Rhabdopeptides from Xenorhabdus budapestensis SN84 and Their

Mar 30, 2018 - For decades, plant parasitic nematodes have caused serious damage to crop production. Most nematicides are banned due to their negative...
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Bioactive Constituents, Metabolites, and Functions

Rhabdopeptides from Xenorhabdus budapestensis SN84 and Their Nematicidal Activities against Meloidogyne incognita Yuhui Bi, Chunzhi Gao, and Zhiguo Yu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b00253 • Publication Date (Web): 30 Mar 2018 Downloaded from http://pubs.acs.org on March 31, 2018

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Journal of Agricultural and Food Chemistry

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Rhabdopeptides from Xenorhabdus budapestensis SN84 and Their Nematicidal Activities against Meloidogyne incognita

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Yuhui Bi †, Chunzhi Gao †, and Zhiguo Yu*,†,‡

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6

People’s Republic of China

7



8

Shenyang 110866, People’s Republic of China

1 2

College of Plant Protection, Shenyang Agricultural University, Shenyang 110866,

Engineering & Technological Research Center of Biopesticide for Liaoning Province,

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ABSTRACT:

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For decades, plant parasitic nematodes have caused serious damage to crop production.

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Most nematicides are banned due to their negative impacts on the environment and

12

public health. The repeated application of the few commercially available nematicides

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has caused more incidences of nematicide resistance. To seek novel nematicides, seven

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linear peptides named rhabdopeptides I-O, 1-7, were isolated from culture broth of

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Xenorhabdus budapestensis SN84. The structures of the peptides were elucidated on the

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basis of extensive MS, and NMR analyses. 3, 4 and 7 were novel compounds. 1, 2, 5, and

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6 were isolated and purified for the first time, despite being previously elucidated from

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an extract mixture based on labeling and MS experiments. All seven compounds were

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tested for their nematicidal activities against the second-stage juveniles (J2) of

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Meloidogyne incognita using 24-microwell plates. Rhabdopeptide J, 2, demonstrated a

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strong inhibitory activity with LC50 value of 27.8 µg/mL. Rhabdopeptide K, 3, and M, 5,

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showed moderate inhibitory activity with LC50 values of 46.3 and 42.4 µg/mL,

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respectively.

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KEYWORDS: Xenorhabdus budapestensis, rhabdopeptides, Meloidogyne incognita,

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nematicidal activity

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INTRODUCTION

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For decades, plant parasitic nematodes have caused serious damage to crop production.

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The annual crop losses caused by them were estimated to have reached $125 billion

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globally.1 Root knot nematodes (Meloidogyne sp.) are probably the most notorious plant

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parasitic nematodes in greenhouse. While causing significant economic damage,2

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Meloidogyne incognita has a very broad host range and infects especially the roots of

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vegetables.3, 4 Plant parasitic nematodes are generally controlled by cultural practices,

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crop rotation, and resistant cultivars, combined with chemical nematicides. However,

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many chemical nematicides are banned due to their negative impacts on the environment

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and public health. Moreover, the repeated application of the few commercially available

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nematicides contributed to the increased incidences of nematicide resistance.5-7 Thus,

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novel and environmentally friendly nematicides are constantly in demand.

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Entomopathogenic bacteria of the genus Xenorhabdus are one of the underexplored

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sources of natural products.8 Xenorhabdus are both symbiotic mutualists of Steinernema

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nematodes and pathogens against the host insect larvae. After infection, Xenorhabdus

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kills the host insect. The cadaver must be protected from food competitors such as other

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fungi or bacteria.9-12 As a result of this interesting lifecycle, a range of structurally

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diverse metabolites have been discovered from various Xenorhabdus strains. These

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metabolites have shown different bioactivities including antibacterial, insecticidal, and

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antifungal activities.13, 14 Since other soil-living nematodes are potential food competitors,

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Xenorhabdus could be a promising source of lead molecules for nematicidal chemicals.

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In our search for novel bioactive natural products,15-17 Xenorhabdus budapestensis

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SN84 was isolated from a sample of soil collected in Fengcheng City of Liaoning

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Province, China. The strain was subsequently cultured in the laboratory. From the

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CH2Cl2 extract of the fermentation broth of X. budapestensis SN84, we have isolated

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seven linear peptides, which belong to a group of virulence-associated small molecules.18

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The structures of the peptides were determined on the basis of MS and NMR analyses.

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All seven linear peptides were then tested for their nematicidal activities against the

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second-stage juveniles (J2) of Meloidogyne incognita using 24-microwell plates, leading

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to information on their structure-activity relationships.

