Environment Health Techniques Metabolites of Bacillus sp. and their nematicidal activities
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Short Communication Isolation and identification of chemical constituents from the bacterium Bacillus sp. and their nematicidal activities Liming Zeng1,2, Hui Jin1,3, Dengxue Lu4, Xiaoyan Yang1, Le Pan1,2, Haiyan Cui1,2, Xiaofeng He1, Hongdeng Qiu1 and Bo Qin1 1
2 3
4
Key Laboratory of Chemistry of Northwestern Plant Resources of CAS and Key Laboratory for Natural Medicine of Gansu Province, Lanzhou Institute of Chemical Physics, Chinese Academy of Sciences, Lanzhou, China University of Chinese Academy of Sciences, Beijing, China Key Laboratory of Tobacco Diseases and Insect Pests Monitoring Controlling and Integrated Management, Institute of Tobacco Research, Chinese Academy of Agricultural Sciences, Qingdao, China Institute of Biology, Gansu Academy of Sciences, Lanzhou, China
A strain SMrs28 was isolated from the rhizosphere soil of a toxic plant Stellera chamaejasme and identified as Bacillus sp. on the basis of morphological and partial 16S rRNA gene sequence analysis. The crude extract of SMrs28 fermentation broth showed strong nematocidal activities in preliminary test. To define the active nematocidal metabolites of SMrs28, a novel compound (1), 4-oxabicyclo[3.2.2]nona-1(7), 5,8-triene, along with five known compounds (2–6), were isolated from the strain by various column chromatographic techniques and characterized on the basis of spectroscopic analysis. Results of the in vitro nematicidal tests showed that the metabolites presented different levels of activity at certain exposure conditions. Compounds (1–3) displayed LC50 values of 904.12, 451.26, 232.98 mg/ml and 1594.0, 366.62, 206.38 mg/ml against Bursaphelenchus xylophilus and Ditylenchus destructor at 72 h, respectively. This is the first report of the nematicidal activity of the compounds as constituents of Bacillus sp.. Our findings help to find potential chemical structures to develop nematicides from microbial source for the management of nematode-infected plant diseases.
: Additional supporting information may be found in the online version of this article at the publisher’s web-site. Keywords: Bacillus sp. SMrs28 / Novel compound / Secondary metabolites / Nematicidal activity Received: October 21, 2014; accepted: March 25, 2015 DOI 10.1002/jobm.201400798
Introduction Plant-parasitic nematodes have become serious problems to agriculture and forestry worldwide. Reportedly, nematodes are responsible for the annual global damage on agriculture, being estimated as ca. 100 billion [1]. In past years, synthetic chemical pesticides have been commonly used to control the nematode-infested plant diseases [2]. However, the application of chemical pesticides in long term and wide scale caused Correspondence: Bo Qin, Key Laboratory of Chemistry of Northwestern Plant Resources of CAS and Key Laboratory for Natural Medicine of Gansu Province, Lanzhou Institute of Chemical Physics, Chinese Academy of Sciences, Lanzhou 730000, China E-mail: [email protected] Phone: þ86-931-4968372 Fax: þ86-931-8277088 ß 2015 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim
environmental pollution and enhancement of resistance in nematodes [3]. Meanwhile, toxic pesticide residues can also affect the safety of agricultural products and endanger human health. In view of the safety to nontarget organisms and the side-effects on environment, the conventional chemical nematicides have been increasingly de-registered or prohibited to use. It is urgent to seek for safe, effective, and eco-friendly nematocidal alternatives from natural resources [4, 5]. Numbers of investigations have shown that the secondary metabolites produced by bacteria were proved to possess a wide range of biological activities including killing nematode activity for extensive applications [6–8]. For example, substances produced by strains of Bacillus megaterium, B. cereus, B. thuringiensis, and B. pumilus caused high lethality against Meloidogyne exigua J2s [9]. In recent
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several decades, large numbers of commercial pesticidal products derived from microorganisms have been exploited and marketed as bio-pesticides [10]. Stellera chamaejasme L. is a dominant toxic plant species widely distributed in the grasslands of northern and western China, its nematocidal active secondary metabolites were isolated and identified [11]. In the process of studies on the structure of microbial communities, a strain was isolated from the rhizospheric soil of S. chamaejasme and identified as Bacillus sp., labeled as SMrs28. Since the pinewood nematode Bursaphelenchus xylophilus is a major threat to forestry leading to a billion US dollars economic losses per year [12], and tuber rot nematode Ditylenchus destructor is one of the most damaging species for agriculture [13], we used these two nematode species as target organisms in the test. The pre-test results have shown that the crude extract of the fermentation broth of SMrs28 had strong nematocidal activities. In this work, the metabolites of SMrs28 were isolated and characterized; their nematicidal activities against B. xylophilus and D. destructor were evaluated to find potential compounds for the development of nematicides from microbial source to manage the nematodes infected plant diseases.
