Ambrosia beetles farm their own food fungi within tunnel systems in wood and are among the three insect lineages performing agriculture (the others are fungus-farming ants and termites). In ambrosia beetles, primary ambrosia fungus cultivars have been regarded essential, whereas other microbes have been more or less ignored. Our KEGG analyses suggest so far unknown roles of yeasts and bacterial symbionts, by preparing the tunnel walls for the primary ambrosia fungi. This preparation includes enzymatic degradation of wood, essential amino acid production, and nitrogen fixation. The latter is especially exciting because if it turns out to be presentin vivoin ambrosia beetles, all farming animals (including humans) are dependent on atmospheric nitrogen fertilization of their crops. As previous internal transcribed spacer (ITS) metabarcoding approaches failed on covering the primary ambrosia fungi, our 18S metabarcoding approach can also serve as a template for future studies on the ambrosia beetle-fungus symbiosis.

Copper nanoparticles (Cu-NPs) have shown great antifungal activity against phytopathogenic fungi, making them a promising and affordable alternative to conventional fungicides. In this study, we evaluated the antifungal activity of Cu-NPs against Fusarium kuroshium, the causal agent of Fusarium dieback, and this might be the first study to do so. The Cu-NPs (at different concentrations) inhibited more than 80% of F. kuroshium growth and were even more efficient than a commercial fungicide used as a positive control (cupric hydroxide). Electron microscopy studies revealed dramatic damage caused by Cu-NPs, mainly in the hyphae surface and in the characteristic form of macroconidia. This damage was visible only 3 days post inoculation with used treatments. At a molecular level, the RNA-seq study suggested that this growth inhibition and colony morphology changes are a result of a reduced ergosterol biosynthesis caused by free cytosolic copper ions. Furthermore, transcriptional responses also revealed that the low- and high-affinity copper transporter modulation and the endosomal sorting complex required for transport (ESCRT) are only a few of the distinct detoxification mechanisms that, in its conjunction, F. kuroshium uses to counteract the toxicity caused by the reduced copper ion.

Somatic embryogenesis (SE) is a valuable model for understanding the mechanism of plant embryogenesis and a tool for the mass production of plants. However, establishing SE in avocado has been complicated due to the very low efficiency of embryo induction and plant regeneration. To understand the molecular foundation of the SE induction and development in avocado, we compared embryogenic (EC) and non-embryogenic (NEC) cultures of two avocado varieties using proteomic and metabolomic approaches. Although Criollo and Hass EC exhibited similarities in the proteome and metabolome profile, in general, we observed a more active phenylpropanoid pathway in EC than NEC. This pathway is associated with the tolerance of stress responses, probably through the reinforcement of the cell wall and flavonoid production. We could corroborate that particular polyphenolics compounds, including p-coumaric acid and t-ferulic acid, stimulated the production of somatic embryos in avocado. Exogen phenolic compounds were associated with the modification of the content of endogenous polyphenolic and the induction of the production of the putative auxin-a, adenosine, cellulose and 1,26-hexacosanediol-diferulate. We suggest that in EC of avocado, there is an enhanced phenylpropanoid metabolism for the production of the building blocks of lignin and flavonoid compounds having a role in cell wall reinforcement for tolerating stress response. Data are available at ProteomeXchange with the identifier PXD019705.

Ambrosia beetles, along with termites and leafcutter ants, are the only fungus-farming lineages within the tree of life. Bacteria harbored by ambrosia beetles may play an essential role in the nutritional symbiotic interactions with their associated fungi; however, little is known about the impact of rearing conditions on the microbiota of ambrosia beetles. We have used culture-independent methods to explore the effect of rearing conditions on the microbiome associated with Xyleborus affinis, Xyleborus bispinatus, and Xyleborus volvulus, evaluating different media in laboratory-controlled conditions and comparing wild and laboratory conditions. Our results revealed that rearing conditions affected the fungal and bacterial microbiome structure and had a strong influence on bacterial metabolic capacities. We propose that the rearing conditions influence the ambrosia-associated fungal and bacterial communities. Furthermore, bacterial microbiome flexibility may help beetles adapt to different substrates.

