Rhabdopeptides from Xenorhabdus budapestensis SN84 and Their

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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

4

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

5

†

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,

ACS Paragon Plus Environment


9

ABSTRACT:

10

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

11

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

13

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

14

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

15

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

16

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

17

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

18

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

19

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

20

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

21

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,

23

respectively.

24 25

KEYWORDS: Xenorhabdus budapestensis, rhabdopeptides, Meloidogyne incognita,

26

nematicidal activity

1


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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

31

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

33

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

34

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

35

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

36

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

37

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

38

novel and environmentally friendly nematicides are constantly in demand.

39

Entomopathogenic bacteria of the genus Xenorhabdus are one of the underexplored

40

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

42

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

43

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

50

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

51

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

59

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

60

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

61

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

62

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

63

was used to collected high-resolution electrospray ionization mass spectrometry

64

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

65

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

66

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

69

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

70

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

72

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

78

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

79

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

83

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

84

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

85

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

87

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

88

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%

90

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

91

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

92

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

4



93

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

96

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

97

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

98

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

99

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

100

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

101

concentration. The concentrated extract was further purified using silica gel

102

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

103

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

104

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

105

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

106

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

107

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

109

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

110

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

111

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

112

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.

114

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]+

116

(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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119

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

120 121

13

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

122

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

123 124

13


125

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

126 127

13


128

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

129 130

13


131

(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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134

(calcd: C42H74N7O6, 772.5700).

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

136

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

6



137

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

138

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

139

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

140

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

141

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

142

were used as standards.

143

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

146

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

147

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

148

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

149

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

150

further experiments at 4 °C.3, 20

151

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

152

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

153

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

154

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

155

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

156

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

157

the corresponding positive control. All prepared solutions were distributed into

158

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

160

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

161

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

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three times under the same conditions.

163

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

164

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

165

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

166

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

167

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

168

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

169

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

170

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

171

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

172

identical conditions.

173

RESULTS AND DISCUSSION

174

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

175

budapestensis SN84 was subjected to silica gel column chromatography and further

176

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

177

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

178

rhabdopeptides I-O, 1-7.

179

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

180

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

8



181

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

182

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

183

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

184

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

185

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

186

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

187

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

188

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

189

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

190

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

191

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

192

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

193

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

194

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

195

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

196

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

197

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

198

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,

199

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

200

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

201

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

202

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

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Consequently,

the

structure

of

204

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

205

C-terminus (Figure 1).

1

was

established peptide

to

with

a

be

a

tryptamide

206

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

207

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

208

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

209

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

210

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

211

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

212

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

213

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,

215

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

216

(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

217

(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

219

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

220

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

10



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225

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

226

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

227

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

229

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,

236

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

11


22

Thus, the

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247


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

248

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

12



269

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

13


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291

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

303

(1) Chitwood, D. J. Research on plant-parasitic nematode biology conducted by the

304

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18



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.

19


Page 20 of 31

Page 21 of 31


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


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.

21


Page 22 of 31

Page 23 of 31


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

22


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


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

23


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


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


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


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

25


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

Page 27 of 31


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.



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

27


R2=CH3, R3=CH3

R3=CH3

Page 29 of 31


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

28


O N O

N H


Figure 3

29


Page 30 of 31

Page 31 of 31


Graphic for table of contents

30


Rhabdopeptides from Xenorhabdus budapestensis SN84 and Their

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