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MATERIALS AND METHODS

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General Experimental Procedures. Optical rotations were acquired using an AP-300

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polarimeter (Atago, Tokyo, Japan). NMR spectra were obtained on an Avance-600 NMR

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spectrometer (Bruker, Karlsruhe, Germany) at 25 °C. Carbon signals and the residual

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proton signals of d6-DMSO (δC 39.5 and δH 2.50) were used for calibration. A 6500

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series quadrupole-time-of-flight (Q-TOF) mass instrument (Agilent, Santa Clara, CA)

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was used to collected high-resolution electrospray ionization mass spectrometry

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(HRESIMS) spectra data. The killed nematodes were observed with the aid of an EZ4 W

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stereomicroscope (Leica, Wetzlar, Germany). A 1260 Infinity LC system (Agilent) was

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used for high-performance liquid chromatography (HPLC) analysis with a 250 mm × 4.6

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mm i.d., 5μm, ZORBAX Eclipse XDB (Agilent, Santa Clara, CA) column.

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Semi-preparative HPLC was performed using a 1260 series system with a 250 mm × 9.4

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mm i.d., 5μm, ZORBAX Eclipse XDB (Agilent) column. Column chromatography was

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performed using either silica gel (100-200 mesh) (Qingdao Ocean Chemical Co. Ltd.,

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Qingdao, China) or Sephadex LH-20 (GE Healthcare, Uppsala, Sweden). All chemical

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reagents were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China)

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and used without further purification.

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Bacterium Material. X. budapestensis SN84 was isolated from a sample of soil

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collected in Fengcheng City of Liaoning Province, China (40° 36′ 25.8″ N, 124° 32′

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44.3″ E) with an elevation of 139 m, in June, 2014. The bacterium was identified after a

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phylogenetic analysis, comparing to 16S rRNA sequences available on the EzTaxon

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database. The strain had genetic resemblances to X. budapestensis and was subsequently

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named X. budapestensis SN84 (Genbank accession no. KU556153). X. budapestensis

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SN84 was stored in the Laboratory of Microbial Metabolites, College of Plant Protection,

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at Shenyang Agricultural University, China.

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Fermentation and Extraction. X. budapestensis SN84 was stored in glycerol

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suspensions (10%, v/v) at -80 °C. Fermentation process was performed in two stages. In

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stage 1, 50 mL of LB medium (i.e., 1% tryptone, 0.5% yeast extract, 1% NaCl, pH 7.0)

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was put into a 250 mL Erlenmeyer flask and inoculated with 2.5 mL of X. budapestensis

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SN84 bacterial suspension. The inoculated flask was subsequently incubated with

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shaking (180 rpm) at 28 °C for 18 h to prepare the seed culture. In stage 2, twenty-four

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2-L Erlenmeyer flasks, each containing 400 mL of M medium (i.e., 0.6% glucose, 2%

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peptone, 0.15% MgSO4•7H2O, 0.25% (NH4)2SO4, 0.09% KH2PO4, 0.1% K2HPO4, 0.17%

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Na2SO4, pH 7.2), were each inoculated with 20 mL of the seed culture and were

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subsequently left for fermentation for 6 d under identical conditions. Centrifugation at

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8000 rpm and 4 °C was applied to the fermentation cultures for 30 min in order to

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remove the bacteria. The leftover broth was extracted with 3% Amberlite XAD 16 resin

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at 25 °C for 4 h with agitation. Resin was subsequently collected using centrifugation and

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was extracted four times using methanol. The methanol extracts were combined and then

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concentrated under reduced pressure, yielding 10.56 g of crude extract.

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Isolation and Purification. The crude extract was re-dissolved in 600 mL of 50%

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MeOH. The solution was subjected to extraction for four times using equal volume of

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CH2Cl2. The resulted extract was collected and put on a rotary evaporator to be

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concentrated under vacuum at 28 °C. 5.52 g of solid brown residue was obtained after the

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concentration. The concentrated extract was further purified using silica gel

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chromatography (350 mm × 30 mm i.d.) eluted stepwise with CH2Cl2-MeOH (100:0,

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50:1, 25:1, 25:2, 25:3, 25:4, 1:1, and 0:100, 2 L each) as the mobile phase to yield five

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fractions, F1 to F5. Fraction F4 was then subjected to repeated chromatography on a

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Sephadex LH-20 gel column (1000 mm × 20 mm i.d.) with CH2Cl2-MeOH (1:1) as

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eluent. It was further purified using a reverse-phase semi-preparative HPLC with a

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CH3CN‒H2O gradient (0.1% trifluoroacetic acid added to both solvents) from 31-40% of

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CH3CN and a flow rate of 3.0 mL/min for 35 min. UV detection was set to 210 nm. Pure

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compounds 1 (10.1 mg), 2 (9.3 mg), 3 (29.2 mg), 4 (11.1 mg), and 5 (10.9 mg), were

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eluted at 11.49 min, 18.50 min, 9.33 min, 27.01 min, and 13.65 min, respectively.

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Fraction F3 was isolated using the same chromatographic separation as F4, affording

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compounds 6 (7.6 mg) and 7 (8.5 mg) at 16.32 min and 22.32 min during reverse-phase

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semi-preparative HPLC, respectively.

