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Diversity and Antibacterial and Antioxidant Activities of Fungal Endophytes from the Roots of Eucalyptus deglupta

In this study, 45 endophytic fungal strains were isolated from the roots of Eucalyptus deglupta . Among them, 16 distinct strains were identified and classified into 14 different genera ( Celoporthe, Aspergillus, Castanediella, Chaetomium, Biscogniauxia, Sordariales, Pestalotiopsis, Clitopilus, Cylindrocladiella, Calonectria, Trichoderma, Xylaria, Neofusicoccum and Pleosporales) according to their morphological characteristics and molecular information. The genera Aspergillus and Calonectria were the dominant endophytic fungi in the roots of E. deglupta. In addition, the antibacterial and antioxidant activities of the 16 endophytic fungi isolated from the roots of E. deglupta were evaluated. All the strains displayed inhibitory activities against Agrobacterium tumefaciens, Bacillus subtilis, Escherichia coli, and Xanthomonas vesicatoria . Strains Edf-1 to Edf-4, Edf-11 and Edf-12 demonstrated strong inhibitory activity against R. solanacearum with plaque diameters between 5 and 10 mm. The crude extract of Edf-14 had inhibitory activity against all tested bacteria. Five strains, Edf-1 to Edf-5, demonstrated a strong scavenging capacity for 2,2-diphenyl-1-picrylhydrazyl (DPPH), with IC 50 values of 0.26 ± 0.04, 0.11 ± 0.03, 0.20 ± 0.05, 0.10 ± 0.04 and 0.14 ± 0.02 mg/mL, respectively. Hence, endophytic fungi isolated from the roots of E. deglupta showed antibacterial and antioxidant activities, providing a theoretical foundation for further isolation and identification of specific active components.


INTRODUCTION
Plant endophytic fungi are microorganisms present in living plant tissues that do not harm host plants but rather play a vital role in their protection against pathogens, animals and harsh environmental conditions. They are considered a source of abundant secondary metabolites with significant biological activities (Mollaei et al. 2019). Endophytic fungi produce identical secondary metabolites as the host plants with similar biological activities (Zhao et al. 2017). To some extent, studies on the biological activity of endophytic fungi could reduce the large-scale utilisation of plants to protect the natural environment. Endophytic fungi have many prospects because of their biodiversity and bioactive metabolites (Tiwari et al. 2018;Wu et al. 2019). Various research has shown that endophytic fungi are a vital resource of bioactive secondary metabolites which possess antibacterial, anticancer, antiviral, insecticidal and antioxidant activities and phytoremediation potential Ferdous et al. 2019;Palanichamy et al. 2018;Pansanit & Pripdeevech 2018;Ye et al. 2019). Diverse endophytic fungi have been reported to produce bioactive compounds such as alkaloids (Mohamadi et al. 2018;Patel et al. 2012), terpenoids (Yuan et al. 2018), phenols (Jacobo-Velázquez & Cisneros-Zevallos 2020; Sahni et al. 2020), peptides (Jiang et al. 2013;Song et al. 2019), steroids (Kim & Shim 2019;Miao et al. 2019), lactones (Kaaniche et al. 2019) and pyrenes (Kim & Shim 2019).
Bacterial wilt caused by Ralstonia solanacearum is one of the most serious diseases of Eucalyptus spp. in China (Wang et al. 2014). However, efficient control measures for this disease remain limited. Eucalyptus spp. are important fast-growing timber and economical tree species that possess antibacterial, cytotoxic, antifungal, and high allelopathic activities (El-Rokiek et al. 2019;Miguel et al. 2017;Syukri et al. 2020;Tiwari et al. 2018). Eucalyptus spp. are rich in flavonoids and phenolics, also a potential source of novel antibacterial and antioxidant compounds (Nasr et al. 2019;Nwabor et al. 2019;Shang et al. 2019). In the previous study, four new depsidones together with nine known compounds were isolated from the endophytic fungus Chaetomium sp. Eef-10 associated with E. exserta, and demonstrated different antibacterial, cytotoxic, anticancer, and antioxidant activities (Ouyang et al. 2020(Ouyang et al. , 2018. Ten endophytic fungal strains were isolated from E. citriodora where Ecf-4 (Rhytidhysteron sp., MK211261) and Ecf-1 (Epicoccum sp., MK211258) displayed the most vigorous activity against R. solanacearum (Shan et al. 2019).
However, the secondary metabolites of endophytic fungi in E. deglupta have rarely been studied. In this study, the endophytic fungi from the roots of E. deglupta were isolated and identified, and the antibacterial and antioxidant activities of secondary metabolites produced by the endophytic fungi were also evaluated. This study aimed to screen endophytic fungal strains with significant biological activity and provides theoretical guidance for the prevention and control of bacterial wilt of Eucalyptus spp. as well as prospects for further separation and identification of specific active components. The endophytic fungi were isolated using the method from our previous report (Shan et al. 2019). After separation and purification, the purified strains were stored at 4 °C on the inclined surface of potato dextrose agar (PDA) in a 5 mL cryopreservation tube. The colonisation frequency (CF) of each isolate was determined as CF = (N COL /N t ) × 100, where N COL is the number of each fungus and N t is the total number of segments, according to the method of Hata and Futai (1995).

