| Summary: | <i>Pseudomonas putida</i> MCCC 1A00316 was originally isolated from an Antarctic soil and has demonstrated potential nematicidal activity. Thus, it has promising applications for the biological control of <i>Meloidogyne incognita</i>. The larval mortality and egg-hatching inhibition rates of <i>M. incognita</i> will increase with the rising concentration of culture filtrates of <i>P. putida</i> MCCC 1A00316 and the duration of exposure. Thus, this study aimed to separate, purify, and identify nematicidal compounds from <i>P. putida</i> MCCC 1A00316 and to validate their anti-<i>M. incognita</i> activities. Compounds were purified through silica gel column chromatography and thin-layer chromatography combined with high-performance liquid chromatography (HPLC). Structural identification was conducted through liquid chromatography time-of-flight mass spectrometry, <sup>1</sup>H nuclear magnetic resonance (NMR) spectroscopy, <sup>13</sup>C-NMR, and Marfey’s method. The isolated compounds were identified as cyclo(<span style="font-variant: small-caps;">l</span>-Pro⁻<span style="font-variant: small-caps;">l</span>-Leu) on the basis of the results of the above analyses and previously reported data. The effects of various concentrations of cyclo(<span style="font-variant: small-caps;">l</span>-Pro⁻<span style="font-variant: small-caps;">l</span>-Leu) on the mortality rates of second-stage juveniles (J2) of <i>M. incognita</i> were investigated. Results showed that HPLC-purified cyclo(<span style="font-variant: small-caps;">l</span>-Pro⁻<span style="font-variant: small-caps;">l</span>-Leu) displayed nematicidal activities. The mortality rate of <i>M. incognita</i> J2 reached 84.3% after 72 h of exposure to 67.5 mg/L cyclo(<span style="font-variant: small-caps;">l</span>-Pro⁻<span style="font-variant: small-caps;">l</span>-Leu). The lowest egg-hatching rate (9.74%) was observed after 8 days of incubation with 2000 mg/L cyclo(<span style="font-variant: small-caps;">l</span>-Pro⁻<span style="font-variant: small-caps;">l</span>-Leu). An egg-hatching rate of 53.11% was obtained under the control treatment (sterile distilled water). However, cyclo(<span style="font-variant: small-caps;">l</span>-Pro⁻<span style="font-variant: small-caps;">l</span>-Leu) did not elicit chemotaxis activity to <i>M. incognita</i>. This is the first work to investigate the anti-<i>M. incognita</i> characteristics of cyclo(<span style="font-variant: small-caps;">l</span>-Pro⁻<span style="font-variant: small-caps;">l</span>-Leu).
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