Biological Control of Meloidogyne hapla Using an Antagonistic Bacterium

Abstract

We examined the efficacy of a bacterium for biocontrol of the root-knot nematode (RKN) Meloidogyne hapla in carrot (Daucus carota subsp. sativus) and tomato (Solanum lycopersicum). Among 542 bacterial isolates from various soils and plants, the highest nematode mortality was observed for treatments with isolate C1-7, which was identified as Bacillus cereus based on cultural and morphological characteristics, the Biolog program, and 16S rRNA sequencing analyses. The population density and the nematicidal activity of B. cereus C1-7 remained high until the end of culture in brain heart infusion broth, suggesting that it may have sustainable biocontrol potential. In pot experiments, the biocontrol efficacy of B. cereus C1-7 was high, showing complete inhibition of root gall or egg mass formation by RKN in carrot and tomato plants, and subsequently reducing RKN damage and suppressing nematode population growth, respectively. Light microscopy of RKN-infected carrot root tissues treated with C1-7 showed reduced formation of gall cells and fully developed giant cells, while extensive gall cells and fully mature giant cells with prominent cell wall ingrowths formed in the untreated control plants infected with RKNs. These histopathological characteristics may be the result of residual or systemic biocontrol activity of the bacterium, which may coincide with the biocontrol efficacies of nematodes in pots. These results suggest that B. cereus C1-7 can be used as a biocontrol agent for M. hapla.

Keywords: Bacillus cereus; Meloidogyne hapla; biological control; giant cell.

Fig. 1

First (A) and second (B) in vitro screening for nematicidal activities of bacterial isolates against Meloidogyne hapla in different concentrations of the bacterial cultures (A) and bacterial cell suspensions (B). Bars and vertical lines represent the means and standard deviations of three replications for M. hapla J2 mortality. *, Means with the same letters in each figure denote no significant difference at P ≤ 0.05 based on Duncan’s multiple range test (DMRT).

Fig. 2

Cultural (A) and morphological (B) characteristics of the bacterial isolate C1-7 showing (A) large circular, cream-colored, flat colonies with an undulate margin after 3 days of culture on BHI agar, and (B) rod-shaped morphology with more than 4 peritrichous flagella revealed by electron microscopy. Bar = 1.0 μm.

Fig. 3

Population growth curves of the bacterial isolate C1-7 with the time after culturing in different media including brain heart infusion broth (BHIB), Luria–Bertani broth (LBB), tryptic soy broth (TSB), and nutrient broth (NB). Vertical lines denote standard deviations of three replications.

Fig. 4

Effect of C1-7 on root galling caused by Meloidogyne hapla on carrot (A–D) and tomato (E–H). (A and E) No nematode inoculation and no bacterial treatment. (B and F) Nematode inoculation only. (C and G) Nematode inoculation and brain heart infusion broth (BHIB) treatment. (D and H) Nematode inoculation and bacterial treatment. Bars = 2.0 cm.

Fig. 5

Light microscopy of structural changes in carrot (A–D) and tomato (E–H) root tissues with no nematode inoculation and bacterial treatment (A, E), influenced by the infection of Meloidogyne hapla alone (B, F), treatments with brain heart infusion broth (BHIB; C, G), and the bacterial culture (D, H) showing intact root tissues with no nematode infection and no treatment (A, E); formation of large giant cells (Gt) with feeding plugs (FP), cell wall ingrowths (WI), and numerous nuclei (N) surrounded by proliferated gall cells (Gc; B, F); formation of Gt sometimes degenerative-looking because of cytoplasmic vacuoles (asterisk) with the proliferation of gall cells similar to or less than the nematode inoculation alone (C, G); and formation of Gt within the stele, which are degenerative-looking because of the chromatin materials (Ch) scattered in Gt, or no Gt formation with no or minimum formation of Gc (D, H). Cr = cortex, Ne = nematode, X = xylem vessel. Bars = 10 μm.