Materials and methods Bacterial isolation, identification and screening of rhizobacteria with nematicidal activity In our previous study, 72 bacterial strains were isolated and identified from the rhizosphere soil of S. chamaejasme [14, 15]. The screening of bacteria with nematicidal activity was conducted following the procedures previously reported with some modifications [16]. In brief, after the bacterial strains were grown in the medium (per liter:15 g glucose, 10 g yeast extract, 6 g KH2PO4, 4 g K2HPO4 3H2O, 0.2 g MgSO4 7H2O, pH 7.2) for 48 h at 180 rpm, and 28 °C, the suspensions were centrifuged to obtain the supernatants. To test the nematicidal activity, 300 ml of each bacteria supernatants were incubated with about 100 nematodes for 72 h at 25 °C. The ratio of dead nematodes was assessed under light microscope. All tests were performed in triplicate and repeated twice. Nematodes incubated in water under the same conditions were used as the control. After testing for the nematicidal activity of their fermentation supernatants, seven strains showed capacity to produce metabolites with nematicidal activity. Among the seven strains, the SMrs28 exhibited the most potent activity. Thus the SMrs28 was selected to further study the nematicidal activity of the compounds. ß 2015 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim
Isolation of compounds from strain SMrs28 After activated on solid medium, the strain was subjected to fermentation. The harvested fermentation broth was isolated and purified with serial chromatographic techniques. Eventually, six compounds were obtained (details on procedures see Supporting Information). Nematicidal activity assay The plant-parasitic nematodes, B. xylophilus and D. destructor were selected as the target nematodes. The second stage juveniles (J2s) was collected based on the protocol reported previously [17]. An aqueous suspension of nematodes was prepared for the subsequent assay. The effect of compounds on B. xylophilus and D. destructor was assessed by the previously described method with some modifications [18]. The nematodes was considered dead when presenting “I” or “L”-shaped body, even did not recover after delivering to clean water [17]. The mortality was calculated as the ratio of dead nematodes to the tested ones. According to the Schneider-Orelli formula, the toxicity values were expressed as the proof mortality standard derivation [19] (details see Supporting Information). Statistical analysis The corrected values were subjected to one-way analysis of variation (ANOVA) with Duncan post hoc multiple comparisons at p ¼ 0.05 using SPSS software for Windows (Version 14.0, SPSS Inc., Chicago, USA). To evaluate the nematicidal effects of the compounds against B. xylophilus and D. destructor, the half lethal concentration (LC50) values were calculated by undergoing probit analysis.
Results and discussion Identification of strain SMrs28 After blasted to the Genbank database, there has been one identical to the targeting sequence deposited with the accession number FJ215794. Therefore, the strain SMrs28 was identified as Bacillus sp. Structure determination of compounds Compound 1 was obtained as a colorless needle crystal. Melting point: 70–75 °C. UV (MeOH) lmax 201, 224, 278 nm. IR (KBr) nmax 3024, 2955, 2928, 2879, 1514, 1365, 1233, 1051, 867, 817, 731, 556 cm1. The molecular formula was determined as C8H8O by high-resolution electrospray ionization mass spectrometry (HR-ESI-MS) (m/z 121.0645 [M þ H]þ, calcd. for C8H9O, 121.0648). The 1 H and 13C NMR spectrum showed typical signals of one
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Metabolites of Bacillus sp. and their nematicidal activities