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1 Rhabdopeptide I, 1. Amorphous solid; [α]24 D -130.84 (c 1.07, CH3OH); H NMR and

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C NMR spectroscopic data (d6-DMSO) (Table 1); HRESIMS m/z 599.4275 [M + H]+

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(calcd: C33H55N6O4, 599.4284). Rhabdopeptide J, 2. Amorphous solid; [α]24D -149.12 (c 1.14, CH3OH); 1H NMR and

117 118

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C NMR spectroscopic data (d6-DMSO) (Table 1); HRESIMS m/z 712.5122 [M + H]+

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(calcd: C39H66N7O5, 712.5125). 1 Rhabdopeptide K, 3. Amorphous solid; [α]24 D -88.61 (c 3.16, CH3OH); H NMR and

120 121

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C NMR spectroscopic data (d6-DMSO) (Table 2); HRESIMS m/z 568.3836 [M + Na]+

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(calcd: C30H51N5NaO4, 568.3838). Rhabdopeptide L, 4. Amorphous solid; [α]24D -104.58 (c 1.53, CH3OH); 1H NMR and

123 124

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C NMR spectroscopic data (d6-DMSO) (Table 2); HRESIMS m/z 659.4858 [M + H]+

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(calcd: C36H63N6O5, 659.4859). Rhabdopeptide M, 5. Amorphous solid; [α]24D -93.75 (c 0.96, CH3OH); 1H NMR and

126 127

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C NMR spectroscopic data (d6-DMSO) (Table 2); HRESIMS m/z 659.4865 [M + H]+

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(calcd: C36H63N6O5, 659.4859). 1 Rhabdopeptide N, 6. Amorphous solid; [α]24 D -72.92 (c 0.96, CH3OH); H NMR and

129 130

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C NMR spectroscopic data (d6-DMSO) (Table 3); HRESIMS m/z 659.4852 [M + H]+

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(calcd: C36H63N6O5, 659.4859). 1 Rhabdopeptide O, 7. Amorphous solid; [α]24 D -218.18 (c 0.55, CH3OH); H NMR and

132 133

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C NMR spectroscopic data (d6-DMSO) (Table 3); HRESIMS m/z 772.5688 [M + H]+

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(calcd: C42H74N7O6, 772.5700).

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Using the Marfey’s Method to Confirm the Configurations of the Valine Residues.

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Approximately 0.5 mg of 1-7 were each dissolved in 6 M HCl (1 mL) and hydrolyzed for

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12 h at 110 °C. The derivatization reactions were performed as described before.19 20 μL

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of each 1-fluoro-2, 4-dinitrophenyl-5-L-alaninamide (FDAA) derivatized product was

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used for HPLC analysis with a CH3CN‒H2O gradient (0.1% trifluoroacetic acid added to

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both solvents) from 20-60% of CH3CN and a flow rate of 1 mL/min at 25 °C for 28 min.

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UV detection was set to 340 nm. The L-N-methylvaline and L-valine derivatized samples

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were used as standards.

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Nematodes. M. incognita nematodes were used in the experiment. The nematode

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population was collected from cucumber roots found in a greenhouse at Yingpan village,

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Tieling City, Liaoning Province, China (123°55'12" E, 42°10'48" N). Nematode eggs

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were extracted from the infected roots of cucumber plants into 1% NaOCl solutions for 4

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min and then rinsed with distilled water for three times. Surface-sterilized eggs were then

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placed in a petri dish with water and incubated at 25 °C to prepare second-stage juveniles

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(J2). Each day, newly emerged J2 individuals were gathered. They were stored for

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further experiments at 4 °C.3, 20

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Nematicidal Activity Bioassay. All seven peptides were tested against the J2s of M.

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incognita using 24-microwell plates for their nematicidal activities. The compounds were

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dissolved in DMSO. The solutions were then diluted using 1% (v/v) Tween-80/distilled

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water solution to prepare a 50 μg/mL final solution with concentration of DMSO at 1%

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(v/v). The same amount of DMSO dissolved in 1% (v/v) Tween 80/distilled water

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solution to prepare the negative control, and 50 μg/mL abamectin solution was used as

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the corresponding positive control. All prepared solutions were distributed into

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24-microwell plates. About 100 J2s per 1 mL of solution were added to each well. The

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plates were covered and maintained at 25 °C. A stereomicroscope was used to observe

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the mortality of the nematodes at 48 h. Nematodes were defined as killed if their bodies

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became straight and did not react to mechanical touches.21 The experiment was repeated

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three times under the same conditions.

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Corrected mortality of each compound was calculated based on the data from

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nematicidal bioassays, using the following formula: corrected mortality % = 100 ×

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[(mortality % in treatment − mortality % in control) / (100 − mortality % in control)].

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Compounds possessing good nematicidal activities (J2 corrected mortality > 50% at 50

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μg/mL) were subjected to more evaluations using the above-mentioned method at

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concentrations of 6.25, 12.5, 25, 50, and 100 μg/mL.3 Abamectin was chosen as the

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positive control and was prepared with the same concentrations as the tested compounds.

