Isolation and Identification of Secondary Metabolites Produced by Phytopathogenic Fungus Corynespora cassiicola from Hevea brasiliensis

The secondary metabolites of the phytopathogenic fungus Corynespora cassiicola CC01 from Hevea brasiliensis were investigated. As a result, two new compounds, 5-acetyl-7-hydroxy-6- methoxybenzofuran-2(3H)-one (1) and (S)-2-(2,3-dihydrofuro [3,2-c]pyridin-2-yl)propan-2-ol (2), together with seven known compounds, 4,6,8-trihydroxy-3,4-dihydronaphthalen-1(2H)-one (3), 3,6,8-trihydroxy-3,4-dihydronaphthalen-1(2H)-one (4), curvulin acid (5), 2-methyl-5-carboxymethyl- 7-hydroxychromone (6), tyrosol (7), p-hydroxybenzoic acid (8) and cerevisterol (9), were isolated from the fermentation extract by comprehensive silica gel, reverse phase silica gel, Sephadex-LH20 column chromatography and high-performance liquid chromatography (HPLC). The structures of these compounds were identified by using high-resolution electrospray mass spectrometry (HRESIMS), nuclear magnetic resonance spectroscopy (NMR), optical rotation, ultraviolet and infrared spectroscopy techniques and a comparison of NMR data with those reported in the literature. Compounds 1 and 2 were new compounds, and compounds 3–9 were discovered from this phytopathogenic fungus for the first time. Compounds 1–9 were tested for phytotoxicity against the fresh tender leaf of Hevea brasiliensis, and the results show that none of them were phytotoxic. Additionally, these compounds were subjected to an antimicrobial assay against three bacteria (E. coli, methicillin-resistant Staphylococcus aureus and Micrococcus luteus), but they showed no activity.


Introduction
Natural rubber is both an important industrial raw material and a strategic material [1]. For a long time, rubber leaf diseases threatened the safety of rubber production. Rubber tree Corynespora leaf fall (CLF) disease, caused by the fungus Corynespora cassiicola, is a devastating rubber tree leaf disease in many countries in Asia and Africa, and also threatens rubber production in China [2]. Cassiicolin, a small 27 amino acid glycoprotein with six cysteines engaged in three disulphide bonds, is the only phytotoxin characterised in C. cassiicola. Based on the amino acid residues and the sequence of the cassicolin-encoding gene, there are six different toxin classes (Cas1-6). Some strains lack the cassicolin-encoding gene, called Cas0 isolates, which can also infect Hevea brasiliensis and other host plants, indicating C. cassiicola can produce other phytotoxic compounds [3]. At present, abundant phytotoxic secondary metabolites, such as polyketides, peptides, terpenoids and nitrogenous metabolites, were isolated from many plant pathogenic fungi and have played an important role in pathogenicity [4,5]. For example, botryoisocoumarinn A and neoisocoumarin, previously isolated from Neufusicoccum batangarum, induced necrotic lesions around the inoculation points in a host (cactus pear) [6]. Both nectriapyrone and methylphomapyrone C were toxic to a number of non-host plants, according to the leaf puncture assay [7]. Polanrazines A, B, C, D and E, isolated from Polish isolates of Leptosphaeria maculans, showed moderate toxicity on leaves of brown mustard [8]. In addition, some secondary metabolites were isolated from C. cassiicola. Herbarin is a naphthoquinone congener, previously isolated from Corynespora sp. BA-10763 [9]. Twelve new chromone derivatives, corynechromones A-L, were previously reported from the sponge-derived fungus C. cassiicola XS-200900I7, collected from the Xisha Islands coral reef in the South China Sea [10]. Additionally, coryoctalactones A-E were isolated from the endolichenic fungal strain C. cassiicola (JCM 23.3). All these compounds were evaluated for their antimicrobial, cytotoxic and antitrypanosomal activities. Unfortunately, none of these isolated natural products proved to be active in any of the bioassays performed [11]. These previous results revealed that the C. cassiicola species had the potential to produce structurally diverse metabolites.
In our previous study, we analyzed the population and pathogenicity diversity of C. cassiicola collected from tropical plants in China and found the Cas5 and Cas2 isolates could cause CLF disease in rubber trees in China, with Cas5 isolates being the dominant strains [12]. We further determined gapless genome sequences of Cas5 (strain CC01) and Cas2 (strain YN49) isolates and found C. cassiicola contained abundant secondary metabolite gene clusters, and these clusters significantly differed among the different Cas types and exhibited a different virulence. These data indicate that the phytogenic C. cassiicola can synthesize abundant secondary metabolites and may contain important pathogenic factors [13]. In order to uncover the secondary metabolites and phytotoxic metabolites produced by C. cassiicola, we investigated the fermentation extract of CC01 strain, a representative strain of Cas5 isolates in this study.
All of the isolated compounds were evaluated for their phytotoxicity against the fresh tender leaves of Hevea brasiliensis. The results show that none of them were phytotoxic. Additionally, the compounds were subjected to antimicrobial assay against three bacteria (E. coli, Methicillin-resistant Staphylococcus aureus and Micrococcus luteus). However, no compounds showed antimicrobial activity.
All of the isolated compounds were evaluated for their phytotoxicity against the fresh tender leaves of Hevea brasiliensis. The results show that none of them were phytotoxic. Additionally, the compounds were subjected to antimicrobial assay against three bacteria (E. coli, Methicillin-resistant Staphylococcus aureus and Micrococcus luteus). However, no compounds showed antimicrobial activity.