1,4-disubstituted benzene ring at dH ¼ 7.02 (d, 2H, J ¼ 8.4 Hz, 2H-7, 8), 6.69 (d, 2H, J ¼ 8.4Hz, 2H-6, 9), and dC ¼ 156.70 (C-5), 130.94 (C-1), 130.85 (d, C-7,8), 116.08 (C6,9). This was further supported by the 1H–1H COSY correlations of H-6/H-7 and H-8/H-9. The mutual single correlation between H-2 (dH 2.70) and H-3 (dH 3.67) in the 1 H-1H COSY as well as the alkyl secondary carbon signals at dC 64.54 (C-3), 39.36 (C-2) indicated the presence of a oxygenated dimethylene group. The HMQC spectrum supports the presence of two pairs of CH signals at dC 130.85(C-7, 8), 116.08(C-6, 9), and two CH2 signals at dC 39.36(C-2), 65.54(C-3). In the HMBC spectrum, obvious correlations from H-7 (dH 7.02) to C-2 (dC 39.36), C-8 (dC 130.85) and C-5 (dC 156.70), from H-6 (dH 6.69) to C-9 (dC 116.08), C-1 (dC 130.94), and C-5 (dC 156.70), from H-3 (dH 3.67) to C-2 (dC 39.36), C-3 (dC 65.54), and C-1 (dC 130.94), and from H-2 (dH 2.70) to C-2 (dC 39.36), C-3 (dC 65.54), C-7, or C-8 (dC 130.85) can be found, which established the novel compound as 4-oxabicyclo[3.2.2]nona-1(7), 5,8triene (Fig.1). Based on the spectroscopic data and comparison with the previously reported data, compounds (2–6) were identified as (3S, 8aS)-hexahydro-3methylpyrro[1,2-a]pyrazine-1, 4-dione, Phenylacetamide, Cyclo (L-Pro-L-Val), Lauric acid, methyl elaidate, respectitively (Fig. 1) [20–24] (detailed spectroscopic data see Supporting Information). Nematicidal activity In vitro, the nematicidal assay was carried out using direct contact method. As the results showed, the three compounds exhibited different levels of activity against B. xylophilus and D. destructor at certain time point and concentration. Compound 1 and 2 showed moderate activity against the test nematodes at 800 ppm after incubation for 72 h, respectively, while the compound 3 displayed strong activity targeting the tested organisms
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(Table 1, Fig. 2). Overall, the mortality of the three compounds against D. destructor tends to be higher than the latter one. The test of compound 4 against nematodes failed due to the limited amount. The LC50 values of the compounds (1–3) against B. xylophilus and D. destructor were 904.12, 451.26, 232.98 mg/ml and 1594.0, 366.62, 206.38 mg/ml, respectively, as listed in Table 2. So far, many nematicidal agents have been discovered from microorganisms such as bacteria, and fungi [25, 26], which suggested that the microorganism-based resources could serve as an abundant reservoir to explore the effective nematicidal agents. In this work, several compounds were obtained from SMrs28. The compound 2 has been isolated from plant, animal, and bacterium-based sources and has antifeedant, antifungal, and toxic activity at varying levels [27–30]. There’s been no data concerning its nematicidal activity yet. However, from the available literature, in which the compound with a hydroxyl at C-10 exhibited much remarkable fungal activity than other two compounds either with the hydroxyl group attached to the proline ring or without any substitutions, we can propose that the nematicidal activity of compound 2 could be possible partly related to the substitution at C-3 of the prolinebased diketopiperazines [31]. The nematicidal activity of compound 3 was also described here for the first time. Hudgins has reported that phenylacetates possess cytostatic activity due to their lipophilicities against human prostate carcinoma DU145 cells in vitro [32]. Considering the ease for compound entering into the lipid bilayer of cell membrane, the lipophilicity may be essential in some degrees for the nematicidal activity of compound 3 [32]. It is worth noting that the compound 1 was isolated for the first time as a new structure. Although a moderate nematicidal activity of compound 1 was merely observed, however, it has an unusual
Figure 1. Structures of compounds 1–6. ß 2015 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim
