Interactions between pinewood nematodes and the fungal community of pine trees

Abstract

The pinewood nematode Bursaphelenchus xylophilus, is the pathogenic agent of pine wilt disease and a globally notable pine pest. Despite being a plant pathogen, B. xylophilus has a mycophagous phase during its life cycle. We assessed the capacity for polyphagy of mycetophagous pinewood nematodes, testing which of the common species of fungi in pine trees provide better food and higher population growth rates. B. xylophilus performed particularly well on airborne fungi, namely the endophytes Botrytis cinerea and Cladosporium herbarum, and the pathogens Sirococcus conigenus and Sphaeropsis sapinea. Surprisingly, growth performance was not as good on the blue stain species (Ophiostoma spp. and Leptographium spp.) which are considered natural associates of B. xylophilus in the wild. Most of the fungi nonetheless permitted positive population growth of B. xylophilus, which is polyphagous and capable of feeding on numerous fungal species with diverse ecological niches.

Introduction

There is an astonishing diversity of fungi colonizing all tissues of forest trees (Baldrian, 2017), with equally complex and diverse fungal-tree interactions, ranging from mutualistic to antagonistic (Carrol, 1988). Adding to the complexity, it is difficult to categorize all tree-dwelling fungi into discrete categories such as pathogens or endophytes, since many fungal species can easily switch their lifestyle from one to the other (Sieber, 2007). In fact, a healthy forest tree supports a diverse fungal community that includes latent pathogens, dormant saprotrophs and mutualists (Carrol, 1988; Saikkonen et al., 1998; Sieber, 2007; Zamora et al., 2008). When the tree weakens and dies, some of these fungi switch life styles (Flowers et al., 2001; Smith et al., 1996; Smith and Stanosz, 2008), while the tree is invaded by a wide range of other airborne and insect-vectored pathogens and saprotrophs (Eckhardt et al., 2004).

Tree inhabiting fungal species can also form complex interactions, not only with the trees, but also with other tree dwelling organisms, such as insects, mites and nematodes. For example, some fungal species change the quality of food for insect herbivorous (Eberl et al., 2019), while others support a rich community of mites and nematodes (Massey, 1974; Hofstetter and Moser, 2014). These interactions arise naturally because the organic matter within living trees is mostly unavailable to potential animal consumers due to indigestible and toxic phytochemicals such as lignin, cellulose, hemicellulose and secondary metabolites. Fungi tend to transform living and dead plant matter into more digestible fungal tissues (Ayres et al., 2000; Schigel, 2016). In the other direction, several groups of fungi have evolved phoretic associations with insects and mites that facilitate the colonization of new tree hosts (Hofstetter and Moser, 2014). Tripartite or multipartite associations that include fungi have resulted in some of the most damaging disease systems and biological disturbances affecting forests worldwide. Some notorious examples are symbiotic associations between Ophiostomatoid fungi and bark beetles (Ayres et al., 2000; Hofstetter et al., 2006; Moser et al., 2010; Zhao et al., 2019).

Bursaphelenchus xylophilus, the pinewood nematode, is the pathogenic agent of pine wilt disease (PWD), and presently is the major invasive pest threat to Eurasian pine forests. It is native to North America (Togashi and Jikumaru, 2007; Vicente et al., 2012). It is vector-borne in that it is transmitted among trees by pine sawyer beetles (genus Monochamus). Nematodes travel on the adult sawyer beetles to move from depleted pine tree hosts to infect new ones (Giblin-Davis et al., 2003). The pinewood nematode is one of the few plant pathogenic species within the genus Bursaphelenchus, which are mostly fungal feeders in dead and dying pine trees, and usually phoretic on bark beetles or wood borers (Giblin-Davis et al., 2003; Ryss et al., 2005). B. xylophilus has evolved an additional phytophagous phase during its life cycle, while retaining the mycetophagous phase. This is unusual within the plant-parasitic nematodes, which are generally obligatory parasites (Espada et al., 2016). In fact, B. xylophilus can be maintained in laboratory cultures either on fungi or pine tissue (Espada et al., 2016; Pimentel et al., 2017).

Thus, development of PWD is dependent of a multipartite system where fungi are a nutritional substrate for potential pathogenic nematodes. The currently accepted view of the B. xylophilus life cycle is that its phytophagous phase starts when the nematodes enter a living pine through maturation feeding of insect vectors in the canopy. Then nematodes migrate within the tree while feeding on the contents of parenchyma cells. Later when the tree is weakened or freshly dead, it is colonized by a succession of fungi species, and the nematode populations switch to the mycetophagous phase before boarding a pupa of its insect vector (Mamiya, 2012; Espada et al., 2016). B. xylophilus can, however, skip the phytophagous phase when inoculated in an already dead or dying pine tree through oviposition of female pine sawyer beetles. It has been proposed that this is most frequent in areas where healthy pines are resistant to infection by the pathogen, mainly in its native range in North America, which precludes the phytophagous phase (Wingfield et al., 1984).

The importance of fungi in the population dynamics of B. xylophilus has been recognized for some time (Wingfield et al., 1984). Surveys of the suitability of different fungi species for the pinewood nematode have focused on fungi identified from the stems of recently dead pine trees infested with the nematode (Fukushige, 1991; Maehara and Futai, 2000; Futai et al., 2007). Special attention has been paid to fungi within the Ophiostomataceae (frequently known as blue stain fungi) that are carried by bark and wood boring insects into the same phloem tissues as pinewood nematodes (Niu et al., 2012; Wang et al., 2018). It has been suggested that B. xylophilus may have specific or even mutualistic relations with some of these fungal species (Zhao et al., 2013, 2018). However, fungal feeding nematodes are commonly regarded as generalists, and surveys of nematode growth on different species of fungi always shows them as polyphagous, although some fungal resources may support higher nematode reproduction rates and population levels (Ruess et al., 2000; Okada et al., 2005; Hasna et al., 2007). B. xylophilus has also been reported to be polyphagous in its mycetophagous phase (Futai et al., 2007).

The complexity of the pinewood nematode life cycle makes likely that it will encounter a diverse array of fungi species during its time within a host pine tree. When entering healthy trees through the canopy, B. xylophilus can encounter dormant saprotrophs and pathogens, and endophytic fungi living asymptomatically within leaves and stems. As the tree weakens and dies, its fungal community changes dramatically as it is invaded by a wide range of other airborne and insect-vectored pathogens and saprotrophs, entering through the canopy and stem. Thus, being polyphagous in its mycetophagous phase may be advantageous for the pinewood nematode. However, the ecological significance of interactions between pinewood nematode populations and the fungal community of pine trees has not been explored.

In the present work, we assessed how polyphagous the pinewood nematode is during its mycetophagous phase and tested whether ophiostomatoid fungi are better substrates for B. xylophilus development than other groups of fungi growing on pine hosts. We used replicated standardized bioassays to compare nematode growth when feeding on insect-vectored fungi of the Ophiostomataceae versus other airborne, endophytic, pathogenic and/or saprotrophs fungi that they may encounter in pine trees. We tested B. xylophilus populations from both Portugal and North America because they have been hypothesized to be ecologically different in their resource use (Wingfield et al., 1984). Our objective was to study the interaction between B. xylophilus and the fungal community of their host trees, and to clarify the importance of these interactions to our understanding of pine wilt disease in general.