Tolerance to host-mediated OS is an essential characteristic of plant-associated organisms. In this study, we tested if B. xylophilus-associated bacteria could tolerate prolonged oxidative stress conditions with or without the nematode, in an attempt to understand their behaviour in the oxidative burst conditions of the host tree in the early stages of PWD.
Plant-associated bacteria, beneficial or pathogenic, have developed efficient detoxification systems to cope with host-ROS [19, 32]. This study demonstrates that Serratia spp. LCN-4 and LCN-16 (S. proteamaculans, 100% identity) and PWN-146 (S. marcescens, 99% identity) associated to B. xylophilus could sustain growth independently, and promote the survival of the nematodes under strong OS conditions. This result indicates, again, a beneficial and a potential helper effect to B. xylophilus. Vicente et al.  reported that some B. xylophilus-associated bacteria displayed plant pathogenic traits potentially related with PWD symptoms and B. xylophilus pathogenicity such as high cellulolytic activity, biofilm formation, EPS exudation and siderophores production. In fact, some of these traits are used by environmental bacteria as protectants against OS (i.e. EPS or biofilm). More recently, Chen et al.  showed that B. xylophilus-associated bacteria could support the nematode in the degradation of host xenobiotics. Based on our results, we suggest that B. xylophilus-associated Serratia spp. has evolved an elaborate detoxifying system to express several antioxidant enzymes to cope with H2O2-mediated OS.
In this study, we measured the transcript levels of two catalases in B. xylophilus in the presence of H2O2. PWN catalase genes presented a high protein similarity with other nematode catalases, evidencing the conserved nature of this enzyme . Cap’n’collar (Cnc) transcription factors are broadly conserved in eukaryotes except for plant and fungi . C. elegans CnC transcription factor SKN-1 regulates cellular differentiation of the pharynx and intestine during early embryogenesis, and also controls expression of many antioxidative and detoxification enzymes such as CTLs, GPXs and GSTs [34, 35]. In C. elegans four pathways (p38 MAPK, Insulin/IGF-1 pathway, WDR-23 ubiquitin pathway, and GSK-3 pathway) are known to control SKN-1 activity and the genomic structures of these pathways are fully conserved in B. xylophilus. Bacterial effect was transversal to virulent and avirulent B. xylophilus. Relative gene expression of catalase genes in B. xylophilus show that without bacteria, the basal expression of the both non-secreted Bxy-ctl-1 and secreted Bxy-ctl-2 genes in the virulent isolate Ka4, were higher than the avirulent C14-5 by 2.5-fold, which explains their differential tolerance level to H2O2. Further investigation on the detoxifying system of B. xylophilus is imperative. When interacting with Serratia spp. PWN-146, both virulent and avirulent B. xylophilus catalase levels decreased to levels comparable to non-stress condition, which is also in agreement with mortality test results (Figure 2).
The correlation between virulence and the ability to cope with oxidative stress has been found in the plant parasitic nematode Melodoigyne incognita[15, 29]. Virulent B. xylophilus Ka4 was more tolerant to H2O2 than the avirulent B. xylophilus strain C14-5. Hirao et al.  reported that the susceptible P. thunbergii reacts to PWN invasion with a strong oxidative burst, which implies that virulent B. xylophilus must possess an efficient antioxidant system to cope with these conditions. Shinya et al. suggested that potential ROS scavengers GST and GAPDH are localized on the surface coat of B. xylophilus. Li et al.  proposed 2-cysteine peroxiredoxin on the nematode cuticle of B. xylophilus, as another antioxidant agent in opposing oxidative burst. Recently, 12 anti-oxidant proteins were identified in the B. xylophilus secretome after plant extract stimuli, namely peroxiredoxin, catalase, glutathione peroxidase, nucleoredoxin-like protein, SOD, and thioredoxin . In this context, it is essential to further investigate the possible relation between virulence of B. xylophilus and its tolerance to oxidative stress, which was shown for the first time in this study.
To explore the bacterial interaction with B. xylophilus, we have studied bacteria attachment to the nematode cuticle, an important characteristic that, to our knowledge, has not been reported before. In our experiments, the associated-bacteria were not found to strongly attach to the cuticle of B. xylophilus. After 24 h contact with a high concentration of GFP-tagged Serratia spp. LCN-16, only a few bacteria could be detected on PWN cuticle (Figure 3). Shinya et al. have shown the presence of few bacteria on the nematode cuticle even after vigorous washing by scanning electron microscopy (SEM). B. xylophilus associated bacteria are reported to be carried on the nematode’s surface, and in average 290 were counted on the cuticle of PWN isolated from diseased trees . If bacteria are not attached to the nematode surface, how can they be transported by B. xylophilus from and into a pine tree? A possible explanation could be that these bacteria are transported within the nematode . However, the possible point of entry in B. xylophilus, the stylet opening, is very small compared with the bacteria size.
Serratia is an environmental ubiquitous Gram-negative bacterium, mostly free-living with an opportunistic lifestyle but also a pathogenic agent to plants, insects and humans . In the plant context, S. proteamaculans is usually identified as an endophytic bacterium living in poplar trees , characterized by colonizing in harmony and even expresses PGP (plant growth promoting) traits to promote host health. S. marcescens is also reported as a pathogenic agent of curcubit yellow vine disease . In both cases, these Serratia species are well adapted to the host plant (or tree) conditions, either as endophytes or pathogens, and are able to evade or suppress plant defences . We could not ascertain a strong attachment of associated-Serratia and B. xylophilus. It is not unlike that these bacteria may assist the nematode in an opportunistic or facultative way, and that perhaps these bacteria could be indeed host endophytes. This hypothesis can explain why diverse bacterial communities are associated to B. xylophilus, and why they possess such interesting traits and host-related lifestyle. Moreover, it can help to explain the contrasting results obtained in pathogenicity tests conducted previously . In this scenario, these multi-species consortia that present some in vitro plant-pathogenic traits that could aid the nematode inside the tree and contribute to PWD development as well , they could be asymptomatic endophytes that can become pathogenic as soon as the host tree is weakened . Nevertheless, the host-colonizing ability of these bacteria requires further investigation.