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Table 1. Nematicidal activity of three compounds(1–3) from Bacillus sp. SMrs28 against Bursaphelenchus xylophilus and Ditylenchus destructor. Data are given as mean standard deviation. Proof mortality (%,B. xylophilus/ D. destructor) Compounds
Concentrations (ppm)
24 h
48 h
72 h
50 100 200 400 800 50 100 200 400 800 50 100 200 400 800
9.15 4.54/13.51 5.05 15.78 4.06/16.44 5.17 21.08 3.36/17.70 4.34 27.46 5.37/30.51 4.52 36.37 4.09/38.49 5.66 9.04 4.41/11.61 5.70 13.37 5.49/16.31 1.93 19.65 5.52/17.13 3.05 24.32 3.41/19.43 4.68 33.43 6.88/49.34 5.96 18.36 3.68/21.15 2.33 21.20 3.67/21.87 1.26 21.81 2.77/25.05 5.54 34.52 3.97/37.38 5.12 68.90 6.47/76.51 6.09 3.66 4.30/4.04 4.61
12.60 5.10/17.35 5.70 19.23 3.42/24.50 7.22 26.75 6.32/25.69 6.25 29.78 2.76/40.89 5.65 43.07 4.87/39.83 4.55 18.15 5.89/19.77 2.10 20.19 3.99/20.06 3.85 23.66 1.70/23.40 4.81 27.71 3.81/27.01 4.21 50.71 5.44/62.94 2.61 20.68 2.71/27.46 2.29 23.64 3.18/33.06 2.41 29.48 3.91/40.09 3.07 37.84 5.26/40.31 3.07 74.12 6.08/78.34 3.96 3.98 0.45/4.81 3.97
17.88 6.27/27.70 6.45 25.23 4.95/33.31 5.67 33.48 6.13/37.28 4.56 40.49 5.78/42.10 6.23 47.57 7.82/44.37 6.63 26.64 3.05/22.73 1.61 32.68 4.14/28.67 3.74 37.36 2.94/37.48 5.27 45.04 3.45/50.07 5.53 60.55 6.39/65.19 1.37 25.11 5.64/36.73 4.36 33.58 2.65/40.61 4.72 42.03 5.56/43.76 6.07 57.28 3.76/47.97 2.05 78.05 6.31/78.51 5.71 6.11 5.31/6.52 4.39
1
2
3
Control (1% DMSO, v/v)
Figure 2. Plant-parasitic nematodes after treatment with compound 3 isolated from Bacillus sp. SMrs28 (A, non-treated D. destructor; B, D. destructor after treated with at 800 ppm for 72 h; C, Non-treated B. xylophilus; D, B. xylophilus after treated with at 800 ppm for 72 h). ß 2015 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim
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Table 2. Effect of compounds on the mortality of two nematodes, Bursaphelenchus xylophilus and Ditylenchus destructor within 72 h in vitro. LC50(ppm) Compounds
B. xylophilus
D. destructor
904.12 451.26 232.98
1594.0 366.62 206.38
1 2 3
carbon skeleton containing an oxygen bridge linking to the 1, 4-position of benzene ring, which can be potentially developed as a lead compound to make effective candidates for nematicides. As yet, litter is known about the mode of action of active compounds against nematodes. The interference of nervous system was the main one discussed on such as commercial nematicides, carbamates, and orgnophosphrous [33]. Inhibition of acetylcholinesterase and glutathione S-transferase of nematodes by compounds were also reported [34]. The observed activity may be attributed to one mechanism or synergetic effect of several ones, therefore, further work is required to shed light on the action mechanism.
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Acknowledgments We are grateful to Professor Frank Stermitz for editorial assistance. This work was supported by the National Natural Science Foundation of China (grant no. 31070386), Basic Research Program of Lanzhou Institute of Chemical Physics, Chinese Academy of Sciences (080423SYR1), the associate scholar program for talents cultivation plan of “Western Light” of Chinese Academy of Sciences, the Province-academy Cooperation Program of Henan Province of China (grant no. 102106000021), and the open project of Key Laboratory of Tobacco Diseases and Insect Pests Monitoring Controlling and Integrated Management, Institute of Tobacco Research, Chinese Academy of Agricultural Sciences (IPM 201404).
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Conflict of interest: The authors declare no competing financial interest.
[16] Paiva, G., ProenSc a, D.N., Francisco, R., Verissimo, P., et al., 2013. Nematicidal bacteria associated to pinewood nematode produce extracellular proteases. PLoS One, 8, e79705.
