Abstract
Pine Wilt Disease (PWD) is caused by Bursaphelenchus xylophilus, the pinewood nematode, and affects several species of pine trees worldwide. The ecosystem of the Pinus pinaster trees was investigated as a source of bacteria producing metabolites affecting this ecosystem: P. pinaster trees as target-plant, nematode as disease effector and its insect-vector as shuttle. For example, metals and metal-carrying compounds contribute to the complex tree-ecosystems. This work aimed to detect novel secondary metabolites like metallophores and related molecules produced under iron limitation by PWD-associated bacteria and to test their activity on nematodes. After screening 357 bacterial strains from Portugal and United States, two promising metallophore-producing strains Erwinia sp. A41C3 and Rouxiella sp. Arv20#4.1 were chosen and investigated in more detail. The genomes of these strains were sequenced, analyzed, and used to detect genetic potential for secondary metabolite production. A combinatorial approach of liquid chromatography-coupled tandem mass spectrometry (LC-MS) linked to molecular networking was used to describe these compounds. Two major metabolites were detected by HPLC analyses and described. One HPLC fraction of strain Arv20#4.1 showed to be a hydroxamate-type siderophore with higher affinity for chelation of Cu. The HPLC fraction of strain A41C3 with highest metal affinity showed to be a catecholate-type siderophore with higher affinity for chelation of Fe. LC-MS allowed the identification of several desferrioxamines from strain Arv20#4.1, in special desferrioxamine E, but no hit was obtained in case of strain A41C3 which might indicate that it is something new. Bacteria and their culture supernatants showed ability to attract C. elegans. HPLC fractions of those supernatant-extracts of Erwinia strain A41C3, enriched with secondary metabolites such as siderophores, were able to kill pinewood nematode. These results suggest that metabolites secreted under iron limitation have potential to biocontrol B. xylophilus and for management of Pine Wilt Disease.
Highlights
The pine wilt disease (PWD) is one of the most devastating diseases of forests pine trees in the world, with tremendous ecological, environmental, and economic damage
Two hundred and seventy-one bacterial strains isolated as wood colonizers/endophytes of P. pinaster trees from Portugal (Proença et al, 2017a), 48 bacterial strains carried by pinewood nematode (PWN) from Portugal (Proença et al, 2010), and 38 bacterial strains carried by PWN from United States (Proença et al, 2014) were evaluated for their production of siderophores on Iron-CAS agar plates (Schwyn and Neilands, 1987)
Numerous bacterial strains were positive on CAS agar medium that serves to detect siderophores: 103 endophytic strains (38.0%), 39 strains carried by B. xylophilus from Portugal (81.2%) and 20 strains carried by B. xylophilus from United States (52.6%) (Supplementary Figure S1)
Summary
The pine wilt disease (PWD) is one of the most devastating diseases of forests pine trees in the world, with tremendous ecological, environmental, and economic damage. Some of the relevant and more tree-related bacteria are endophytic opportunistic, living in soil and may enter the tree through the roots (Proença et al, 2017a). They are believed to be plant growth promoting rhizobacteria (PGPR) or do at least affect and interact with plants. It was reported that under iron limitation not lipopolysaccharides but siderophores as pseudobactin induce systemic resistance towards plant pests (Saikia et al, 2005; Romera et al, 2019) This response seems to be complementary to other defense mechanisms in dependence on the environmental conditions
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