MORPHOLOGICAL IDENTIFICATION
The morphological characteristics and identification were conducted according to a standard taxonomic key including colony diameter, texture, color and the dimensions and morphology of hyphae and conidia (Ainsworth et al. 1973). First, the edge hyphae of the endophytic fungi were inoculated onto the PDA medium, and cultures were grown in darkness at 28 ℃ for 3-20 days. Then, the cultures were identified based on the colony morphology.

ANALYSIS
Genomic DNA of the endophytic fungal strains was extracted using a DNA extraction kit. A total of 3 µL of DNA extract, 1 µL of ITS4 and ITS5, and 25 µL of DNA were mixed, and 20 µL of sterile deionised water were added to the PCR tube. Universal primers for endophytic fungi, ITS4 (5´-TCCTCCGCTTATTGATATGC-3´) and ITS5 (5´-GGAAGTAAAAGTCGTAACAAGG-3´), were used for ITS-rDNA amplification as reported previously (Shan et al. 2019). The PCR amplification procedure started with pre-denaturation at 94 ℃ for 3 min, followed by 30 cycles of denaturation at 94 °C for 30 s, annealing at 52 ℃ for 30 s, and extension at 72 °C for 8 min. The amplification was completed by holding the reaction mixture at 72 °C for 10 min to allow complete extension of PCR products. The PCR products were stored at 4 °C. The sequences were sequenced and analysed using the BLAST program against the National Center for Biotechnology Information database and submitted to GenBank, where the accession numbers were obtained. After MAFTT (version 7) processing, MEGA7 software was used to construct a phylogenetic tree with the maximum likelihood method.

EXTRACTS
Different crude extracts of endophytic fungi were prepared according to the method of Ouyang et al. (2020). Briefly, the endophytic fungi were cultivated on PDA in Petri dishes for 3 to 5 days. The mycelia were inoculated into 250 mL Erlenmeyer flasks containing 100 mL of potato glucose liquid medium (5 flasks for each fungal culture). Seed cultures were injected into sterilized rice medium (25 g per flask) and stored for 60 days at 25 °C. Later, the fermentation product was soaked in EtOAc for 7 days. This process was repeated three times. Finally, the fermentation liquid was filtered using vacuum with reduced pressure until concentrated to dryness. The crude extract was collected into a clean penicillin bottle for future use and stored at 4 to 5 °C.

