Assessment of the Molecular Responses of an Ancient Angiosperm against Atypical Insect Oviposition: The Case of Hass Avocados and the Tephritid Fly Anastrepha ludens.

Anastrepha spp. (Diptera: Tephritidae) infestations cause significant economic losses in commercial fruit production worldwide. However, some plants quickly counteract the insertion of eggs by females by generating neoplasia and hindering eclosion, as is the case for Persea americana Mill., cv. Hass (Hass avocados). We followed a combined transcriptomics/metabolomics approach to identify the molecular mechanisms triggered by Hass avocados to detect and react to the oviposition of the pestiferous Anastrepha ludens (Loew). We evaluated two conditions: fruit damaged using a sterile pin (pin) and fruit oviposited by A. ludens females (ovi). We evaluated both of the conditions in a time course experiment covering five sampling points: without treatment (day 0), 20 min after the treatment (day 1), and days 3, 6, and 9 after the treatment. We identified 288 differentially expressed genes related to the treatments. Oviposition (and possibly bacteria on the eggs' surface) induces a plant hypersensitive response (HR), triggering a chitin receptor, producing an oxidative burst, and synthesizing phytoalexins. We also observed a process of cell wall modification and polyphenols biosynthesis, which could lead to polymerization in the neoplastic tissue surrounding the eggs.

Transcriptional Basis for Haustorium Formation and Host Establishment in Hemiparasitic Psittacanthus schiedeanus Mistletoes.

The mistletoe Psittacanthus schiedeanus, a keystone species in interaction networks between plants, pollinators, and seed dispersers, infects a wide range of native and non-native tree species of commercial interest. Here, using RNA-seq methodology we assembled the whole circularized quadripartite structure of P. schiedeanus chloroplast genome and described changes in the gene expression of the nuclear genomes across time of experimentally inoculated seeds. Of the 140,467 assembled and annotated uniGenes, 2,000 were identified as differentially expressed (DEGs) and were classified in six distinct clusters according to their expression profiles. DEGs were also classified in enriched functional categories related to synthesis, signaling, homoeostasis, and response to auxin and jasmonic acid. Since many orthologs are involved in lateral or adventitious root formation in other plant species, we propose that in P. schiedeanus (and perhaps in other rootless mistletoe species), these genes participate in haustorium formation by complex regulatory networks here described. Lastly, and according to the structural similarities of P. schiedeanus enzymes with those that are involved in host cell wall degradation in fungi, we suggest that a similar enzymatic arsenal is secreted extracellularly and used by mistletoes species to easily parasitize and break through tissues of the host.

Metagenomic Survey of the Highly Polyphagous Anastrepha ludens Developing in Ancestral and Exotic Hosts Reveals the Lack of a Stable Microbiota in Larvae and the Strong Influence of Metamorphosis on Adult Gut Microbiota.

We studied the microbiota of a highly polyphagous insect, Anastrepha ludens (Diptera: Tephritidae), developing in six of its hosts, including two ancestral (Casimiroa edulis and C. greggii), three exotic (Mangifera indica cv. Ataulfo, Prunus persica cv. Criollo, and Citrus x aurantium) and one occasional host (Capsicum pubescens cv. Manzano), that is only used when extreme drought conditions limit fruiting by the common hosts. One of the exotic hosts ("criollo" peach) is rife with polyphenols and the occasional host with capsaicinoids exerting high fitness costs on the larvae. We pursued the following questions: (1) How is the microbial composition of the larval food related to the composition of the larval and adult microbiota, and what does this tell us about transience and stability of this species' gut microbiota? (2) How does metamorphosis affect the adult microbiota? We surveyed the microbiota of the pulp of each host fruit, as well as the gut microbiota of larvae and adult flies and found that the gut of A. ludens larvae lacks a stable microbiota, since it was invariably associated with the composition of the pulp microbiota of the host plant species studied and was also different from the microbiota of adult flies indicating that metamorphosis filters out much of the microbiota present in larvae. The microbiota of adult males and females was similar between them, independent of host plant and was dominated by bacteria within the Enterobacteriaceae. We found that in the case of the "toxic" occasional host C. pubescens the microbiota is enriched in potentially deleterious genera that were much less abundant in the other hosts. In contrast, the pulp of the ancestral host C. edulis is enriched in several bacterial groups that can be beneficial for larval development. We also report for the first time the presence of bacteria within the Arcobacteraceae family in the gut microbiota of A. ludens stemming from C. edulis. Based on our findings, we conclude that changes in the food-associated microbiota dictate major changes in the larval microbiota, suggesting that most larval gut microbiota is originated from the food.

