Identification of Distinct Bacillus thuringiensis 4A4 Nematicidal Factors Using the Model Nematodes Pristionchus pacificus and Caenorhabditis elegans

Abstract

Bacillus thuringiensis has been extensively used for the biological control of insect pests. Nematicidal B. thuringiensis strains have also been identified; however, virulence factors of such strains are poorly investigated. Here, we describe virulence factors of the nematicidal B. thuringiensis 4A4 strain, using the model nematodes Pristionchus pacificus and Caenorhabditis elegans. We show that B. thuringiensis 4A4 kills both nematodes via intestinal damage. Whole genome sequencing of B. thuringiensis 4A4 identified Cry21Ha, Cry1Ba, Vip1/Vip2 and β-exotoxin as potential nematicidal factors. Only Cry21Ha showed toxicity to C. elegans, while neither Cry nor Vip toxins were active against P. pacificus, when expressed in E. coli. Purified crystals also failed to intoxicate P. pacificus, while autoclaved spore-crystal mixture of B. thuringiensis 4A4 retained toxicity, suggesting that primary β-exotoxin is responsible for P. pacificus killing. In support of this, we found that a β-exotoxin-deficient variant of B. thuringiensis 4A4, generated by plasmid curing lost virulence to the nematodes. Thus, using two model nematodes we revealed virulence factors of the nematicidal strain B. thuringiensis 4A4 and showed the multifactorial nature of its virulence.

Detection of β-exotoxin synthesis in Bacillus thuringiensis using an easy bioassay with the nematode Caenorhabditis elegans

“…; Burgerjon ; Noel ; Iatsenko et al . ). The thuringiensin has a teratogenic and mutagenic effect in mammalian cells, and the use of bioinsecticide of B. thuringiensis strains able to synthesize β‐exotoxins, is prohibited in different countries, including USA and Europe (Lüthy et al .…”

Insecticidal Activity and Synergistic Combinations of Ten Different Bt Toxins against Mythimna separata (Walker)

“…Bacillus thuringiensis (Bt) is a gram-positive bacterium that produces pesticidal crystal proteins which are active against insects, nematodes and other invertebrates [ 1 , 2 , 3 ]. Since Hannay discovered the crystalline inclusion in sporulating cells of Bt [ 4 ], the crystal proteins have been studied extensively to control the lepidopteran pest [ 1 , 5 , 6 ].…”

Nematicidal spore-forming Bacilli share similar virulence factors and mechanisms

“…Thuringiensin (β-exotoxin) is an adenine nucleoside derivative produced by B. thuringiensis that contributes to insecticidal abilities through the inhibition of RNA polymerases by competing with ATP for binding sites 16 . Thuringiensin can be used to control Heterodera glycines in soybeans and shows toxicity to C. elegans and Pristionchus pacificus 31 32 . Nematicidal strains containing the thuringiensin synthesizing cluster killed nematodes with a mortality of 74.86 ± 13.68%, which was higher than the 39.93 ± 16.06% mortality for thuringiensin non-synthesizing strains ( Fig.…”