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[29] Li, T., Wang, G.C., Huang, X.J., Ye, W.C., 2013. Whitmanoside A, a new a-pyrone glycoside from the leech Whitmania pigra. Heterocycles, 87, 1537–1543. [30] Li, X.J., Zhang, Q., Zhang, A.L., Gao, J.M., 2012. Metabolites from Aspergillus fumigatus, an endophytic fungus associated with Melia azedarach, and their antifungal, antifeedant, and toxic activities. J. Agric. Food Chem., 60, 3424– 3431. [31] Li, X.J., Tang, H.Y., Duan, J.L., Gao, J.M., et al., 2013. Bioactive alkaloids produced by Pseudomonas brassicacearum subsp. Neoaurantiaca, an endophytic bacterium from Salvia miltiorrhiza. Nat. Prod. Res., 27, 496–499. [32] Hudgins, W.R., Shack, S., Myers, C.E., Samid, D., 1995. Cytostatic activity of phenylacetate and derivatives against tumor cells. Correlation with lipophilicity and inhibition of protein prenylation. Biochem. Pharmacol., 50, 1273–1279. [33] Musilek, K., Holas, O., Pohanka, M., Opletalova, V., et al., 2010. Progress of acetylcholinesterase reactivators against organophosphorus pesticides-almost 60 years since pralidoxime. Toxicol. Lett., 196, S314–S314. [34] Kang, J. S., Kim, E., Lee, S. H., Park, I.K., 2013. Inhibition of acetylcholinesterases of the pinewood nematode, Bursaphelenchus xylophilus, by phytochemicals from plant essential oils. Pestic. Biochem. Phys., 105, 50–56.
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Short Communication Isolation and identification of chemical constituents from the bacterium Bacillus sp. and their nematicidal activities Liming Zeng1,2, Hui Jin1,3, Dengxue Lu4, Xiaoyan Yang1, Le Pan1,2, Haiyan Cui1,2, Xiaofeng He1, Hongdeng Qiu1 and Bo Qin1 1
2 3
4
Key Laboratory of Chemistry of Northwestern Plant Resources of CAS and Key Laboratory for Natural Medicine of Gansu Province, Lanzhou Institute of Chemical Physics, Chinese Academy of Sciences, Lanzhou, China University of Chinese Academy of Sciences, Beijing, China Key Laboratory of Tobacco Diseases and Insect Pests Monitoring Controlling and Integrated Management, Institute of Tobacco Research, Chinese Academy of Agricultural Sciences, Qingdao, China Institute of Biology, Gansu Academy of Sciences, Lanzhou, China
A strain SMrs28 was isolated from the rhizosphere soil of a toxic plant Stellera chamaejasme and identified as Bacillus sp. on the basis of morphological and partial 16S rRNA gene sequence analysis. The crude extract of SMrs28 fermentation broth showed strong nematocidal activities in preliminary test. To define the active nematocidal metabolites of SMrs28, a novel compound (1), 4-oxabicyclo[3.2.2]nona-1(7), 5,8-triene, along with five known compounds (2–6), were isolated from the strain by various column chromatographic techniques and characterized on the basis of spectroscopic analysis. Results of the in vitro nematicidal tests showed that the metabolites presented different levels of activity at certain exposure conditions. Compounds (1–3) displayed LC50 values of 904.12, 451.26, 232.98 mg/ml and 1594.0, 366.62, 206.38 mg/ml against Bursaphelenchus xylophilus and Ditylenchus destructor at 72 h, respectively. This is the first report of the nematicidal activity of the compounds as constituents of Bacillus sp.. Our findings help to find potential chemical structures to develop nematicides from microbial source for the management of nematode-infected plant diseases.
: Additional supporting information may be found in the online version of this article at the publisher’s web-site. Keywords: Bacillus sp. SMrs28 / Novel compound / Secondary metabolites / Nematicidal activity Received: October 21, 2014; accepted: March 25, 2015 DOI 10.1002/jobm.201400798
Introduction Plant-parasitic nematodes have become serious problems to agriculture and forestry worldwide. Reportedly, nematodes are responsible for the annual global damage on agriculture, being estimated as ca. 100 billion [1]. In past years, synthetic chemical pesticides have been commonly used to control the nematode-infested plant diseases [2]. However, the application of chemical pesticides in long term and wide scale caused Correspondence: Bo Qin, Key Laboratory of Chemistry of Northwestern Plant Resources of CAS and Key Laboratory for Natural Medicine of Gansu Province, Lanzhou Institute of Chemical Physics, Chinese Academy of Sciences, Lanzhou 730000, China E-mail: [email protected] Phone: þ86-931-4968372 Fax: þ86-931-8277088 ß 2015 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim
