Phylogenetic characterization of bacterial endophytes from four Pinus species and their nematicidal activity against the pine wood nematode

Recently, bacterial endophytes (BEs) have gained importance in the agricultural sector for their use as biocontrol agents to manage plant pathogens. Outbreak of the pine wilt disease (PWD) in Korea has led researchers to test the feasibility of BEs in controlling the pine wood nematode (PWN) Bursaphelenchus xylophilus. In this study, we have reported the diversity and biocontrol activity of BEs against the PWN. By employing a culture-dependent approach, 1,622 BEs were isolated from the needle, stem, and root tissues of P. densiflora, P. rigida, P. thunbergii, and P. koraiensis across 18 sampling sites in Korea. We classified 389 members based on 16S rDNA analysis and taxonomic binning, of which, 215 operational taxonomic units (OTUs) were determined. Using Shannon’s indices, diversity across the Pinus species and tissues was estimated to reveal the composition of BEs and their tissue-specific preferences. When their ethyl acetate crude extracts were analysed for biocontrol activity, 44 candidates with nematicidal activity were obtained. Among these, Stenotrophomonas and Bacillus sp. exhibited significant inhibitory activity against PWN during their developmental stages. Altogether, our study furnishes a basic comprehension of bacterial communities found in the Pinus species and highlights the potential of BEs as biocontrol agents to combat PWD.


Supplementary
Movie S1. Control 1: Treatment of water on egg. After 24 h, 100% of egg was hatched to a L1/L2 juvenile stage of nematodes.
Movie S3. Positive control: Treatment of abamectin at 1,000 ppm. After 24 h, egg embryo was arrested, and the least hatching rate of egg was 31%.
Movie S4. Treatment of crude extract of Stenotrophomonas sp. at 1,000 ppm. After 24 h, movement of active egg embryo was arrested, and the least hatching rate of egg was 57%.
Movie S5. Treatment of crude extract of Bacillus sp. at 1,000 ppm. After 24 h, movement of active egg embryo was arrested, and the least hatching rate of egg was 64%.
Movie S6. Movie S10. Treatment of crude extract of Bacillus sp. at 1,000 ppm. After 24 h, 61% of L3/L4 juvenile stage nematodes was confirmed as dead and paralyzed.
Movie S11. Control 1: Treatment of water on adult stage. After 24 h, 100% of L3/L4 adult stage of nematodes was alive.
Movie S12. Control 2: Treatment of 3% DMSO in methanol on adult stage. After 24 h, 98% of adult stage of nematodes was alive.
Movie S13. Positive control: Treatment of abamectin at 1,000 ppm. After 24 h, 86% of adult stage nematodes was confirmed as dead and paralyzed.
adult stage nematodes was confirmed as dead and paralyzed.
Movie S15. Treatment of crude extract of Bacillus sp. at 1,000 ppm. After 24 h, 70% of adult stage nematodes was confirmed as dead and paralyzed.