ANTIBACTERIAL ACTIVITY ASSAY
Thin-layer chromatography (TLC)-bioautography assays for different endophytic fungal crude extracts were carried out as described previously ). All the crude extracts were dissolved with a mixture of reagents, and the appropriate solution was extracted using a 0.5 mm capillary tube and sampled on a TLC plate. The developing agent used for TLC was petroleum ether:acetone (4:1, V/V). Then, 0.2 mg/mL streptomycin sulfate at the origin point on one side of the TLC plate was used as the positive control. A certain amount of prepared bacterial solution (150 mL LB + 10 mL bacterial solution) was added to sterilised LB semisolid medium (agar concentration of 0.5%), and the mixture was oscillated and homogenised. The prepared bacterial suspension was evenly inoculated onto a silica gel plate with a pipette. Bacterial inoculated TLC plates were placed at 28 ℃ for 12 h. After 12 h, color developing agent MTT was sprayed uniformly on the TLC plates. The experimental results were observed after approximately 2 min. The bacterial growth inhibition area was white and the other areas were blue and purple. Antibacterial activity was detected as white inhibition zones against a purple background and the R f value of the antibacterial area was determined. R f = D 1 /D 2 where D 1 is the distance between the antimicrobial area and the initial sample point, and D 2 is the distance between the developing solvent front and initial sample point on a TLC plate. At the same time, the diameter of each antibacterial area was measured.

ANTIOXIDANT ACTIVITY ASSAY
All the crude extracts were tested for the antioxidant activity based on the reduction of a methanol solution of 2, 2-diphenyl-1-picrylhydrazyl (DPPH) as described previously (Ouyang et al. 2018). First of all, 20 mg of DPPH was accurately weighed and dissolved in 100 mL of absolute ethanol. Dimethyl sulfoxide (DMSO) was used to configure the samples of different crude extracts with an initial concentration of 20 mg/mL, and then the solutions with concentrations of 10 mg/mL to 0.15625 mg/mL were successively diluted by the half dilution method. The initial concentration of the positive control BHT was 2 mg/mL, and later, the solution was diluted from 1 to 0.15625 mg/mL using the same method. Second, 80 µL of DPPH and 20 µL of sample solution were added to a 96 microwell plate, and each concentration gradient was replicated three times. Sealing film was used to create an airtight cover, the light absorption value was measured at 517 nm by horizontal oscillation for 10 min, and then the mixtures were heated in a water bath at 37 ℃ for 30 min. A total of 20 µL DMSO solution was used as a negative control instead of sample solution, and the treatment was repeated three times. The inhibition (%) of free radicals (DPPH) was calculated as [(A control / A sample )/ A control ] × 100, where A control is the absorbance of the negative control, and A sample is the absorbance of each crude extracts. Then, the results from all replicates were plotted and analysed using Excel. The logarithm (X) of the sample concentration was taken, and the clearance rate was converted to a probability (Y). Finally, the regression equation (Y = aX+ B) and IC 50 value of antioxidant activity were obtained.

RESULTS AND DISCUSSION
Forty-five endophytic fungal strains were isolated from the roots of E. deglupta. According to morphological characteristics and molecular identification results, the 16 strains shown in Figure 1 and Table 1 were identified successfully and assigned to 14 genera namely Aspergillus, Celoporthe, Castanediella, Chaetomium, Biscogniauxia, Sordariales, Pestalotiopsis, Clitopilus, Cylindrocladiella, Calonectria, Trichoderma, Xylaria, Neofusicoccum, and Pleosporales. After submitting their ITS4-5.8S-ITS5 partial sequences to the GenBank, accession numbers (KX960786 -KX960801) and the closest related species were obtained. All the sequenced strains had more than 99% similarity with the closest related species in GenBank (Table 1). The primary fungal isolates were Aspergillus sp. and Calonectria sp., with CFs of 20.0 and 13.3%, respectively, as shown in Table 1.
All the genera were grouped into clades in the phylogenetic tree based on ITS sequences. They were categorised into 8 groups which are Xylariomycetidae,

