Alcaligenes faecalis ZD02, a Novel Nematicidal Bacterium with an Extracellular Serine Protease Virulence Factor

Abstract

Root knot nematodes (RKNs) are the world's most damaging plant-parasitic nematodes (PPNs), and they can infect almost all crops. At present, harmful chemical nematicides are applied to control RKNs. Using microbial nematicides has been proposed as a better management strategy than chemical control. In this study, we describe a novel nematicidal bacterium named Alcaligenes faecalis ZD02. A. faecalis ZD02 was isolated from Caenorhabditis elegans cadavers and has nematostatic and nematicidal activity, as confirmed by C. elegans growth assay and life span assay. In addition, A. faecalis ZD02 fermentation broth showed toxicity against C. elegans and Meloidogyne incognita. To identify the nematicidal virulence factor, the genome of strain ZD02 was sequenced. By comparing all of the predicted proteins of strain ZD02 to reported nematicidal virulence factors, we determined that an extracellular serine protease (Esp) has potential to be a nematicidal virulence factor, which was confirmed by bioassay on C. elegans and M. incognita. Using C. elegans as the target model, we found that both A. faecalis ZD02 and the virulence factor Esp can damage the intestines of C. elegans. The discovery that A. faecalis ZD02 has nematicidal activity provides a novel bacterial resource for the control of RKNs.

FIG 1

Growth assay and life span assay when wild-type N2 worms were fed strain ZD02 or the normal food E. coli OP50. (A) Wild-type N2 L1-stage worms were fed strain ZD02 or E. coli OP50 and, after incubation at 20°C for 72 h, were photographed using a light microscope. (B) The average size of the worms fed strain ZD02 or E. coli OP50 was calculated using the software NIH Image J 1.33. (C) A. faecalis ZD02 shortened the longevity of C. elegans. Wild-type L4 worms were washed with M9 buffer, transferred to A. faecalis ZD02 plates or normal-food (E. coli OP50) plates and incubated at 20°C. The survival rate of the worms on each plate was counted at 0.5-day intervals. These results are from three independent experiments. More than 60 worms were used in each well or on each plate. Bar, 0.2 mm. Significance was determined using two-sample t tests: ***, P < 0.001 (a lack of any symbol indicates no significant difference).

FIG 2

Phylogenetic tree highlighting the position of strain ZD02 relative to other strains within the genus Alcaligenes. A neighbor-joining phylogenetic tree showing the phylogenetic relationship of A. faecalis ZD02 (triangle) was generated using MEGA 6-based 16S rRNA gene sequence alignments.

FIG 3

A. faecalis ZD02 fermentation broth killed C. elegans (A) and M. incognita J2 (B). Wild-type L4 C. elegans (A) and M. incognita (B) J2s were incubated in A. faecalis ZD02 fermentation broth in 96-well plates at 20°C. The mortality rate of the worms in each well was then counted; LB medium was used as the control. Percent mortality is the corrected mortality, which is the mortality in the fermentation broth minus the mortality in the LB medium. Heat inactivation (“heat”) was performed by heat treating the 56-hour fermentation broth for 30 min at 80°C. More than 60 worms were used in each plate. The results are from three independent experiments. Significance was determined using two-sample t tests: *, P < 0.05; **, P < 0.01 (a lack of any symbol indicates no significant difference).

FIG 4

The Esp protein inhibited the growth of C. elegans and had toxicity against the nematode C. elegans and M. incognita. (A) Transcript levels of esp mRNA of A. faecalis ZD02 when cultured for 12 h, 24 h, 36 h, and 48 h in LB medium at 30°C. The 16S rRNA gene was used as a reference. (B) Wild-type N2 L1-stage worms were exposed to E. coli ESP or E. coli CK and, after incubation at 20°C for 72 h, photographed at a magnification of ×100. E. coli ESP is E. coli BL21(DE3) transformed with the recombinant vector pET28a(+)-Esp, and E. coli CK is E. coli BL21(DE3) transformed with empty vector pET28a(+), which was used as the control. (C) The average size of the worms fed E. coli ESP or E. coli CK was calculated using the software NIH Image J 1.33. More than 60 worms were photographed. Bar, 0.2 mm. Significance was determined using two-sample t tests: **, P < 0.01. (D) Mortality assays of C. elegans that were incubated with various concentrations of purified Esp protein. Wild-type L4 worms were washed with M9 buffer, transferred to 48-well plates, and incubated at 20°C with different concentrations of Esp protein, with E. coli OP50 as the food source; the mortality rate of the worms in each well was determined after 7 days. (E) Mortality assays of M. incognita J2s that were incubated with different concentrations of purified Esp protein. These results are from three independent experiments.

FIG 5

A. faecalis ZD02 and the Esp protein damaged the intestines of C. elegans. Wild-type N2 L4-stage worms were fed the normal food E. coli OP50 (A), A. faecalis ZD02 (B), BSA (as the control) (D), and Esp protein (E) at 20°C for 24 h. The anterior intestines of the nematodes were photographed using a differential interference contrast (DIC) microscope. Arrows indicate intestinal damage by A. faecalis ZD02. (C) Frequency of intestinal damage in worms fed E. coli OP50 or A. faecalis ZD02. (F) Frequency of intestinal damage in worms fed BSA or Esp. Values are the percentage of worms with damaged intestines. More than 60 worms were inspected by microscopy. Bar, 20 μm. Significance was determined using two-sample t tests: **, P < 0.01; ***, P < 0.001.

FIG 6

The anterior intestines were observed in GFP-labeled transgenic FT63 (dlg::gfp) worms by epifluorescence microscopy after feeding with E. coli OP50 (A), A. faecalis ZD02 (B), BSA (C), or Esp protein (D). Twenty to 30 worms were incubated for 24 h at 20°C. More than 60 worms were inspected by microscopy. Bar, 200 μm.