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Then, the J2 corrected mortalities were calculated, and the LC50 values were determined

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using probit analysis. The experiment was repeated individually three times under

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identical conditions.

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RESULTS AND DISCUSSION

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Structure Elucidation. The CH2Cl2 extract of the fermentation broth of X.

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budapestensis SN84 was subjected to silica gel column chromatography and further

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purified by gel chromatography on Sephadex LH-20, and by HPLC to afford seven

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compounds, 1-7 (Figure 1). All of them were linear peptides and named as

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rhabdopeptides I-O, 1-7.

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Rhabdopeptide I, 1, was assigned the molecular formula C33H54N6O4 in accordance to

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its HRESIMS data, indicating 10 degrees of unsaturation. The 1H NMR spectrum of 1

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exhibited signals characteristic of a peptide including four α-proton signals at δH 5.11

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(1H, d, J = 10.7 Hz), 4.60 (2H, m), and 3.68 (1H, d, J = 5.3 Hz), along with eight methyl

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groups at δH 0.6-1.0 and four methine proton signals [δH 2.21 (1H, m), 2.11 (1H, m), 2.05

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(1H, m) and 1.98 (1H, m)] (Table 1). These signals were deduced to be four spin systems

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in the COSY spectrum, which were characteristics of valine residues (Figure 2).

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Additionally, the 1H NMR spectrum of 1 also revealed three methyl groups [δH 3.06 (3H,

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s), 3.01 (3H, s), and 2.46 (3H, s)] attached to nitrogen atoms and two NH protons [δH

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8.81 (2H, d, J = 8.0 Hz)]. These results suggested three of four valine building blocks

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were N-methylated valines in 1, which was confirmed by careful analysis of the HMBC

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and COSY spectra of 1. Each carbonyl carbon [δC 171.6, 170.1, 169.0, and 166.3] was

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assigned to one of four valine building blocks according to long-range couplings in the

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HMBC spectrum. Positions of three methyl groups attached to nitrogen atoms were

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evident from HMBC correlations between methyl protons to α-carbons of the respective

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amino acids. The 13C NMR spectrum of 1 showed signals from 33 carbons: twenty-three

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carbon signals were attributed to four valine building blocks, and the remaining ten

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carbon signals were deduced as follows. The 1H NMR data also showed two methylene

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protons [δH 3.33 (2H, m) and 2.77 (2H, t, J = 7.4 Hz)], an indole residue [δH 7.51 (1H, d,

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J = 7.9 Hz), 7.32 (1H, d, J = 8.1 Hz), 7.11 (1H, d, J = 2.2 Hz), 7.05 (1H, m), 6.96 (1H,

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m), and 10.80 (1H, s)], and one NH proton [δH 8.06 (1H, t, J = 5.7 Hz)], which were

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attributed to a C-terminal tryptamide residue by analysis of COSY and HMBC data

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(Figure 2). The connectivity of these building blocks was determined by sequential

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HMBC correlations of the NH, α-H or N-methyl protons to carbonyl carbons.

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Consequently,

the

structure

of

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N-methylvaline-valine-N-methylvaline-N-methylvaline

205

C-terminus (Figure 1).

1

was

established peptide

to

with

a

be

a

tryptamide

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Rhabdopeptide J, 2, was determined to be C39H65N7O5 based on the HRESIMS data,

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with 11 degrees of unsaturation. Careful study of the NMR spectroscopic data of 1 and 2

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revealed that 2 has one more N-methylated valine at the C-terminus, i.e. Valine-5 (Table

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1, Figures 1 and 2), which was in accordance with the 113 Da mass increase.

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Rhabdopeptide K, 3, was determined to be C30H51N5O4 based on its HRESIMS data,

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indicating 8 degrees of unsaturation. Analysis of the NMR spectroscopic data revealed 3

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to be an analogue of 1 with a phenyl group [δH 7.25 (2H, m), and 7.18 (3H, m)] in place

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of the indole residue in 1 (Table 1, and Table 2). The phenyl group was predicted to be

214

attributed to a phenethylamine residue by the 1D (1H,

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(Table 2, Figure 2). The 1H NMR spectrum of 3 showed four α-proton signals [δH 4.57

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(1H, d, J = 11.0 Hz), 4.45 (1H, t, J = 8.9 Hz), 4.37 (1H, dd, J = 8.5, 7.1 Hz), and 3.72

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(1H, d, J = 5.6 Hz)] and two N-methyl groups [δH 3.05 (3H, s), and 2.45 (3H, s)], which

218

suggested two of four valine building blocks were N-methylated in 3. Valine-1 and

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Valine-4 were verified to be N-methylated based on HMBC correlations of δH 2.45 with

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δC 65.7 and δH 3.05 with δC 61.0, respectively. Using the same correlations that were

221

used to determine the connectivity of building blocks in 1, compound 3 was assigned as a

222

N-methylvaline-valine-valine-N-methylvaline

223

C-terminus (Figure 1).

