The fungal genus Collemopsidium comprises species that develop so-called borderline lichen symbioses with algae or cyanobacteria. Together with morphologically similar pyrenocarpous fungi it has been assigned to the family Xanthopyreniaceae. The adscription of this family to higher taxonomic ranks remain uncertain. Using sequence data of five nuclear genomic regions (nuLSU, nuSSU, tef1-α, rpb1 and rpb2) and one mitochondrial locus (mtSSU) we found that the studied representatives of this family are placed in the Dothideomyceta, yet relationships with the classes Dothideomycetes and Arthoniomycetes remain uncertain. We describe the new order Collemopsidiales to accommodate the genus Collemopsidium (paraphyletic as currently understood) and the lichenicolous genus Zwackhiomyces. Using five fungal fossils as calibrations points, we infer an age of c. 230 Mya for the crown of Collemopsidiales. Based on two molecular markers, we also provide insight into the global diversity of marine species of the genus Collemopsidium. According to the species delimitation algorithm GMYC, c. 26 putative species exist, far more than the six species recognized hitherto. We have confirmed this result by comparing the two alternative species models by means of Bayes factors, using path sampling and stepping-stone sampling algorithms to estimate the marginal likelihood of each model. Finally, our observations suggest rock-boring ability evolved in parallel in the different lineages within this group of fungi.

Developmental plasticity is one of the most striking features of plant morphogenesis, as plants are able to vary their shapes in response to environmental cues. Biotic or abiotic stimuli often promote organogenesis events in plants not observed under normal growth conditions. Root-knot nematodes (RKNs) are known to parasitize multiple species of rooting plants and to induce characteristic tissue expansion called galls or root-knots on the roots of their hosts by perturbing the plant cellular machinery. Galls contain giant cells (GCs) and neighboring cells (NCs), and the GCs are a source of nutrients for the parasitizing nematode. Highly active cell proliferation was observed in galls. However, the underlying mechanisms that regulate the symptoms triggered by the plant-nematode interaction have not yet been elucidated. In this study, we deciphered the molecular mechanism of gall formation with an in vitro infection assay system using RKN Meloidogyne incognita, and the model plant Arabidopsis thaliana. By taking advantages of this system, we performed next-generation sequencing (NGS)-based transcriptome profiling, and found that the expression of procambium identity-associated genes were enriched during gall formation. Clustering analyses with artificial xylogenic systems, together with the results of expression analyses of the candidate genes, showed a significant correlation between the induction of gall cells and procambium-associated cells. Furthermore, the promoters of several procambial marker genes such as ATHB8, TDR and WOX4 were activated not only in M. incognita-induced galls, but similarly in M. javanica induced-galls and Heterodera schachtii-induced syncytia. Our findings suggest that phytoparasitic nematodes modulate the host’s developmental regulation of the vascular stem cells during gall formation.

Most effective nematicides for the control of root-knot nematodes are banned, which demands a better understanding of the plant-nematode interaction. Understanding how gene expression in the nematode-feeding sites relates to morphological features may assist a better characterization of the interaction. However, nematode-induced galls resulting from cell-proliferation and hypertrophy hinders such observation, which would require tissue sectioning or clearing. We demonstrate that a method based on the green auto-fluorescence produced by glutaraldehyde and the tissue-clearing properties of benzyl-alcohol/benzyl-benzoate preserves the structure of the nematode-feeding sites and the plant-nematode interface with unprecedented resolution quality. This allowed us to obtain detailed measurements of the giant cells’ area in an Arabidopsis line overexpressing CHITINASE-LIKE-1 (CTL1) from optical sections by confocal microscopy, assigning a role for CTL1 and adding essential data to the scarce information of the role of gene repression in giant cells. Furthermore, subcellular structures and features of the nematodes body and tissues from thick organs formed after different biotic interactions, i.e., galls, syncytia, and nodules, were clearly distinguished without embedding or sectioning in different plant species (Arabidopsis, cucumber or Medicago). The combination of this method with molecular studies will be valuable for a better understanding of the plant-biotic interactions.

Root-knot nematodes (RKN) are polyphagous plant-parasitic roundworms that produce large crop losses, representing a relevant agricultural pest worldwide. After infection, they induce swollen root structures called galls containing giant cells (GCs) indispensable for nematode development. Among efficient control methods are biotechnology-based strategies that require a deep knowledge of underlying molecular processes during the plant-nematode interaction. Methods of achieving this knowledge include the application of molecular biology techniques such as transcriptomics (massive sequencing or microarray hybridization), proteomics or metabolomics. These require aseptic experimental conditions, as undetected contamination with other microorganisms could compromise the interpretation of the results. Herein, we present a simple, efficient and long-term method for nematode amplification on cucumber roots grown in vitro. Amplification of juveniles (J2) from the starting inoculum is around 40-fold. The method was validated for three Meloidogyne species (M. javanica, M. incognita and M. arenaria), producing viable and robust freshly hatched J2s. These can be used for further in vitro infection of different plant species such as Arabidopsis, tobacco and tomato, as well as enough J2s to maintain the population. The method allowed maintenance of around 90 Meloidogyne spp. generations (one every two months) from a single initial female over 15 years.

Root-knot nematodes (RKNs; Meloidogyne spp.) induce feeding cells (giant cells; GCs) inside a pseudo-organ (gall) from still unknown root cells. Understanding GCs ontogeny is essential to the basic knowledge of RKN-plant interaction and to discover novel and effective control strategies. Hence, we report for the first time in a model plant, Arabidopsis, molecular and cellular features concerning ectopic de novo organogenesis of RKNs GCs in leaves. RKNs induce GCs in leaves with irregular shape, a reticulated cytosol and fragmented vacuoles as GCs from roots. Leaf cells around the nematode enter G2-M shown by ProCycB1;1:CycB1;1(NT)-GUS expression, consistent to multinucleated GCs. In addition, GCs nuclei present irregular and varied sizes. All these characteristics mentioned, being equivalent to GCs in root-galls. RKNs complete their life cycle forming a gall/callus-like structure in the leaf vascular tissues resembling auxin-induced callus with an auxin-response maxima, indicated by high expression of DR5::GUS that is dependent on leaf auxin-transport. Notably, induction of leaves calli/ GCs requires molecular components from roots crucial for lateral roots (LRs), auxin-induced callus and root-gall formation, i.e. LBD16. Hence, LBD16 is a xylem pole pericycle specific and local marker in LR primordia unexpectedly induced locally in the vascular tissue of leaves after RKN infection. LBD16 is also fundamental for feeding site formation as RKNs could not stablish in 35S::LBD16-SRDX leaves, and likely it is also a conserved molecular hub between biotic and developmental signals in Arabidopsis either in roots or leaves. Moreover, RKNs induce the ectopic development of roots from leaf and root-galls, also formed in mutants compromised in LR formation, arf7/arf19, slr and alf4. Therefore, nematodes must target molecular signatures to induce post-embryogenic de novo organogenesis through the LBD16 callus formation pathway partially different from those prevalent during normal LR development.