environmental pollution and enhancement of resistance in nematodes [3]. Meanwhile, toxic pesticide residues can also affect the safety of agricultural products and endanger human health. In view of the safety to nontarget organisms and the side-effects on environment, the conventional chemical nematicides have been increasingly de-registered or prohibited to use. It is urgent to seek for safe, effective, and eco-friendly nematocidal alternatives from natural resources [4, 5]. Numbers of investigations have shown that the secondary metabolites produced by bacteria were proved to possess a wide range of biological activities including killing nematode activity for extensive applications [6–8]. For example, substances produced by strains of Bacillus megaterium, B. cereus, B. thuringiensis, and B. pumilus caused high lethality against Meloidogyne exigua J2s [9]. In recent
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several decades, large numbers of commercial pesticidal products derived from microorganisms have been exploited and marketed as bio-pesticides [10]. Stellera chamaejasme L. is a dominant toxic plant species widely distributed in the grasslands of northern and western China, its nematocidal active secondary metabolites were isolated and identified [11]. In the process of studies on the structure of microbial communities, a strain was isolated from the rhizospheric soil of S. chamaejasme and identified as Bacillus sp., labeled as SMrs28. Since the pinewood nematode Bursaphelenchus xylophilus is a major threat to forestry leading to a billion US dollars economic losses per year [12], and tuber rot nematode Ditylenchus destructor is one of the most damaging species for agriculture [13], we used these two nematode species as target organisms in the test. The pre-test results have shown that the crude extract of the fermentation broth of SMrs28 had strong nematocidal activities. In this work, the metabolites of SMrs28 were isolated and characterized; their nematicidal activities against B. xylophilus and D. destructor were evaluated to find potential compounds for the development of nematicides from microbial source to manage the nematodes infected plant diseases.
Materials and methods Bacterial isolation, identification and screening of rhizobacteria with nematicidal activity In our previous study, 72 bacterial strains were isolated and identified from the rhizosphere soil of S. chamaejasme [14, 15]. The screening of bacteria with nematicidal activity was conducted following the procedures previously reported with some modifications [16]. In brief, after the bacterial strains were grown in the medium (per liter:15 g glucose, 10 g yeast extract, 6 g KH2PO4, 4 g K2HPO4 3H2O, 0.2 g MgSO4 7H2O, pH 7.2) for 48 h at 180 rpm, and 28 °C, the suspensions were centrifuged to obtain the supernatants. To test the nematicidal activity, 300 ml of each bacteria supernatants were incubated with about 100 nematodes for 72 h at 25 °C. The ratio of dead nematodes was assessed under light microscope. All tests were performed in triplicate and repeated twice. Nematodes incubated in water under the same conditions were used as the control. After testing for the nematicidal activity of their fermentation supernatants, seven strains showed capacity to produce metabolites with nematicidal activity. Among the seven strains, the SMrs28 exhibited the most potent activity. Thus the SMrs28 was selected to further study the nematicidal activity of the compounds. ß 2015 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim
Isolation of compounds from strain SMrs28 After activated on solid medium, the strain was subjected to fermentation. The harvested fermentation broth was isolated and purified with serial chromatographic techniques. Eventually, six compounds were obtained (details on procedures see Supporting Information). Nematicidal activity assay The plant-parasitic nematodes, B. xylophilus and D. destructor were selected as the target nematodes. The second stage juveniles (J2s) was collected based on the protocol reported previously [17]. An aqueous suspension of nematodes was prepared for the subsequent assay. The effect of compounds on B. xylophilus and D. destructor was assessed by the previously described method with some modifications [18]. The nematodes was considered dead when presenting “I” or “L”-shaped body, even did not recover after delivering to clean water [17]. The mortality was calculated as the ratio of dead nematodes to the tested ones. According to the Schneider-Orelli formula, the toxicity values were expressed as the proof mortality standard derivation [19] (details see Supporting Information). Statistical analysis The corrected values were subjected to one-way analysis of variation (ANOVA) with Duncan post hoc multiple comparisons at p ¼ 0.05 using SPSS software for Windows (Version 14.0, SPSS Inc., Chicago, USA). To evaluate the nematicidal effects of the compounds against B. xylophilus and D. destructor, the half lethal concentration (LC50) values were calculated by undergoing probit analysis.
