeability of E. turcicum mycelia cytomembrane are shown in Fig. 3. The relative permeability was positively correlated with the treatment time. During the 30-360 min intervals, the relative permeability of the lasiodiplodin-treated group increased by 0.39-2.74 times, significantly higher than that of the control group (0-1.21 times). The sharp increase in the relative permeability in the test group indicated that lasiodiplodin could induce destruction of the cytoplasmic membranes and cause efflux of the intercellular content, ultimately leading to fungal death.

Optimization of cultural conditions
Results of the orthogonal test are shown in Table 3. A comparative analysis of the R values of the orthogonal test revealed that the most important factor for the optimal production of lasiodiplodin in E. turcicum was the C: N ratio, followed by the fermentation time; the effects of initial pH value, temperature, and liquid volume were comparatively low. In addition, the optimal fermentation conditions were obtained as follows: A 3 B 2 C 4 D 1 E 4 , i.e., initial  Table 3 Results of L 16 (4 5 ) orthogonal test Note: K represents the total yield of lasiodiplodin for every factor at each level and R stands for range (max-min). A − E represent initial pH, temperature (℃), liquid volumea (mL), carbon:nitrogen ratio (C:N, g:g), culture duration, respectively pH, 7; temperature, 28 ℃; liquid volume, 300/500 mL; C: N (sucrose:yeast extract) ratio, 1:0; and fermentation time, 20 d. Following these fermentation conditions, lasiodiplodin yield increased to 52.33 mg/L, much higher than that at pre-optimization (0.59 mg/L). It is worth noting that A 4 B 3 C 2 D 1 E 2 could be more suitable for practical application, considering the shorter fermentation period and low fermentation cost, i.e., initial pH, 8; temperature, 31 ℃; liquid volume, 200/500 mL; C: N ratio, 1:0; fermentation time, 10 d, with the lasiodiplodin yield reaching 45.10 mg/L.

Discussion
This is the first report on the isolation of lasiodiplodin from the EF L. pseudotheobromae J-10 associated with the medicinal plant S. glabra. Lasiodiplodin is a known macrolide antibiotic (Yang et al. 2006a) that is ubiquitous in plants and microorganisms and demonstrates important biological activities, including antileukemic (Lee et al. 1982), cytotoxic (Cao et al. 2011;Sobreira et al. 2016), and antimicrobial activities (Yang et al. 2006b;Li et al. 2016). In this study, we demonstrated the broad-spectrum antifungal activity of lasiodiplodin against six tested plant pathogenic fungi, with the highest activity against E. turcicum, C. capsici, and P. theae. The antifungal activity of lasiodiplodin was mediated via the alteration in the colony morphology and disruption of the cell membrane integrity of the tested fungal pathogens. Yang et al. (2006b) first reported the antimicrobial activity of lasiodiplodin and its antifungal activity against phytopathogen, F. oxysporum f. sp. cubense. Additionally, Li et al. (2016) found that lasiodiplodin demonstrates inhibition against phytopathogen, Ralstonia solanacearum. These results support that lasiodiplodin is a natural compound with high potential bioactivity against phytopathogens. Though the antifungal activity of lasiodiplodin was lower than the chemical fungicide carbendazim, its usage could reduce the use of traditional chemical agents. Lasiodiplodin could disturb the cell membrane integrity of E. turcicum. These results were similar to that found for the hyphae of other fungi (Pan et al. 2019a;Wang et al. 2021). The cell membrane plays a primary role in protecting the cellular components from the extracellular environment and encountering antimicrobial agents Xing et al. 2017). The leakage of cellular components could indicate damage of the fungal membrane (Marinho et al. 2005). Most of antifungal compounds reported so far primarily target the cell wall or cell membrane of fungi (Mandal et al. 2013;Kinoshita et al. 2016;Han et al. 2017). Lasiodiplodin induces the destruction of cytoplasmic membranes and cause efflux of the intercellular content, which could lead to the death of the fungi and could be used to prevent the plant disease, while further research using plants infected with the plant pathogenic fungi could give further clarifications of the potential application of lasiodiplodin in agriculture.
Efficient production of lasiodiplodin is critical for its application. Based on previous single factor tests, the optimal combination of the five influencing factors, temperature, pH, fermentation time, C:N ratio, and liquid volume, to improve lasiodiplodin yield in L. pseudotheobromae J-10 was obtained by orthogonal experiment. To the best of our knowledge, there is no report on the optimization of culture conditions for lasiodiplodin production, although it is commonly used in other fungal metabolites (He et al. 2012;Wang et al. 2022;Peng et al. 2022). The results of this study indicate that C: N ratio had the greatest effect on lasiodiplodin yield in L. pseudotheobromae J-10. Moreover, Adetunji et al. (2020b) found that C: N ratio significantly affected the herbicidal activity of L. pseudotheobromae C1136 metabolite, and the phytotoxic metabolite amended with C: N at 10:1 exhibited maximum disease severity on Chromolaena odorata and Echinochloa crus-galli weeds. C and N are popularly used as the basic components for the optimization of media to permit mass production of spores or vegetative propagules (Gao et al. 2007). There are several reports regarding the influence of the C: N ratio on the development, sporulation, and mycelia biomass of fungi with bio-activity potential (Kalim and Ali 2016;Adetunji et al. 2020b). Furthermore, the addition of other factors, such as metal ions, host extracts, precursors, epigenetic modifiers, and enzyme inhibitors, to the fermentation media has been widely reported to enhance secondary metabolite production in fungi (Pan et al. 2019b).  found that the addition of the host extract in the culture medium could enhance the anti-MRSA activity of the EF L. pseudotheobromae IBRL OS-64 against MRSA ATCC 33,591. Therefore, several factors are yet to be screened to enhance the production of lasiodiplodin and other active metabolites in L. pseudotheobromae J-10 of S. glabra.
Previously, S. glabra, an important medicinal plant, was reported to demonstrate significant antifungal activity against phytopathogens (Tong et al. 2022). Notably, the EF L. pseudotheobromae J-10 (used in this study), and the other EF from S. glabra , also displayed broad-spectrum antifungal activity against phytopathogenic fungi. These results underpin that EF could possess activities similar to the host, and also provide a basis for further study of EF in S. glabra.

Conclusion
It is the first report on the isolation of lasiodiplodin from the EF L. pseudotheobromae J-10 associated with the medicinal plant S. glabra, as well as the optimization of L. pseudotheobromae J-10 culture conditions to increase lasiodiplodin yield. Lasiodiplodin demonstrated broadspectrum antifungal activity against six phytopathogenic fungi, with the highest efficacy against E. turcicum, C. capsici, and P. theae. Its mode of action includes alteration of the colony morphology and disruption of the cell membrane integrity of the fungus. The optimal culture conditions to increase lasiodiplodin yield were as follows: initial pH, 7; temperature, 28 ℃; liquid volume, 300/500 mL; C: N ratio (sucrose: yeast extract), 1:0; fermentation time, 20 d, following which lasiodiplodin yield increased to 52.33 mg/L. The results of this study support that lasiodiplodin is a natural compound with high potential bioactivity against phytopathogens, and provided a basis for further study of EF in S. glabra.