224

13

peptide

C) and 2D NMR spectra of 3

with

an

N-phenethylamine

Rhabdopeptide L, M, and N, 4-6, share the same molecular formula of C36H62N6O5 as

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established by their HRESIMS data, indicating 9 degrees of unsaturation for each one.

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4-6 were predicted to be extended by one more N-methylated valine than 3 based on the

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113 Da mass increase and the analysis of NMR spectroscopic data (Tables 2 and 3,

228

Figure 2). There are three N-methyl groups in each of them. The significant difference

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between compounds 4-6 is the positions of N-methyl groups, which was consistent with

230

HMBC correlations between methyl protons and α-carbons of the respective amino acids

231

(Figure 2).

232

Rhabdopeptide O, 7, was established to be C42H73N7O6 from its HRESIMS data, with 1

233

10 degrees of unsaturation. The

H NMR spectrum of 7 also showed signals

234

characteristic of a peptide, such as six α-proton signals [δH 5.08 (1H, d, J = 10.7 Hz),

235

4.68 (1H, d, J = 11.0 Hz), 4.60 (1H, t, J = 8.1 Hz), 4.57 (1H, d, J = 11.0 Hz), 4.37 (1H, t,

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J = 8.8 Hz), and 3.69 (1H, s)] and four N-methyl groups [δH 3.05 (3H, s), 3.04 (3H, s),

237

2.99 (3H, s), and 2.46 (3H, s)] (Table 3), which suggested four of six valine building

238

blocks were N-methylated in 7. A careful analysis of the 1D (1H,

239

spectroscopic data of 7 revealed the same amino acid sequence as 5 (Tables 2 and 3,

240

Figure 2), but extended by one more N-methylated valine at C-terminus, i.e. Valine-6,

241

corresponding to a 113 Da mass increase (Figure 1).

13

C) and 2D NMR

242

The constitutions of 1-7 were similar to those of rhabdopeptides. All the

243

configurations of amino acids of rhabdopeptides were determined to be L-configuration

244

deduced from detailed bioinformatic analysis in a previous research.18,

245

configurations of N-methylated and non-methylated valines in the isolated compounds

246

were all assigned as L-configuration on the basis of their biosynthetic origin, which were

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Thus, the

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confirmed by Marfey’s method analyses of 1-7.

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In general, seven linear peptides were isolated from culture broth of X. budapestensis

249

SN84. Their structures were elucidated based on extensive MS, and NMR analyses. 1, 2,

250

5, and 6 were isolated and purified for the first time, despite being previously elucidated

251

from an extract mixture based on labeling and MS experiments.22, 23 3, 4 and 7 were

252

novel compounds.

253

Nematicidal Activity Bioassay. 1-7 were preliminarily evaluated for their nematicidal

254

activities against M. incognita. All of the evaluated compounds demonstrated nematicidal

255

activities at the concentration of 50µg/mL, and their preliminarily nematicidal activities

256

were shown in Figure 3. 2 had much stronger activity than the other compounds with a J2

257

corrected mortality of 74.5% and an LC50 value of 27.8 µg/mL (Table 4). 3 and 5 showed

258

moderate activities with their J2 corrected mortality > 50% at 50 µg/mL, and their LC50

259

values were estimated to be 46.3 and 42.4 µg/mL, respectively. Abamectin was used as

260

positive control and had an LC50 value of 9.9 µg/mL.

261

By comparing the structures of 1-7, we deduced that the composition of building

262

blocks, especially the terminal amine, chain length, and position of N-methylation, had a

263

strong influence on the nematicidal activity against M. incognita. Both 1 and 2 had a

264

tryptamide C-terminus, but the nematicidal activity of 2 was much stronger than that of 1,

265

as 2 had one more N-methylated valine extended in the chain. However, the nematicidal

266

activity of rhabdopeptides with an N-phenethylamine C-terminus was decreased with the

267

extension of N-methylated valine as the activity of 7 was much lower than that of 5.

268

Although 4, 5, and 6 had the same molecular formula, but they have demonstrated

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different activities with the difference in positions of N-methyl groups. Due to the

270

paucity of 1-7, other aspects of bioactivity were not tested.

271

Plant parasitic nematodes cause significant global crop loss annually and many

272

chemical nematicides disappeared from market due to their negative impact on the

273

environment and public health.3,

274

resistance has increased due to the repeated applications of the few commercially

275

available nematicides.5-7 Thus, new nematicidal compounds are always in demand.

276

Xenorhabdus are underexplored as a source of natural products.8 Compounds 3, 4, and 7

277

are reported for the first time, demonstrating the structural diversity of natural products

278

from X. budapestensis. Given that natural products are usually environmentally benign,

279

they remain the most promising source of lead molecules for agricultural chemicals.25-30

280

The discovery of nematicidal activity against M. incognita for rhabdopeptides J, 2, K, 3,

281

and M, 5 may provide a new template for nematicides discovery to create effective and

282

environmentally safe alternatives to the presently used toxic synthetic nematicides.

