Gene sdaB Is Involved in the Nematocidal Activity of Enterobacter ludwigii AA4 Against the Pine Wood Nematode Bursaphelenchus xylophilus

Abstract

Bursaphelenchus xylophilus, a plant parasitic nematode, is the causal agent of pine wilt, a devastating forest tree disease. Essentially, no efficient methods for controlling B. xylophilus and pine wilt disease have yet been developed. Enterobacter ludwigii AA4, isolated from the root of maize, has powerful nematocidal activity against B. xylophilus in a new in vitro dye exclusion test. The corrected mortality of the B. xylophilus treated by E. ludwigii AA4 or its cell extract reached 98.3 and 98.6%, respectively. Morphological changes in B. xylophilus treated with a cell extract from strain AA4 suggested that the death of B. xylophilus might be caused by an increased number of vacuoles in non-apoptotic cell death and the damage to tissues of the nematodes. In a greenhouse test, the disease index of the seedlings of Scots pine (Pinus sylvestris) treated with the cells of strain AA4 plus B. xylophilus or those treated by AA4 cell extract plus B. xylophilus was 38.2 and 30.3, respectively, was significantly lower than 92.5 in the control plants treated with distilled water and B. xylophilus. We created a sdaB gene knockout in strain AA4 by deleting the gene that was putatively encoding the beta-subunit of L-serine dehydratase through Red homologous recombination. The nematocidal and disease-suppressing activities of the knockout strain were remarkably impaired. Finally, we revealed a robust colonization of P. sylvestris seedling needles by E. ludwigii AA4, which is supposed to contribute to the disease-controlling efficacy of strain AA4. Therefore, E. ludwigii AA4 has significant potential to serve as an agent for the biological control of pine wilt disease caused by B. xylophilus.

Engineering the Maize Root Microbiome: A Rapid MoClo Toolkit and Identification of Potential Bacterial Chassis for Studying Plant–Microbe Interactions

“…Additionally, Clo4 contains the nonreplicative origin R6Kγ plus an origin of transfer (oriT) sequence. While the nonreplicative origin enables the rapid cloning protocol, the oriT sequence on Clo4 enables its use for conjugation and has been employed in genomic editing methods such as transposon mutant libraries . These are important functionalities for plasmid systems as a deep understanding of plant–microbe interactions can come from genetic knockouts/knock-in experiments. , …”

“…Microbiomes have diverse impacts on plant health, including nutrient acquisition, − stress response, ,, and disease resistance. ,− Determining the mechanisms behind these plant growth-promoting phenotypes has been largely due to advancements in ‘omics technologies and in high-throughput phenotypic measurements. For example, 16S amplicon sequencing has greatly expanded our understanding of which microbes associate with plant hosts and which are correlated with growth promotion, while high-throughput assays such as nutrient profiling, , functional genomics/metagenomics, , and transposon mutant libraries − have helped uncover the genetic bases of these properties. Many of these studies are facilitated by the use of simplified systems, such as synthetic communities or single microbes inoculated with their plant host, − , which greatly reduces system complexity and allows individual interactions to be studied.…”

“…For example, 16S amplicon sequencing has greatly expanded our understanding of which microbes associate with plant hosts and which are correlated with growth promotion, while high-throughput assays such as nutrient profiling, , functional genomics/metagenomics, , and transposon mutant libraries − have helped uncover the genetic bases of these properties. Many of these studies are facilitated by the use of simplified systems, such as synthetic communities or single microbes inoculated with their plant host, − , which greatly reduces system complexity and allows individual interactions to be studied. However, the study of plant–microbe interactions is still limited by a lack of genetic tools and methods for most members of the plant microbiome. , …”

“…To start defining genetic tools for plant microbiomes, we focused on the 7-member synthetic maize root microbiome (SynCom) developed by Niu et al This SynCom was chosen for its simplicity, its representation of the complex and diverse maize root microbiome, and its ability to model disease resistance and community assembly. Currently, there are 6 studies ,− that have utilized this SynCom, indicating that it is an emerging standard for plant microbiome research. This SynCom has enabled studies of community stability/dynamics, , microbe-dependent heterosis in maize, development of protein extraction protocols for meta-proteomics in maize, identification of biocontrol strains against nematodes, and metabolic analysis of the SynCom strains .…”

“…Currently, there are 6 studies ,− that have utilized this SynCom, indicating that it is an emerging standard for plant microbiome research. This SynCom has enabled studies of community stability/dynamics, , microbe-dependent heterosis in maize, development of protein extraction protocols for meta-proteomics in maize, identification of biocontrol strains against nematodes, and metabolic analysis of the SynCom strains . Genetic tools are available for two SynCom species: Enterobacter ludwigii strain AA4 ( Elu , formerly E.…”