Lophophora williamsii (commonly named peyote) is a small, spineless cactus with psychoactive alkaloids, particularly mescaline. Peyote utilizes crassulacean acid metabolism (CAM), an alternative form of photosynthesis that exists in succulents such as cacti and other desert plants. Therefore, its transcriptome can be considered an important resource for future research focused on understanding how these plants make more efficient use of water in marginal environments and also for research focused on better understanding of the overall mechanisms leading to production of plant natural products and secondary metabolites. In this study, two cDNA libraries were generated from L. williamsii. These libraries, representing buttons (tops of stems) and roots were sequenced using different sequencing platforms (GS-FLX, GS-Junior and PGM, respectively). A total of 5,541,550 raw reads were generated, which were assembled into 63,704 unigenes with an average length of 564.04 bp. A total of 25,149 unigenes (62.19 %) was annotated using public databases. 681 unigenes were found to be differentially expressed when comparing the two libraries, where 400 were preferentially expressed in buttons and 281 in roots. Some of the major alkaloids, including mescaline, were identified by GC-MS and relevant metabolic pathways were reconstructed using the Kyoto encyclopedia of genes and genomes database (KEGG). Subsequently, the expression patterns of preferentially expressed genes putatively involved in mescaline production were examined and validated by qRT-PCR. High throughput transcriptome sequencing (RNA-seq) analysis allowed us to efficiently identify candidate genes involved in mescaline biosynthetic pathway in L. williamsii; these included tyrosine/DOPA decarboxylase, hydroxylases, and O-methyltransferases. This study sets the theoretical foundation for bioassay design directed at confirming the participation of these genes in mescaline production.

Fusarium kuroshium is a novel member of the Ambrosia Fusarium Clade (AFC) that has been recognized as one of the symbionts of the invasive Kuroshio shot hole borer, an Asian ambrosia beetle. This complex is considered the causal agent of Fusarium dieback, a disease that has severely threatened natural forests, landscape trees, and avocado orchards in the last 8 years. Despite the interest in this species, the molecular responses of both the host and F. kuroshium during the infection process and disease establishment remain unknown. In this work, we established an in vitro pathosystem using Hass avocado stems inoculated with F. kuroshium to investigate differential gene expression at 1, 4, 7 and 14 days post-inoculation. RNA-seq technology allowed us to obtain data from both the plant and the fungus, and the sequences obtained from both organisms were analyzed independently. The pathosystem established was able to mimic Fusarium dieback symptoms, such as carbohydrate exudation, necrosis, and vascular tissue discoloration. The results provide interesting evidence regarding the genes that may play roles in the avocado defense response to Fusarium dieback disease. The avocado data set comprised a coding sequence collection of 51,379 UniGenes, from which 2,403 (4.67%) were identified as differentially expressed. The global expression analysis showed that F. kuroshium responsive UniGenes can be clustered into six groups according to their expression profiles. The biologically relevant functional categories that were identified included photosynthesis as well as responses to stress, hormones, abscisic acid, and water deprivation. Additionally, processes such as oxidation-reduction, organization and biogenesis of the cell wall and polysaccharide metabolism were detected. Moreover, we identified orthologues of nucleotide-binding leucine-rich receptors, and their possible action mode was analyzed. In F. kuroshium, we identified 57 differentially expressed genes. Interestingly, the alcohol metabolic process biological category had the highest number of upregulated genes, and the enzyme group in this category may play an important role in the mechanisms of secondary metabolite detoxification. Hydrolytic enzymes, such as endoglucanases and a pectate lyase, were also identified, as well as some proteases. In conclusion, our research was conducted mainly to explain how the vascular tissue of a recognized host of the ambrosia complex responds during F. kuroshium infection since Fusarium dieback is an ambrosia beetle-vectored disease and many variables facilitate its establishment.