283

ASSOCIATED CONTENT

284

Supporting Information

285

Spectra of NMR and HRESIMS, and Marfey’s method analyses for compounds 1-7

286

AUTHOR INFORMATION

287

Corresponding Author

288

* Tel: +86 24 88487148. Fax: +86 24 88487038.

289

E-mail: [email protected].

290

Funding

24

Moreover, number of incidences of nematicide

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This work was financially supported by National Key R&D Program of China

292

(2017YFD0201100).

293

Notes

294

The authors declare that there are no conflicts of interest.

295

ACKNOWLEDGMENTS

296

We are grateful to Dr. Xiaofeng Zhu and Dan Zhao of the Nematology Institute of

297

Northern China, Shenyang Agricultural University, for technical assistance with

298

collecting the infected cucumber roots and J2 of M. incognita. We are grateful to

299

Shenyang Pharmaceutical University and Dalian Institute of Chemical Physics, Chinese

300

Academy of Sciences for their technical assistance with NMR and MS spectra,

301

respectively.

302

REFERENCES

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to 2014. J. Nat. Prod. 2016, 79, 629−661.

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A.; Developing global leaders for research, regulation, and stewardship of crop

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protection chemistry in the 21st century. J. Agric. Food Chem. 2016, 64, 52−60.

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Figure captions Figure 1. Structures of compounds 1-7 Figure 2. Key 1H-1H COSY (bold) and HMBC (arrows) correlations of compounds 1-7 Figure 3. Nematicidal effects of compounds 1-7 at 50 μg/mL on J2s of Meloidogyne incognita with abamectin as positive control. The biological data presented are the mean values for each treatment across replicates.

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Table 1. 1H (600 MHz) and 13C (150 MHz) NMR Data of Compounds 1 and 2 in d6-DMSOa subunit

position

Val-1

1 2 3 4b 5b HN-CH3 NH 1 2 3 4c 5c NH 1 2 3 4d 5d N-CH3 1 2 3 4e 5e N-CH3 1 2 3 4f 5f N-CH3 1 2 3 4 5 6 7 8 9 11 NH NH (Ind)

Val-2

Val-3

Val-4

Val-5

tryptamine

a

1 δC, type 166.3, s 65.5, d 29.5, d 18.1, q 17.7, q 32.0, q 171.6, s 54.5, d 29.8, d 18.2, q 18.7, q 170.1, s 57.0, d 27.0, d 18.7, q 19.3, q 30.1, q 169.0, s 61.2, d 26.0, d 18.1, q 18.2, q 30.3, q

39.1, t 25.1, t 111.5, s 127.1, s 118.1, d 118.1, d 120.9, d 111.3, d 136.2, s 122.5, d

2 δH,mult(J in Hz) 3.68, d (5.3) 2.05, m 0.88, overlap 0.93, overlap 2.46, s 8.81, d (8.0) 4.60, m 1.98, m 0.93, overlap 0.88, overlap 8.81, d (8.0) 5.11, d (10.7) 2.21, m 0.78, d (6.5) 0.70, d (6.7) 3.06, s 4.60, m 2.11, m 0.66, d (6.7) 0.82, d (6.5) 3.01, s

3.33, m 2.77, t (7.4)

7.51, d (7.9) 6.96, m 7.05, m 7.32, d (8.1) 7.11, d (2.2) 8.06, t (5.7) 10.80, s

δC, type 166.3, s 65.5, d 29.4, d 18.1, q 19.3, q 32.0, q 171.7, s 54.5, d 29.7, d 17.7, q 18.0, q 169.9, s 57.3, d 26.9, d 18.1, q 18.9, q 30.0, q 169.9, s 57.2, d 26.6, d 18.2, q 18.8, q 30.0, q 169.0, s 61.2, d 26.0, d 18.8, s 17.7, s 30.3, s 39.1, t 25.1, t 111.5, s 127.1, s 118.1, d 118.1, d 120.9, d 111.3, d 136.2, s 122.5, d

Assignments were based on COSY, HSQC, HMBC, and NOESY experiments. 20

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δH,mult(J in Hz) 3.69, d (5.2) 2.07, m 0.88, overlap 0.79, overlap 2.45, s 8.81, d (8.1) 4.61, t (8.0) 1.98, m 0.93, overlap 0.68, overlap 8.81, d (8.1) 5.09, d (10.7) 2.22, m 0.68, overlap 0.79, overlap 3.04, s 5.08, d (10.7) 2.22, m 0.88, overlap 0.93, overlap 2.93, s 4.57, d (11.0) 2.12, m 0.82, d (6.5) 0.68, overlap 3.00, s 3.33, m 2.77, t (7.5)

7.51, d (7.9) 6.96, m 7.05, m 7.32, d (8.0) 7.11, d (2.2) 8.03, t (5.7) 10.79, s

Journal of Agricultural and Food Chemistry b~f

Chemical shifts with same superscripts may be interchanged.