Results and discussion Identification of strain SMrs28 After blasted to the Genbank database, there has been one identical to the targeting sequence deposited with the accession number FJ215794. Therefore, the strain SMrs28 was identified as Bacillus sp. Structure determination of compounds Compound 1 was obtained as a colorless needle crystal. Melting point: 70–75 °C. UV (MeOH) lmax 201, 224, 278 nm. IR (KBr) nmax 3024, 2955, 2928, 2879, 1514, 1365, 1233, 1051, 867, 817, 731, 556 cm1. The molecular formula was determined as C8H8O by high-resolution electrospray ionization mass spectrometry (HR-ESI-MS) (m/z 121.0645 [M þ H]þ, calcd. for C8H9O, 121.0648). The 1 H and 13C NMR spectrum showed typical signals of one
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1,4-disubstituted benzene ring at dH ¼ 7.02 (d, 2H, J ¼ 8.4 Hz, 2H-7, 8), 6.69 (d, 2H, J ¼ 8.4Hz, 2H-6, 9), and dC ¼ 156.70 (C-5), 130.94 (C-1), 130.85 (d, C-7,8), 116.08 (C6,9). This was further supported by the 1H–1H COSY correlations of H-6/H-7 and H-8/H-9. The mutual single correlation between H-2 (dH 2.70) and H-3 (dH 3.67) in the 1 H-1H COSY as well as the alkyl secondary carbon signals at dC 64.54 (C-3), 39.36 (C-2) indicated the presence of a oxygenated dimethylene group. The HMQC spectrum supports the presence of two pairs of CH signals at dC 130.85(C-7, 8), 116.08(C-6, 9), and two CH2 signals at dC 39.36(C-2), 65.54(C-3). In the HMBC spectrum, obvious correlations from H-7 (dH 7.02) to C-2 (dC 39.36), C-8 (dC 130.85) and C-5 (dC 156.70), from H-6 (dH 6.69) to C-9 (dC 116.08), C-1 (dC 130.94), and C-5 (dC 156.70), from H-3 (dH 3.67) to C-2 (dC 39.36), C-3 (dC 65.54), and C-1 (dC 130.94), and from H-2 (dH 2.70) to C-2 (dC 39.36), C-3 (dC 65.54), C-7, or C-8 (dC 130.85) can be found, which established the novel compound as 4-oxabicyclo[3.2.2]nona-1(7), 5,8triene (Fig.1). Based on the spectroscopic data and comparison with the previously reported data, compounds (2–6) were identified as (3S, 8aS)-hexahydro-3methylpyrro[1,2-a]pyrazine-1, 4-dione, Phenylacetamide, Cyclo (L-Pro-L-Val), Lauric acid, methyl elaidate, respectitively (Fig. 1) [20–24] (detailed spectroscopic data see Supporting Information). Nematicidal activity In vitro, the nematicidal assay was carried out using direct contact method. As the results showed, the three compounds exhibited different levels of activity against B. xylophilus and D. destructor at certain time point and concentration. Compound 1 and 2 showed moderate activity against the test nematodes at 800 ppm after incubation for 72 h, respectively, while the compound 3 displayed strong activity targeting the tested organisms
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(Table 1, Fig. 2). Overall, the mortality of the three compounds against D. destructor tends to be higher than the latter one. The test of compound 4 against nematodes failed due to the limited amount. The LC50 values of the compounds (1–3) against B. xylophilus and D. destructor were 904.12, 451.26, 232.98 mg/ml and 1594.0, 366.62, 206.38 mg/ml, respectively, as listed in Table 2. So far, many nematicidal agents have been discovered from microorganisms such as bacteria, and fungi [25, 26], which suggested that the microorganism-based resources could serve as an abundant reservoir to explore the effective nematicidal agents. In this work, several compounds were obtained from SMrs28. The compound 2 has been isolated from plant, animal, and bacterium-based sources and has antifeedant, antifungal, and toxic activity at varying levels [27–30]. There’s been no data concerning its nematicidal activity yet. However, from the available literature, in which the compound with a hydroxyl at C-10 exhibited much remarkable fungal activity than other two compounds either with the hydroxyl group attached to the proline ring or without any substitutions, we can propose that the nematicidal activity of compound 2 could be possible partly related to the substitution at C-3 of the prolinebased diketopiperazines [31]. The nematicidal activity of compound 3 was also described here for the first time. Hudgins has reported that phenylacetates possess cytostatic activity due to their lipophilicities against human prostate carcinoma DU145 cells in vitro [32]. Considering the ease for compound entering into the lipid bilayer of cell membrane, the lipophilicity may be essential in some degrees for the nematicidal activity of compound 3 [32]. It is worth noting that the compound 1 was isolated for the first time as a new structure. Although a moderate nematicidal activity of compound 1 was merely observed, however, it has an unusual