Barks from avocado trees of different geographic locations have consistent microbial communities.

Bark is a permanent surface for microbial colonization at the interface of trees and the surrounding air, but little is known about its microbial communities. We used shotgun metagenomic sequencing to analyze the bark microbiomes of avocado trees from two orchards, and compared one of them to rhizospheric soil. It was shown that the microbial communities of avocado bark have a well-defined taxonomic structure, with consistent patterns of abundance of bacteria, fungi, and archaea, even in trees from two different locations. Bark microbial communities were distinct from rhizospheric soil, although they showed overlap in some taxa. Thus, avocado bark is a well-defined environment, providing niches for specific taxonomic groups, many of which are also found in other aerial plant tissues. The present in-depth characterization of bark microbial communities can form a basis for their future manipulation for agronomical purposes.

The Bacterial Microbiome of Meloidogyne-Based Disease Complex in Coffee and Tomato.

The Meloidogyne-based disease complexes (MDCs) are caused by the interaction of different root-knot nematode species and phytopathogenic fungi. These complexes are devastating several important crops worldwide including tomato and coffee. Despite their relevance, little is known about the role of the bacterial communities in the MDCs. In this study 16s rDNA gene sequencing was used to analyze the bacterial microbiome associated with healthy and infested roots, as well with females and eggs of Meloidogyne enterolobii and M. paranaensis, the causal agents of MDC in tomato and coffee, respectively. Each MDC pathosystems displayed a specific taxonomic diversity and relative abundances constituting a very complex system. The main bacterial drivers of the MDC infection process were identified for both crops at order level. While corky-root coffee samples presented an enrichment of Bacillales and Burkholderiales, the corcky-root tomato samples presented an enrichment on Saprospirales, Chthoniobacterales, Alteromonadales, and Xanthomonadales. At genus level, Nocardia was common to both systems, and it could be related to the development of tumor symptoms by altering both nematode and plant systems. Furthermore, we predicted the healthy metabolic profile of the roots microbiome and a shift that may result in an increment of activity of central metabolism and the presence of pathogenic genes in both crops.

Impact of Rearing Conditions on the Ambrosia Beetle's Microbiome.

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.

Genomic Signals of Adaptation towards Mutualism and Sociality in Two Ambrosia Beetle Complexes.

Mutualistic symbiosis and eusociality have developed through gradual evolutionary processes at different times in specific lineages. Like some species of termites and ants, ambrosia beetles have independently evolved a mutualistic nutritional symbiosis with fungi, which has been associated with the evolution of complex social behaviors in some members of this group. We sequenced the transcriptomes of two ambrosia complexes (Euwallacea sp. near fornicatus⁻Fusarium euwallaceae and Xyleborus glabratus⁻Raffaelea lauricola) to find evolutionary signatures associated with mutualism and behavior evolution. We identified signatures of positive selection in genes related to nutrient homeostasis; regulation of gene expression; development and function of the nervous system, which may be involved in diet specialization; behavioral changes; and social evolution in this lineage. Finally, we found convergent changes in evolutionary rates of proteins across lineages with phylogenetically independent origins of sociality and mutualism, suggesting a constrained evolution of conserved genes in social species, and an evolutionary rate acceleration related to changes in selective pressures in mutualistic lineages.

