Javanicin, an Antibacterial Naphthaquinone from an Endophytic ...

Curr Microbiol (2009) 58:233–238 DOI 10.1007/s00284-008-9313-7

Javanicin, an Antibacterial Naphthaquinone from an Endophytic Fungus of Neem, Chloridium sp. Ravindra N. Kharwar Æ Vijay C. Verma Æ Anuj Kumar Æ Surendra K. Gond Æ James K. Harper Æ Wilford M. Hess Æ Emil Lobkovosky Æ Cong Ma Æ Yuhao Ren Æ Gary A. Strobel

Received: 23 July 2008 / Accepted: 13 October 2008 / Published online: 19 November 2008 Ó Springer Science+Business Media, LLC 2008

Abstract The endophytic fungus Chloridium sp. produces javanicin under liquid and solid media culture conditions. This highly functionalized naphthaquinone exhibits strong antibacterial activity against Pseudomonas spp., representing pathogens to both humans and plants. The compound was crystallized and the structure was elucidated by X-ray crystallography. The X-ray structure confirms the previously elucidated structure of the compound that was done under standard spectroscopic methods. The importance of javanicin in establishing symbiosis between Chloridium sp. and its host plant, Azadirachta indica, is briefly discussed.

R. N. Kharwar (&) V. C. Verma A. Kumar S. K. Gond Mycopathology and Microbial Technology Laboratory, Department of Botany, Banaras Hindu University, Varanasi 221005, India e-mail: [email protected] J. K. Harper Department of Chemistry, University of Utah, 315 South 1400 East, Salt Lake City, UT 84112, USA W. M. Hess Department of Plant and Wildlife Sciences, Brigham Young University, Provo, UT 84602, USA E. Lobkovosky Department of Chemistry, Baker Laboratory, Cornell University, Ithaca, NY, USA C. Ma Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06520, USA Y. Ren G. A. Strobel Department of Plant Sciences, Montana State University, Bozeman, MT 59717, USA

Introduction An endophytic fungus, Chloridium sp. (J.F.H. Beyma) W. Gams and Holubova-Jchova [10] (anamorph: Lasiosphaeriaceae), was originally isolated in pure culture from the roots of Azadirachta indica A. Juss. (Meliaceae) obtained in the Varanasi district of India. This plant is commonly known as ‘‘neem’’ or ‘‘Indian Lilac’’ and is found in several natural habitats in various parts of India. This plant is known worldwide for its medicinal properties and incredible insecticidal activities and thus it is extensively harvested. ‘‘Azadirachtin’’ and other limnoides of the tetranortriterpenoids group are the main insecticidal and pharmaceutical compounds extracted from leaves, stems, and fruits of this plant. For hundreds of years, humans used the extracts of this plant for wound-healing and antiseptic purposes in rural and suburban India. In the modern pesticide market, the neem extracts are made into oils, emulsions, and other derivative formulations to control harmful agricultural pests. The most active ingredient among the tetranortriterpenoids group of limnoides is azadirachtin. Recently, azadirachtin has been synthesized chemically from ‘‘epoxide 2.’’ This compound alone can serve as a precursor to all three groups of limnoides, including azadirachtin, azadirachtol, and meliacarpin [25, 26]. In addition to the fungus Chloridium, a large number of other microbial endophytes have been reported from A. indica [18, 21, 27]. Presently, efforts have been made to isolate the bioactive compounds from fungal endophytes of this host plant [12, 13, 22, 24]. Some10-membered bioactive lactones are now known from Phomopsis sp., isolated as an endophyte from the stem of the neem plant. Some of these lactones have very promising activity against such plant pathogens as Ophiostoma minus and Botrytis cinerea

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with minimum inhibitory concentration (MIC) values 31.25 and 62.50 lg/ml, respectively [28]. Furthermore, an endophytic Geotrichum sp., isolated from the leaves of the neem tree, has been reported to produce two new chlorinated epimeric 1,3-oxazinane derivatives that have significant activity against the nematodes Bursaphelenchus xylophilus and Panagrellus redivevus [17]. Thus, in continuation of these works we hereby reported a highly functionalized naphthaquinone ‘‘javanicin,’’ with promising antibacterial activity from an endophytic Chloridium sp. that was isolated from the surface treated root tissues of A. indica A. Juss. This fungus is for the first time reported from this host plant, is characterized at molecular level, and produces javanicin, which has been characterized and its structure confirmed by X-ray crystallography.

