Evidence for succession and putative metabolic roles of fungi and bacteria in the farming mutualism of the ambrosia beetle Xyleborus affinis

The bacterial and fungal community involved in ambrosia beetle fungiculture remains poorly studied compared to the famous fungus-farming ants and termites. Here we studied microbial community dynamics of laboratory nests, adults, and brood during the life cycle of the sugarcane shot hole borer, Xyleborus affinis. We identified a total of 40 fungal and 428 bacterial operational taxonomic units (OTUs), from which only five fungi (a Raffaelea fungus and four ascomycete yeasts) and four bacterial genera (Stenotrophomonas, Enterobacter, Burkholderia, and Ochrobactrum) can be considered the core community playing the most relevant symbiotic role. Both the fungal and bacterial populations varied significantly during the beetle’s life cycle. While the ascomycete yeasts were the main colonizers of the gallery early on, the Raffaelea and other filamentous fungi appeared after day 10, at the time when larval hatching happened. Regarding bacteria, Stenotrophomonas and Enterobacter dominated overall but decreased in foundresses and brood with age. Finally, inferred analyses of the putative metabolic capabilities of the bacterial microbiome revealed that they are involved in (i) degradation of fungal and plant polymers, (ii) fixation of atmospheric nitrogen, and (iii) essential amino acid, cofactor, and vitamin provisioning. Overall, our results suggest that yeasts and bacteria are more strongly involved in supporting the beetle-fungus farming symbiosis than previously thought.Research reported in this publication was supported by CONACyT-FORDECYT number 292399, Spanish Ministry of Economy and Competitiveness under award numbers SAF2015-65878-R and PGC2018-099344-B-I00, cofinanced by the European Regional Development Fund (ERDF), and from Generalitat Valenciana (project Prometeo/2018/A133). P.H.W.B. was supported by the German Research Foundation (DFG Emmy Noether grant BI 1956/1-1).Peer reviewe

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.The research leading to these results has received funding from the Research Projects of High Strategic Value for Society (Project 2003530920) founded by Institute of Ecology A. C (INECOL).Peer reviewe

Molecular evidence of the avocado defense response to Fusarium kuroshium infection: a deep transcriptome analysis using RNA-Seq

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

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.This study was principally financed with resources from the Mexican Ministry of Agriculture (SADER), Dirección del Programa Nacional de Moscas de la Fruta (DGSV – SENASICA) via the National Consultative Phytosanitary Council (CONACOFI) through projects 41011–2017, 41012–2018, 41013–2019, 80124–2020, and 80147–2021 awarded to MA. Additional funds were provided by the Asociación de Productores y Empacadores Exportadores de Aguacate de México (APEAM), Project 41010 to MA, the Universidad de Valencia via a Distinguished Professor Fellowship to MA (UV-INV-EPC17-548793), the Consejo Nacional de Ciencia y Tecnología (CONACyT)–Gobierno del Estado de Veracruz FOMIX grant (Project VER-2017-01-292397) to MA, and by the Instituto de Ecología, AC–INECOL. This project was also financed by Generalitat Valenciana (Valencia, Spain), Prometeo/2018/A/133 grant to AM.Peer reviewe

Design of a diagnostic system based on molecular markers derived from the ascomycetes pan-genome analysis: The case of Fusarium dieback disease.

A key factor to take actions against phytosanitary problems is the accurate and rapid detection of the causal agent. Here, we develop a molecular diagnostics system based on comparative genomics to easily identify fusariosis and specific pathogenic species as the Fusarium kuroshium, the symbiont of the ambrosia beetle Euwallaceae kuroshio Gomez and Hulcr which is responsible for Fusarium dieback disease in San Diego CA, USA. We performed a pan-genome analysis using sixty-three ascomycetes fungi species including phytopathogens and fungi associated with the ambrosia beetles. Pan-genome analysis revealed that 2,631 orthologue genes are only shared by Fusarium spp., and on average 3,941 (SD ± 1,418.6) are species-specific genes. These genes were used for PCR primer design and tested on DNA isolated from i) different strains of ascomycete species, ii) artificially infected avocado stems and iii) plant tissue of field-collected samples presumably infected. Our results let us propose a useful set of primers to either identify any species from Fusarium genus or, in a specific manner, species such as F. kuroshium, F. oxysporum, and F. graminearum. The results suggest that the molecular strategy employed in this study can be expanded to design primers against different types of pathogens responsible for provoking critical plant diseases

Additional file 8: of Identification of candidate genes related to calanolide biosynthesis by transcriptome sequencing of Calophyllum brasiliense (Calophyllaceae)

Coding sequences alignment between C. brasiliense and Arabidopsis 4CL proteins. (TXT 18 kb

Additional file 5: of Identification of candidate genes related to calanolide biosynthesis by transcriptome sequencing of Calophyllum brasiliense (Calophyllaceae)

Coding sequences alignment between PAL-like C. brasiliense unigenes and Arabidopsis PAL1 protein. (TXT 8 kb

Additional file 11: of Identification of candidate genes related to calanolide biosynthesis by transcriptome sequencing of Calophyllum brasiliense (Calophyllaceae)

Coding sequences alignment between CYP-like unigenes from C. brasiliense and CYP proteins from different plant species involved in furanocoumarins biosynthesis. (TXT 74 kb

Additional file 3: Figures S1-S9. of Identification of candidate genes related to calanolide biosynthesis by transcriptome sequencing of Calophyllum brasiliense (Calophyllaceae)

Figure S1: Gene ontology classification of C. brasiliense unigenes. Figure S2: Biological processes assigned to differentially expressed genes. Figure S3: Re-constructed C. brasiliense metabolic network from glucose to trans-cinnamate. Figure S4: Phylogenetic relationships, primary protein structures and identities percent of trans-cinnamate 4-hydroxylases proteins. Figure S5: Structural and phylogenetic analysis of 4-coumarate: CoA ligases (4CL) from C. brasiliense. Figure S6: Alignment of the 4-coumaroyl 2′-hydroxylases from C. brasiliense. Figure S7: Schematic representation of the Wagner-Meerwein rearrangement. Figure S8: C. brasiliense prenyltransferases. Figure S9: Phylogenetic tree of selected C. brasiliense cytochrome P450. (PPTX 1219 kb