Figure 1. Structures of compounds 1–6. ß 2015 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim
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Table 1. Nematicidal activity of three compounds(1–3) from Bacillus sp. SMrs28 against Bursaphelenchus xylophilus and Ditylenchus destructor. Data are given as mean standard deviation. Proof mortality (%,B. xylophilus/ D. destructor) Compounds
Concentrations (ppm)
24 h
48 h
72 h
50 100 200 400 800 50 100 200 400 800 50 100 200 400 800
9.15 4.54/13.51 5.05 15.78 4.06/16.44 5.17 21.08 3.36/17.70 4.34 27.46 5.37/30.51 4.52 36.37 4.09/38.49 5.66 9.04 4.41/11.61 5.70 13.37 5.49/16.31 1.93 19.65 5.52/17.13 3.05 24.32 3.41/19.43 4.68 33.43 6.88/49.34 5.96 18.36 3.68/21.15 2.33 21.20 3.67/21.87 1.26 21.81 2.77/25.05 5.54 34.52 3.97/37.38 5.12 68.90 6.47/76.51 6.09 3.66 4.30/4.04 4.61
12.60 5.10/17.35 5.70 19.23 3.42/24.50 7.22 26.75 6.32/25.69 6.25 29.78 2.76/40.89 5.65 43.07 4.87/39.83 4.55 18.15 5.89/19.77 2.10 20.19 3.99/20.06 3.85 23.66 1.70/23.40 4.81 27.71 3.81/27.01 4.21 50.71 5.44/62.94 2.61 20.68 2.71/27.46 2.29 23.64 3.18/33.06 2.41 29.48 3.91/40.09 3.07 37.84 5.26/40.31 3.07 74.12 6.08/78.34 3.96 3.98 0.45/4.81 3.97
17.88 6.27/27.70 6.45 25.23 4.95/33.31 5.67 33.48 6.13/37.28 4.56 40.49 5.78/42.10 6.23 47.57 7.82/44.37 6.63 26.64 3.05/22.73 1.61 32.68 4.14/28.67 3.74 37.36 2.94/37.48 5.27 45.04 3.45/50.07 5.53 60.55 6.39/65.19 1.37 25.11 5.64/36.73 4.36 33.58 2.65/40.61 4.72 42.03 5.56/43.76 6.07 57.28 3.76/47.97 2.05 78.05 6.31/78.51 5.71 6.11 5.31/6.52 4.39
1
2
3
Control (1% DMSO, v/v)
Figure 2. Plant-parasitic nematodes after treatment with compound 3 isolated from Bacillus sp. SMrs28 (A, non-treated D. destructor; B, D. destructor after treated with at 800 ppm for 72 h; C, Non-treated B. xylophilus; D, B. xylophilus after treated with at 800 ppm for 72 h). ß 2015 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim
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Table 2. Effect of compounds on the mortality of two nematodes, Bursaphelenchus xylophilus and Ditylenchus destructor within 72 h in vitro. LC50(ppm) Compounds
B. xylophilus
D. destructor
904.12 451.26 232.98
1594.0 366.62 206.38
1 2 3
carbon skeleton containing an oxygen bridge linking to the 1, 4-position of benzene ring, which can be potentially developed as a lead compound to make effective candidates for nematicides. As yet, litter is known about the mode of action of active compounds against nematodes. The interference of nervous system was the main one discussed on such as commercial nematicides, carbamates, and orgnophosphrous [33]. Inhibition of acetylcholinesterase and glutathione S-transferase of nematodes by compounds were also reported [34]. The observed activity may be attributed to one mechanism or synergetic effect of several ones, therefore, further work is required to shed light on the action mechanism.
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Acknowledgments We are grateful to Professor Frank Stermitz for editorial assistance. This work was supported by the National Natural Science Foundation of China (grant no. 31070386), Basic Research Program of Lanzhou Institute of Chemical Physics, Chinese Academy of Sciences (080423SYR1), the associate scholar program for talents cultivation plan of “Western Light” of Chinese Academy of Sciences, the Province-academy Cooperation Program of Henan Province of China (grant no. 102106000021), and the open project of Key Laboratory of Tobacco Diseases and Insect Pests Monitoring Controlling and Integrated Management, Institute of Tobacco Research, Chinese Academy of Agricultural Sciences (IPM 201404).
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Conflict of interest: The authors declare no competing financial interest.
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