Materials and Methods

R. N. Kharwar et al.: Javanicin, an Antibacterial Naphthaquinone

pressure thin ranged from 5 to 6 Torr, providing humidity up to 100% at the sample. Molecular Characterization of Endophytic Chloridium The fungus was grown on potato dextrose broth for 7 days and the mycelium was harvested; then the genomic nucleic acid (DNA) was extracted using a DNeasy Plant and Fungi Mini Kit (Qiagen) according to the manufacturer’s directions. Additionally, an ITS-5.8S rDNA analysis was performed followed by a BLAST search [1], the ITS regions of the fungus were amplified using polymerase chain reaction (PCR) and the universal ITS primers ITS1 (50 TCC GTA GGT GAA CCT GCG G 30 ) and ITS4 (50 TCC TCC GCT TAT TGA TAT GC 30 ). All other procedures were carried out as previously described [9]. The DNA was sequenced at the W.M. Keck Facility at Yale University. The sequence data of this fungus are deposited in GenBank as EU 394444.

Isolation of Endophytic Chloridium The fungus discussed in this report was isolated from a symptomless 1 cm-diameter piece of neem root that had been surface treated with 75% ethanol and 5.0% NaOCl (v/ v) for 2 min to decontaminate the surface from bacteria and fungi. The dissected tissues of the root were placed on water agar and the endophytic fungi eventually began to produce hyphal filaments after a few days of incubation [23]. The hyphal tips that did appear were carefully transferred to other fresh Potato Dextrose Agar (PDA) plates. One of the fungi that emerged was Chloridium sp. and this is the first report of its presence in this host plant. After obtaining it in pure culture, the fungus was transferred on to pieces of c-irradiated carnation leaves to induce the formation of asexual fruiting structures that ultimately provided evidence of its taxonomic identity. SEM and ESEM Studies of Chloridium Scanning electron microscopy (SEM) was performed on Chloridium sp., using the techniques described by Ezra et al. [9]. After processing, the fungal material was dried, coated with gold by the sputtering technique, and examined with a JEOL 6100 SEM. However, because the conidia and conidiophores of this fungus appeared fragile and were easily disrupted, the organism was subjected to the relatively unique microscopic application that preserves the sporophore intact (spores attached). Thus, fresh or nontreated specimens were examined by environmental scanning electron microscopy (ESEM) and images were recorded with a Philips XL 30 ESEM FEG. A gaseous secondary electron detector was used with a spot size of 3, at 15 kV. The temperature was 4°C, with a chamber

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Fermentation, Extraction, and Characterization of Javanicin The javanicin (naphthaquinone) was isolated from 4-weekold still cultures of Chloridium sp., grown in 1 L of Potato Dextrose Broth (PDB) in two 2 L flasks at 25°C. The fungal mats were removed from both flasks by filtration and the culture fluid was extracted twice with equal volumes of ethyl acetate. The ethyl acetate fraction was dried by flash evaporation and the yield of crude material was up to 10 g. This material was dissolved in a minimal volume of methylene chloride and was subjected to two successive silica gel chromatographic steps; the first silica gel chromatography step was performed on a 3 9 30-cm column. The organic solvent extract was applied to the column and eluted using 500 ml of chloroform:acetonitrile:water (10:1:0.1 v/v). The first 100 ml eluted from the column was discarded and the next 400 ml eluting from the column (possessing bioactivity) was taken to dryness (350 mg), redissolved in 2.0 ml of methanol, and placed on a second silica gel column (identical in size to the first column). It was eluted with methylene chloride:methanol (20:10 v/v). Fractions (20 ml) were collected and the bioactivity appeared at 150–200 ml. Assessment of the chemical purity of the bioactive compound was carried out in four chromatographic systems using thin-layer chromatography (TLC). TLC was conducted on Merck 0.25-mm silica gel plates (5 9 10 cm). The Rf values recorded for each system are as follows: ethyl acetate:methanol, 5:1 v/v, 0.70; chloroform:methanol, 7:1 v/v, 0.67; ethyl acetate:tetrahydrofuran, 3:1 v/v, 0.58; methylene chloride:acetonitrile, 2:1 v/v, 0.56. Detection of the compound could be made by its fluorescence at 254 nm or by spraying the plate with a