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Page 23 of 31

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Table 2. 1H (600 MHz) and 13C (150 MHz) NMR Data of Compounds 3-5 in d6-DMSOa subunit

position

Val-1

1 2 3 4b 5b HN-CH3 NH 1 2 3 4c 5c NH 1 2 3 4d 5d NH / N-CH3 1 2 3 4e 5e N-CH3

Val-2

Val-3

Val-4

4

3 δC, type 166.2, s 65.7, d 29.5, d 18.41, q 18.0, q 32.0, q 170.2, s 57.5, d 30.7, d 19.2, q 18.4, q 172.0, s 54.1, d 30.2, d 18.5, q 18.6, q 169.3, s 61.0, d 26.0, d 17.8, q 19.0, q 30.4, q

δH,mult(J in Hz)

δC, type 166.2, s 65.8, d 30.2, d 17.8, q 18.4, q 32.0, q

3.72, d (5.6) 2.07, m 0.66, d (6.7) 0.89, d (6.9) 2.45, s 8.82, d (7.7)

170.3, s 57.5, d 30.6, d 18.0, q 18.4, q

4.37, dd (8.5, 7.1) 1.93, m 0.82, overlap 0.87, d (6.7) 8.52, d (8.6)

171.9, s 54.1, d 29.5, d 18.0, q 18.2, q

4.45, t (8.9) 1.93, m 0.82, overlap 0.80, d (6.7) 8.33, d (8.5)

170.0, s 57.1, d 26.9, d 19.0, q 18.8, q 29.9, q

4.57, d (11.0) 2.07, m 0.76, d (6.5) 0.96, d (7.0) 3.05, s

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5

δH,mult(J in Hz) 3.70, d (3.5) 2.06, m 0.89, d (6.8) 0.95, d (6.9) 2.45, s 8.77, m 4.40, t (7.1) 1.94, m 0.84, overlap 0.80, overlap 8.51, d (7.9) 4.50, t (8.6) 1.94, m 0.86, d (6.7) 0.69, d (6.7) 8.35, d (8.4) 5.06, d (10.7) 2.20, m 0.84, overlap 0.80, overlap 3.00, s

δC, type 166.3, s 65.5, d 29.5, d 18.1, q 17.7, q 32.0, q 171.6, s 54.5, d 29.8, d 18.0, q 17.8, q 170.4, s 57.3, d 26.8, d 18.9, q 19.1, q 30.1, q 169.0, s 61.3, d 25.7, d 18.1, q 18.8, q 30.3, q

δH,mult(J in Hz) 3.67, d (5.0) 2.05, m 0.88, overlap 0.93, overlap 2.46, s 8.80, d (8.0) 4.61, t (8.2) 1.99, m 0.88, overlap 0.93, overlap 8.80, d (8.0) 5.09, d (10.7) 2.24, m 0.67, d (6.6) 0.70, d (6.8) 3.04, s 4.66, d (11.1) 2.11, m 0.82, overlap 0.73, d (6.7) 2.95, s

Journal of Agricultural and Food Chemistry

subunit

position

Val-5

1 2 3 4f 5f NH / N-CH3 1 2 3 4/8g 5/7h 6 NH

phenylethylamine

a

4

3 δC, type

40.0, t 34.9, t 139.3, s 128.5, d 128.2, d 126.0, d

Page 24 of 31

δH,mult(J in Hz)

δC, type 169.0, s 61.1, d 26.0, d 18.0, q 19.2, q 30.0, q 40.0, t 34.9, t 139.3, s 128.5, d 128.2, d 126.0, d

3.27, m 2.67, t (7.3) 7.18, m 7.25, m 7.18, m 8.06, t (5.6)

Assignments were based on COSY, HSQC, HMBC, and NOESY experiments. Chemical shifts with same superscripts may be interchanged.

b~h

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5

δH,mult(J in Hz) 4.56, d (11.0) 2.06, m 0.63, d (6.6) 0.77, d (6.2) 2.92, s 3.28, m 2.68, m 7.17, m 7.25, m 7.17, m 8.02, t (5.5)

δC, type 170.2, s 57.7, d 30.5, d 18.0, q 19.2, q 39.6, t 35.0, t 139.3, s 128.6, d 128.3, d 126.1, d

δH,mult(J in Hz) 4.06, dd (9.1, 6.9) 1.87, m 0.70, d (6.8) 0.82, overlap 7.47, d (9.1) 3.30, m 2.70, t (7.2) 7.19, m 7.27, m 7.19, m 7.97, t (5.5)

Page 25 of 31

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Table 3. 1H (600 MHz) and 13C (150 MHz) NMR Data of Compounds 6 and 7 in d6-DMSOa subunit

position

Val-1

1 2 3 4b 5b HN-CH3 NH 1 2 3 4c 5c NH 1 2 3 4d 5d N-CH3 1 2 3 4e 5e NH / N-CH3 1 2 3 4f 5f NH / N-CH3 1 2 3 4g 5g N-CH3 1 2 3 4/8h