Environmental pH modulates transcriptomic responses in the fungus Fusarium sp. associated with KSHB Euwallacea sp. near fornicatus.

BACKGROUND: The Ambrosia Fusarium Clade phytopathogenic Fusarium fungi species have a symbiotic relationship with ambrosia beetles in the genus Euwallacea (Coleoptera: Curculionidae). Related beetle species referred to as Euwallacea sp. near fornicatus have been spread in California, USA and are recognized as the causal agents of Fusarium dieback, a disease that causes mortality of many plant species. Despite the importance of this fungi, no transcriptomic resources have been generated. The datasets described here represent the first ever transcripts available for these species. We focused our study on the isolated species of Fusarium that is associated with one of the cryptic species referred to as Kuroshio Shot Hole Borer (KSHB) Euwallacea sp. near fornicatus. RESULTS: Hydrogen concentration is a critical signal in fungi for growth and host colonization, the aim of this study was to evaluate the effect of different pH conditions on growth and gene expression of the fungus Fusarium sp. associated with KSHB. An RNA-seq approach was used to compare the gene expression of the fungus grown for 2 weeks in liquid medium at three different pH levels (5.0, 6.0, and 7.0). An unbuffered treatment was included to evaluate the capability of the fungus to change the pH of its environment and the impact in gene expression. The results showed that the fungus can grow and modulate its genetic expression at different pH conditions; however, growth was stunted in acidic pH in comparison with neutral pH. The results showed a differential expression pattern in each pH condition even when acidic conditions prevailed at the end of the experiment. After comparing transcriptomics data from the three treatments, we found a total of 4,943 unique transcripts that were differentially expressed. CONCLUSIONS: We identified transcripts related to pH signaling such as the conserved PAL/RIM pathway, some transcripts related to secondary metabolism and other transcripts that were differentially expressed. Our analysis suggests possible mechanisms involved in pathogenicity in this novel Fusarium species. This is the first report that shows transcriptomic data of this pathogen as well as the first report of genes and proteins involved in their metabolism identifying potential virulence factors.

Draft Genome Sequence of the Phytopathogenic Fungus Fusarium euwallaceae, the Causal Agent of Fusarium Dieback.

Here, we report the genome of Fusarium euwallaceae strain HFEW-16-IV-019, an isolate obtained from Kuroshio shot hole borer (a Euwallacea sp.). These beetles were collected in Tijuana, Mexico, from elm trees showing typical symptoms of Fusarium dieback. The final assembly consists of 287 scaffolds spanning 48,274,071 bp and 13,777 genes.

Identification of candidate genes related to calanolide biosynthesis by transcriptome sequencing of Calophyllum brasiliense (Calophyllaceae).

BACKGROUND: Calophyllum brasiliense is highlighted as an important resource of calanolides, which are dipyranocoumarins that inhibit the reverse transcriptase of human immunodeficiency virus type 1 (HIV-1 RT). Despite having great medicinal importance, enzymes involved in calanolide, biosynthesis and the pathway itself, are still largely unknown. Additionally, no genomic resources exist for this plant species. RESULTS: In this work, we first analyzed the transcriptome of C. brasiliense leaves, stem, and roots using a RNA-seq strategy, which provided a dataset for functional gene mining. According to the structures of the calanolides, putative biosynthetic pathways were proposed. Finally, candidate unigenes in the transcriptome dataset, potentially involved in umbelliferone and calanolide (angular pyranocoumarin) biosynthetic pathways, were screened using mainly homology-based BLAST and phylogenetic analyses. CONCLUSIONS: The unigene dataset that was generated in this study provides an important resource for further molecular studies of C. brasiliense, especially for functional analysis of candidate genes involved in the biosynthetic pathways of linear and angular pyranocoumarins.