vanillin sulfuric acid spray reagent followed by gentle heating resulting in a reddish color [6]. The final silica gel step did yield a compound that appeared as a single spot on the TLC plates in each solvent system with Rf values as indicated. In addition, the compound was applied (*30 lg in 40 ll) to a Microsorb-MV-100-5 Varian C-18 reversephase high-performane liquid chromatography (HPLC) column (4.6 9 250 mm) and subjected to a gradient of 0– 100% methanol for the first 10 min, followed by a gradient of methanol to acetonitrile of 100–60% to 0–40% over the next 30 min, respectively. The flow rate was 1 ml/min and detection of the compound could be made by its absorbance at 254 nm. Although some minor peaks appeared, a major peak resulted with a retention time of 7.75 min by all measures. The compound appeared to be identical to the previously reported compound from Fusarium sp. javanicin [2]. Javanicin decomposes in the range 206–210°C. It has four UV absorption peaks at k 237, 300, 472, and 500, with millimolar extinction coefficients in methanol of 2.17, 1.84, 1.16, and 1.26, respectively. High-resolution mass spectroscopy of javanicin on a Bruker Qe FT-FT–ICR MS instrument revealed an (M ? H)? peak at 291.08741. The calculated best-fit empirical formula of this parent compound is C15H14O6. The calculated molecular weight of javanicin –H? = 290.1120. Single-Crystal X-Ray Crystallography of Javanicin A slow evaporation of methanol and acetone 1:1 (v/v) solution of javanicin at 4°C yielded numerous small individual reddish crystals. Crystals were transferred from a crystallization vessel into a drop of viscose oil (polybutenes). Using a nylon loop, a suitable specimen (fineneedle-like crystal of size 0.4 9 0.05 9 0.02 mm3) was chosen and mounted on a Bruker X8 APEX II diffractometer (MoKa radiation) and cooled to -100°C. Data collection and reduction were done using Bruker APEX2 [v.1.0–22 User Manual, Bruker AXS Inc., Madison WI 53719, 2004] and SAINT ? [v.6.02 User Manual, Bruker AXS Inc., Madison WI 53719, 1999] software packages. An empirical absorption correction was applied with SADABS [G.M. Scheldrick, Program for Empirical Absorption Correction of Area Detector Data, University of Go¨ttingen, 1996]. Structures were solved by direct methods and refined on F2 by full-matrix least-squares techniques using SHELXTL [G.M.Scheldrick, v. 5.10 Bruker AXS Inc., Madison WI 53719, 1999 d] software package. All nonhydrogen atoms were refined anisotropically. Hydrogen atoms were found in a difference Fourier map and refined isotropically. Overall, 9476 reflections were collected, 1782 of which were symmetry independent (Rint = 10%); with 890 ‘‘strong’’ reflections (with Fo [ 4rFo), final R1 = 5.16%. The crystal belonged to

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space group P21/c with a = 4.806(7), b = 34.480(5), and ˚ . The molecular structure was determined c = 8.2564(11) A by single-crystal X-ray diffraction (see Fig. 2). Antimicrobial Activity Bioassay A variety of plant pathogenic fungi, bacteria, and a human pathogen (Candida albicans) were selected to assess the biological activity of javanicin. MIC was determined for each test organism. MIC (in lg/mL) values for javanicin were obtained using a standard plate bioassay test method that employed a microtiter plate containing various concentrations of the test compounds against fungal and bacterial test organisms in PDB and Nutrient broth. Each well, after 24 h, was visually observed, and the well showing no growth next to the well having some growth was taken as the MIC values. The bioassay test consisted of the placement of an aliquot (20 ll) of each column fraction on sterile paper disks and allowing the solvent to evaporate to dryness on the surface in a decontaminated (alcoholsprayed) laminar flow hood for 15–20 min. Subsequently, 5-mm plugs of PDA containing a Gram-negative Pseudomonas aeruginosa and P. fluorescens were placed within 1 cm of the test residue previously deposited on the plate. After 3 days of incubation at 25°C, bacterial colony inhibition could be assessed by virtue of its uneven growth, with little or no growth developing toward the dried residue.