Phytopathogenic Fungal Material
The fungal strain C. cassiicola CC01 (CGMCC 3.20258) was isolated from rubber trees collected from the plantations in Yangjiang, Guangdong province. The strain was maintained in our laboratory. The strain of C. cassiicola CC01 was grown on potato glucose medium (potato 200.0 g, glucose 20.0 g, agar 18.0 g, pure water 1.0 L, sterilized at 121 • C for 20 min in a high-pressure steam sterilization pot) and cultured at 28 • C for 4 days. After that, the activated strain was inoculated into a 500 mL flask containing 200 mL PDB liquid medium (potato 200.0 g, glucose 20.0 g, pure water 1.0 L, sterilized at 121 • C for 20 min in a high-pressure steam sterilization pot) and incubated at a constant temperature shaker for 72 h (160 r/min, 28 • C). Then 10 mL of the liquid seed solution was inoculated into a solid medium (rice 40.0 g, 60 mL water, sterilized at 121 • C for 20 min in a high-pressure steam sterilization pot) for fermentation for 30 days.

Extraction and Isolation
The strain C. cassiicola CC01 was extracted three times with an equal volume of ethyl acetate and the organic solvents were combined and evaporated under reduced pressure to produce a crude extract (83.08 g). Then the extract was separated onto a silica gel CC with CH 2 Cl 2 /MeOH (100:0 to 0:100, v/v) to obtain nine fractions (Fr.A-Fr.H). Fr.G, was subjected to an ODS reverse-phase CC under reduced pressure (MeOH-H 2 O, 20% to 80%, v/v), Sephadex LH-20 CC and semi-preparative HPLC to yield compound 1 (30.7 mg, t R = 8.

Electronic Circular Dichroism Calculation
Monte Carlo conformational searches were conducted by means of the Spartan's 14 software using Merck Molecular Force Field (MMFF). The conformers with Boltzmannpopulation of over 5% were chosen for ECD calculations, and then the conformers were initially optimized at B3LYP/6-31g level in gas. The theoretical calculation of ECD was carried out in MeOH using a Time-dependent Density functional theory (TD-DFT) at the B3LYP/6-31+g (d, p) level for all conformers of compound 2. Rotatory strengths for a total of 30 excited states were calculated. ECD spectra were generated using the program SpecDis 1.6 (University of Würzburg, Würzburg, Germany) and GraphPad Prism 5 (University of California San Diego, USA) from dipole-length rotational strengths by applying Gaussian band shapes with sigma = 0.3 eV.

Phytotoxic Assay
The activities of the isolated compounds were measured by leaf puncture [25] to test their phytotoxicity. Healthy and fresh rubber tree leaves in the light green stage (highresistance germplasm of Reyan7-33-97 planted in the Yanfeng Experimental Base of the Chinese Academy of Tropical Agricultural Sciences) were used as inoculum material. The compounds were tested at 0.1 mg/mL and 1 mg/mL (dissolved in 5% methanol-aqueous solution) on rubber tree leaves, using a small insect needle (over 1 mm 2 ). One drop of the test sample (5 µL) was placed on the gently punctured spot, with 10 min vacuum infiltration. Leaflets were maintained in a moist environment at 28 • C under alternate light (photoperiod 12 h/12 h) and the effects of the metabolites on the leaves were observed at 72 h.

Antimicrobial Assay
The antimicrobial activities of the metabolites were measured by the filter paper bacteriostatic method. Compounds 1-9 were evaluated against three bacteria (E. coli, Methicillin-resistant Staphylococcus aureus and Micrococcus luteus). Each compound was tested at 1.0 mg/mL in a DMSO solvent. DMSO solution was used as a negative control and kanamycin solution (10 mg/mL) was used as a positive control. Each test was treated with three replicates and incubated at 28 • C for 12 h. Then the size of the inhibition zone was recorded.

Conclusions
In this study, nine compounds were isolated and purified from the fermentation extract of C. cassiicola CC01, an important phytopathogenic fungus causing rubber leaf diseases. Compounds 1 and 2 were identified as new compounds and compounds 3-9 were discovered from this phytopathogenic fungus for the first time. Compound 3 was previously identified from a freshwater fungus, YMF 1.01029 and showed a weak nematocidal activity against xylocysts. Compound 4 is a novel metabolite with antifungal activity isolated from Phomopsis sp. [26]. Compound 5 was reported to have a weak anti-HIV-1 integrase chain transfer activity, with an IC 50 value of 75.1 µmol/L Compound 6 is a chromone derivative firstly isolated from Rhubarb equinosa in 1984 [27]. Compound 7 can be used for the synthesis of medolol, betalol, salidroside and other drugs [28]. Compound 9 was first isolated from Myriapora truncate in nature in 1985 [29]. All of the isolated compounds were evaluated for their phytotoxicity, and the results show that none of them were phytotoxic at 0.1 mg/mL and 1 mg/mL, indicating that there may be some trace phytotoxic components of this fungus that have not been isolated. The subsequent exploration for phytotoxic metabolites will continue. Although no pathogenic molecules have been found from this phytopathogenic fungus, the new compounds 1 and 2 and the other seven natural products were discovered from the secondary metabolites of this fungus for the first time, which further enriched the chemical diversity produced by this pathogenic fungus.