Val-2

Val-3

Val-4

Val-5

Val-6

phenylethyla mine

7

6 δC, type 166.1, s 65.5, d 29.4, d 18.5, q 17.6, q 32.0, q 171.6, s 54.6, d 30.2, d 18.5, q 18.2, q 169.7, s 60.5, d 26.5, d 18.3, q 18.7, q 30.6, q 172.0, s 54.3, d 29.9, d 18.6, q 18.6, q 169.3, s 61.0, d 26.0, d 18.4, q 19.2, q 30.4, q

40.0, t 35.0, t 139.3, s 128.5, d

δH,mult(J in Hz) 3.67, d (5.6) 2.06, m 0.93, d (6.8) 0.86, d (6.8) 2.46, s 8.80, m 4.56, m 1.98, m 0.93, d (6.8) 0.77, overlap 8.80, m 4.74, d (11.0) 2.06, m 0.66, overlap 0.86, d (6.8) 3.11, s 4.35, t (8.8) 1.98, m 0.77, overlap 0.82, d (6.7) 8.25, d (7.7) 4.56, m 2.06, m 0.66, overlap 0.77, overlap 3.05, s

3.30, m 2.66, t (7.3) 7.17, m 24

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δC, type 166.2, s 65.5, d 29.8, d 18.2, q 18.1, q 32.0, q 171.6, s 54.5, d 29.4, d 17.6, q 18.1, q 170.0, s 57.2, d 26.9, d 18.4, q 18.8, q 30.1, q 169.4, s 60.7, d 26.2, d 18.2, q 19.2, q 30.4, q 172.0, s 54.2, d 30.0, d 18.7, q 18.6, q 169.3, s 61.0, d 26.0, d 18.3, q 18.9, q 30.4, q 40.0, t 34.9, t 139.3, s 128.5, d

δH,mult(J in Hz) 3.69, s 2.07, m 0.88, overlap 0.93, overlap 2.46, s 8.81, d (8.0) 4.60, t (8.1) 1.97, m 0.93, overlap 0.88, overlap 8.81, d (8.0) 5.08, d (10.7) 2.22, m 0.81, overlap 0.70, overlap 3.04, s 4.68, d (11.0) 2.07, m 0.66, overlap 0.77, overlap 2.99, s 4.37, t (8.8) 1.97, m 0.77, overlap 0.81, overlap 8.13, d (8.2) 4.57, d (11.0) 2.07, m 0.66, overlap 0.81, overlap 3.05, s 3.28, m 2.67, t (7.4) 7.18, m

Journal of Agricultural and Food Chemistry

subunit

position i

5/7 6 NH a

Page 26 of 31

6 δC, type 128.2, d 126.0, d

7

δH,mult(J in Hz) 7.25, m 7.17, m 8.03, t (5.4)

δC, type 128.2, d 126.0, d

Assignments were based on COSY, HSQC, HMBC, and NOESY experiments Chemical shifts with same superscripts may be interchanged.

b~i

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δH,mult(J in Hz) 7.25, m 7.18, m 8.04, t (5.6)

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Journal of Agricultural and Food Chemistry

Table 4. Nematicidal Effects of Selected Compounds on Meloidogyne incognita a Compound Rhabdopeptide J, 2 Rhabdopeptide K, 3 Rhabdopeptide M, 5 Abamectin c

LC50b, μg/mL(±SD) 27.8±2.4 46.3±1.3 42.4±1.0 9.9±0.6

a

Data are mean values of three independent experiments, each with three replicates.

b

The lethal concentration values necessary to result in 50% J2s mortality of Meloidogyne

incognita. c

Positive control.

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Page 28 of 31

6

H N

O 2 1

N H

7

5

O N

H N

N

O

3

1

O

H N

8

O

O N H

NH 11

3

N

N

O

O 2

1

N H

3

H N

2

O

H N

N

O

3

H N

1

O

6

3 Val-1 H N

Val-2

Val-3

O

Val-4

O N H

N H

O

1

H N

NH

O N

N

H N

N

O

Val-5

O

Val-6

O

H N

N O

O N H

R1 N

O

O

N R2

R3 N O

O N H

4

R1=H,

5

R1=CH3, R2=CH3, R3=H

6

R1=CH3, R2=H,

7

Figure 1

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R2=CH3, R3=CH3

R3=CH3

Page 29 of 31

Journal of Agricultural and Food Chemistry

H N

O

O N H

N

H N

N

O

H N

O N H

NH

O

O N

H N

O N H

H N

2

O

H N

N

O

H N

O N H

O

O

H N

N H

H N

N

O

O

O N

N

O

N H

O 4

O N

N H

O

3

H N

N

O

1

NH

O N

O

H N

N H

O

O

O N H

N

N H

O

5

6

H N

O

O N H

N

H N

N

O

O

O

H N

N O

7

Figure 2

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O N O

N H

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Figure 3

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Graphic for table of contents

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