Results and Discussion Identity of the Fungal Endophyte The endophytic fungus Chloridium sp. (J.F.H. Beyma) W. Gams and Holubova-Jchova [10] was isolated in pure culture from the roots of A. indica A. Juss. (Meliaceae) obtained in the Varanasi [25.58 N 82.98 E, elevation 279 ft (85 m)] district of India. The fungus used in this report was isolated from a symptomless 1.0-cm-diameter piece of neem root that had been surface treated with 75% ethanol and 5.0% NaOCl (v/v) for 2 min to decontaminate root surfaces from bacteria and fungi. Both SEM and ESEM clearly showed single-celled conidia on conidiophores appearing as ball-like forms, with some swollen structures (chlamydospores) also present in the hyphal masses. With ESEM, there was less hyphal/spore distortions appearing in the images (Fig. 1). This fungus was initially identified on the basis of these structures as Chloridium sp. An ITS-5.8S rDNA analysis followed by a BLAST search [1] revealed that the closest relative to this fungus is an unidentified ascomycete SV13F8 at the 100% level of 545/545 bases. Obviously, the ITS-5.8S rDNA analysis was inconclusive

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Fig. 1 Photographs (SEM) of Chloridium sp. showing spores and hyphae (left) and a conidiophore with an assemblage of spores (S) taken with by ESEM (right)

because the closest BLAST search match was an unidentified ascomycete SV13F8. Although this GenBank entry was an unidentified ascomycete, it possibly could have been a non-spore-producing isolate of Chloridium sp. The sequence data of this Chloridium sp. is deposited in GenBank as EU 394444. The fungus itself is deposited as isolate No. 2343 in the living mycological culture collection of Montana State University, Bozeman, MT, USA. It is also deposited in the Mycopathology and Microbial Technology Laboratory, Department of Botany, Banaras Hindu University, India (RNK-AzR-0142/07). Biological Relationship of Chloridium sp. to its Host An interesting aspect of this fungus is that it does not cause any detectable adverse effects or diseaselike symptoms on the host (neem) from which it was isolated, which confirms that it meets the description of an endophyte. To confirm its relationship to the host, pure cultures of this fungus were inoculated into marked roots of A. indica. Despite 1 year of incubation of the fungus in the roots of neem, there was no evidence of the development of any disease-related symptoms. Thus, it appears that the fungus is successfully living as an endophyte in the root system of this plant, perhaps in a symbiotic state. Because the fungus is living in the internal root tissues of the host, it, therefore, might be acquiring its support and nutrition from the host and, in turn, the fungus might have a role in the host–plant relationship by producing antimicrobials that might ward off disease-causing organisms that might otherwise harm the host. Isolation and Characterization of Javanicin from Chloridium Following the methods described earlier, a biologically active compound was isolated from cultures of Chloridium sp. This compound was identical in all respects to a previously described naphthaquinone, javanicin. However, it was crystallized and its structure was confirmed by X-ray crystallography (Fig. 2). This appears to be the first report of this compound from a fungal genus other than Fusarium

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spp. Biologically active naphthaquinones have been previously reported, and among these, javanicin was isolated for the first time from the soil fungus Fusarium javanicum [2]. Then Gatenbeck and Bentley described the mechanism of javanicin production from soil fungi [11]. Some other workers have also isolated several naphthaquinones from different species of Fusarium, but none was significantly active either against Pseudomonas spp. or to most fungal pathogens [3, 4, 16]. A colored extracellular dimeric naphthaquinone, aurofusarin, is produced by Fusarium decemecellulare from Russia [20]. Aurofusarin shows the naphthazarin structure such as the other naphthaquinones, including javanicin, anhydrojavanicin, fusarubin, anhydrofusarubin, bostricoidin, and novarubin. The biosynthesis of naphthaquinone pigments seems to be responsible for warding off fungi to maintain their growth under stressed conditions [20]. Only a few examples of natural products are known from Chloridium sp. Among them is a new 12-membered macrolide (chloriolide; 1) from Chloridium virescens var. chlaymydosporum (NRRL 37636) [14]. A novel D-glucose6-phosphate phosphohydrolase (G6Pase) inhibitor, CJ21,164, was also isolated from the fermentation broth of Chloridium sp. Compound CJ-21,164 inhibited G6Pase in rat liver microsomes with an IC50 (inhibitory concentration) of 1.6 lM. Glucose output from hepatocytes isolated from rat liver was inhibited when CJ-21,164 was present in the incubation medium, consistent with the role of CJ-21164 as a G6Pase inhibitor [15]. Biological Activity of Javanicin A variety of plant pathogenic fungi, bacteria, and a human pathogen (C. albicans) were selected to assess the biological activity of javanicin. The MIC was determined for each test organism. The javanicin was either slightly active or not active against fungi such as Pythium ultimum, Phytophthora infestans, Botrytis cinerea, and Ceratocystis ulmi, whereas it was active against C. albicans, Escherichia coli, Bacillus sp., and Fusarium oxysporum at higher MIC values ranging from 20 and 40 lg/ml. The activity of javanicin was recorded against Rhizoctonia solani and


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b

a Fig. 2 Computer generated model of the structure of javanicin (a) with the structural formula (b)

Table 1 MIC tests of javanicin for a representative group of plant pathogenic fungi and bacteria Fungus/bacterium

Family

Javanicin (lg/ml)

Bacillus sp.