D i a p o r t h o m y c e t i d a e , H y p o c r e o m y c e t i d a e ,
Sordariomycetidae, Eurotiomycetidae, Botryosphaeriales, Pleosporomycetidae and Agaricomycetidae (Figure 2). It has been proven that endophytic fungi are related to the growing site, climate, tissues, organs and age of the host plant (Miguel et al. 2017 In this study, a TLC-bioautography assay was conducted to evaluate the antibacterial activity of secondary metabolites produced by endophytic fungi. This assay was a rapid screening method for secondary metabolites with antibacterial activity mainly according to the diameters of inhibition spots and the R f values . The inhibitory activity values of 16 different crude extracts are listed in Table 2. All the crude extracts showed antibacterial activity against X. vesicatoria, and the plaque diameters of Edf-7, Edf-9, Edf-11 to Edf-14 and Edf-16 were between 5 and 10 mm. Strain Edf-14 displayed inhibitory activity against all the tested bacteria. Edf-14 belonging to Neofusicoccum sp. exhibited a broad spectrum of antibacterial activity and had excellent inhibitory activity against bacterial strains. Strains Edf-1 to Edf-4, Edf-11 and Edf-12 demonstrated strong inhibitory activity against R. solanacearum with plaque diameters between 5 and 10 mm. Strain Edf-11 (Castanediella sp.) had strong antibacterial activity against R. solanacearum, A. tumefaciens, and X. vesicatoria. To date, there have been no relevant studies on the secondary metabolites of Castanediella sp. Edf-2 displayed the strongest inhibitory activity against A. tumefaciens, and the plaque diameter was greater than 10 mm. Most of the strains lacked inhibitory activity against P. lachrymans, with the exceptions of Edf-11 and Edf-14. Edf-8 showed the weakest antibacterial activity and had only weak inhibitory activity against X.
vesicatoria. The R f value is closely related to the polarity of the compound. In this study, the antibacterial activity in the R f range of 0.20 to 0.50 was stronger than that in the R f range of 0.00 to 0.20. This antibacterial assay result indicated that the secondary metabolites produced by the endophytic fungi of E. deglupta were mainly intermediate-polarity substances. In addition, inhibitory activity of different endophytic fungal crude extracts did not differ between Gram-positive and Gram-negative bacteria.  Note: -, antimicrobial activity was not observed; +, the diameter of the antimicrobial activity area was 0-5 mm; ++, the diameter of the antimicrobial activity area was 5-10 mm; +++, the diameter of the antimicrobial activity area was more than 10 mm; the positive control was streptomycin sulfate which was only sampled on the TLC plate and showed antibacterial activity The antioxidant activity of 15 different crude extracts from all the isolates except Edf-16 was determined by DPPH free radical scavenging assay (Figure 3). Crude extracts of Edf-1 (Aspergillus sp.), Edf-2 (Aspergillus sp.), Edf-3 (Celoporthe sp.), Edf-4 (Castanediella sp.) and Edf-5 (Chaetomium sp.) showed excellent antioxidant activities with the IC 50 values of 0.26 ± 0.04, 0.11 ± 0.03, 0.20 ± 0.05, 0.10 ± 0.04 and 0.14 ± 0.02 mg/mL, respectively. In previous reports, it was found that the secondary metabolites from Chaetomium sp. had strong antioxidant activities (Tantapakul et al. 2020;Wang et al. 2019;Yao et al. 2019). Edf-10, Edf-13 and Edf-14 displayed weak antioxidant activity with high IC 50 values (> 2.00 mg/mL). In addition, the rest of the extracts showed IC 50 values from 0.50 to 2.00 mg/mL, such as that from Edf-6, with a value of 0.50 mg/mL.

CONCLUSIONS
This study presented the identification and biological activities of endophytic fungi from the economic tree E. deglupta for the first time. Endophytic fungi from the roots of E. deglupta distributed in 14 different genera where Aspergillus sp. and Calonectria sp. were the dominant strains. Endophytic fungi Edf-1 to Edf-5, Edf-11 and Edf-12 showed obvious bioactivity. These strains have the potential to be pharmaceuticals that inhibit bacterial wilt of Eucalyptus spp. or other pathogenic bacteria as well as exhibited antioxidant activity. They could be used as candidate strains for further isolation of active compounds. This study provided an important theoretical basis for the large-scale development and application of biological pesticides and biological control of diseases.