Bacillaceae

40

Botrytis cinerea

Moniliaceae

Not active

Candida albicans

Saccharomycetaceae

40

Ceratocystis ulmi

Erysiphaceae

Not active

Cercospora arachidicola

Dematiaceae

5

Escherichia coli

Enterobacteriaceae

40

Fusarium oxysporum Pseudomonas fluorescens

Tuberculariaceae Pseudomonadaceae

20 2

Phytophthora infestans

Pytheaceae

Not active

Pseudomonas aerugenosa

Pseudomonadaceae

2

Pythium ultimum

Pytheaceae

Not active

Rhizoctonia solani

Agonomycetaceae

10

Verticillium dahlae

Moniliaceae

10

Note: The family name of each organism tested is presented next to the specific fungal/bacterial name in the table. Specific details on how the MIC tests were conducted are given in the Material and Methods section. The experiments were twice repeated with essentially the same results

Verticillium dahliae at 10 lg/ml while it was active at 5 lg/ml against Cercospora arachidicola (Table 1). The bacteria that were the most sensitive to the javanicin (2 lg/ ml) were P. aeruginosa and P. fluorescens (Table 1, Fig. 3). This biological activity is impressive given the fact that many antibiotics are ineffective against these Gramnegative bacteria [19]. Several Pseudomonas spp. cause severe diseases to animals, humans, and a variety of plants [5, 7, 8]. Thus, as some species of pseudomonas have developed resistance to many existing drugs, there is good reason to search for new biologically active compounds [19]. Recently, it appears that compounds derived from natural sources have not been the subject of intensive search by the agriculture and medicinal industries throughout the world, especially in developing and underdeveloped countries. Additionally, with the advent of an increased

Fig. 3 The inhibition zones produced by javanicin against P. fluorescens

public concern related to the hazards of synthetic chemicals used to treat agricultural crops, there might be a place for naturally derived compounds to replace the man-made products now in use. Although javanicin shows antibacterial activity against Pseudomonas spp., representing Gramnegative pathogenic bacteria of both human and plants (Table 1), there is some prospect that it can be used to control the diseases caused by this group of bacteria. Given the fact that Chloridium sp. seems to live in an endophytic association with certain higher plants, without causing disease symptoms, it might symbiotically provide protection to its host by warding off potential pathogens. Thus, a biological rationale would suggest that if Chloridium sp. is acting as a symbiotic endophyte providing protection to its plant host, certain plant pathogenic members of the Pseudomonadaceae might be the best target for javanicin. Javanicin is the only chemically characterized antimicrobial produced by this endophytic fungus [14]. Although several phytopathogenic fungi and bacteria, including some human pathogens, are not sensitive to javanicin, this compound has impressive effectiveness against Pseudomonas spp., making it a valuable selective antibiotic (Table 1). In addition, there is efficacy of javanicin against R. solani, V. dahliae, and C. arachidicola, which also cause a variety of diseases among many plant species, suggesting a role for it in plant protection. Being the first endophytic Chloridium sp. isolated from this host, it seems reasonable that other isolates of this organism that exist in other hosts might offer opportunities for the isolation and characterization of other unique antimicrobials.

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238 Acknowledgments One of the authors (R.N.K.) is thankful to the Department of Science and Technology, New Delhi, for award of a ‘‘BOYSCAST fellowship’’ [(SR/BY/L-02/06) 2006-2007] to study at MSU. Support from the NSF, a Howard Hughes professorship to Scott Strobel at Yale University, and the Montana Agricultural Experiment Station are also acknowledged. We thank Dr T.T. Lam at the W.M. Keck Foundation Biotechnology Laboratory at Yale University for the FT-ICR MS analyses. Financial support to other authors (R.N.K., V.C.V., S.K.G., A.K.) from CSIR and UGC is gratefully acknowledged.

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