Antifungal capabilities of gut microbial communities of three dung beetle species (Scarabaeidae: Scarabaeinae).

Gut microbial communities are part of the regulatory array of various processes within their hosts, ranging from nutrition to pathogen control. Recent evidence shows that dung beetle's gut microbial communities release substances with antifungal activity. Because of the enormous diversity of gut microorganisms in dung beetles, there is a possibility of discovering novel compounds with antifungal properties. We tested the antifungal activity mediated by gut microbial communities of female dung beetles against nine phytopathogenic fungi strains (Colletotrichum asianum-339, C. asianum-340, C. asianum-1, C. kahawae-390, C. karstii-358, C. siamense-220, Fusarium oxysporum-ATCC338, Nectria pseudotrichia-232, Verticillium zaelandica-22). Our tests included the gut microbial communities of three species of dung beetles: Canthon cyanellus (roller beetle), Digitonthophagus gazella (burrower beetle), and Onthophagus batesi (burrower beetle), and we followed the dual confrontation protocol, i.e., we challenged each fungal strain with the microbial communities of each species of beetles in Petri dishes containing culture medium. Our results showed that gut microbial communities of the three dung beetle species exhibit antifungal activity against at least seven of the nine phytopathogenic fungal strains. The gut microbial communities of Onthophagus batesi significantly decreased the mycelial growth of the nine phytopathogenic fungi strains; the gut microbial communities of Canthon cyanellus and Digitonthophagus gazella significantly reduced the mycelial growth of seven strains. These results provide a basis for investigating novel antifungal substances within gut microbial communities of dung beetles.

Influence of phylogenetic, environmental, and behavioral factors on the gut bacterial community structure of dung beetles (Scarabaeidae: Scarabaeinae) in a Neotropical Biosphere Reserve.

Gut bacteria help dung beetles metabolize nutrients contained and synthesize those unavailable in their food, depending on the ecological scenario in which they develop. However, less is known about the influence of environmental and behavioral factors on the taxonomic composition of bacterial gut communities in Scarabaeinae beetles. To address this research topic, we analyzed 13 tropical dung beetle species in the Los Tuxtlas Biosphere Reserve, Mexico, to understand how the beetle tribe, habitat, food preference, food relocation, and parental care influence the composition of gut bacterial communities. We found that the beetle tribe is the primary factor impacting the taxonomic composition of gut bacterial communities. Among them, Deltochilini displayed the highest variability in diversity due to the different combinations of habitat and food preferences among its species. On the other hand, the other tribes studied did not exhibit such variable combinations. Habitat emerged as the second most influential factor, with forest-dwelling beetles displaying higher diversity. This can be attributed to the heterogeneous environments within tropical forests, which offer a greater diversity of food resources. In contrast, grassland beetles, living in more homogeneous environments and relying on cow feces as their main food source, exhibited lower diversity. Our findings suggest a correlation between bacterial diversity and food resource availability in complex habitats, such as tropical forests, which offer a wider array of food sources compared to simpler environments like grasslands.

Bitter friends are not always toxic: The loss of acetic acid bacteria and the absence of Komagataeibacter in the gut microbiota of the polyphagous fly Anastrepha ludens could inhibit its development in Psidium guajava in contrast to A. striata and A. fraterculus that flourish in this host.

The gut microbiota is key for the homeostasis of many phytophagous insects, but there are few studies comparing its role on host use by stenophagous or polyphagous frugivores. Guava (Psidium guajava) is a fruit infested in nature by the tephritids Anastrepha striata and A. fraterculus. In contrast, the extremely polyphagous A. ludens infests guava only under artificial conditions, but unlike A. striata and the Mexican A. fraterculus, it infests bitter oranges (Citrus x aurantium). We used these models to analyze whether the gut microbiota could explain the differences in host use observed in these flies. We compared the gut microbiota of the larvae of the three species when they developed in guava and the microbiota of the fruit pulp larvae fed on. We also compared the gut microbiota of A. ludens developing in C. x aurantium with the pulp microbiota of this widely used host. The three flies modified the composition of the host pulp microbiota (i.e., pulp the larvae fed on). We observed a depletion of Acetic Acid Bacteria (AAB) associated with a deleterious phenotype in A. ludens when infesting P. guajava. In contrast, the ability of A. striata and A. fraterculus to infest this fruit is likely associated to a symbiotic interaction with species of the Komagataeibacter genus, which are known to degrade a wide spectrum of tannins and polyphenols. The three flies establish genera specific symbiotic associations with AABs. In the case of A. ludens, the association is with Gluconobacter and Acetobacter, but importantly, it cannot be colonized by Komagataeibacter, a factor likely inhibiting its development in guava.

Plant growth-promoting and non-promoting rhizobacteria from avocado trees differentially emit volatiles that influence growth of Arabidopsis thaliana.

Microbial volatile organic compounds (mVOCs) play important roles in inter- and intra-kingdom interactions, and they are also important as signal molecules in physiological processes acting either as plant growth-promoting or negatively modulating plant development. We investigated the effects of mVOCs emitted by PGPR vs non-PGPR from avocado trees (Persea americana) on growth of Arabidopsis thaliana seedlings. Chemical diversity of mVOCs was determined by SPME-GC-MS; selected compounds were screened in dose-response experiments in A. thaliana transgenic lines. We found that plant growth parameters were affected depending on inoculum concentration. Twenty-six compounds were identified in PGPR and non-PGPR with eight of them not previously reported. The VOCs signatures were differential between those groups. 4-methyl-2-pentanone, 1-nonanol, 2-phenyl-2-propanol and ethyl isovalerate modified primary root architecture influencing the expression of auxin- and JA-responsive genes, and cell division. Lateral root formation was regulated by 4-methyl-2-pentanone, 3-methyl-1-butanol, 1-nonanol and ethyl isovalerate suggesting a participation via JA signalling. Our study revealed the differential emission of volatiles by PGPR vs non-PGPR from avocado trees and provides a general view about the mechanisms by which those volatiles influence plant growth and development. Rhizobacteria strains and mVOCs here reported are promising for improvement the growth and productivity of avocado crop.

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.

Molecular and Environmental Triggering Factors of Pathogenicity of Fusarium oxysporum and F. solani Isolates Involved in the Coffee Corky-Root Disease.

Coffee corky-root disease causes serious damages to coffee crop and is linked to combined infection of Fusarium spp. and root-knot nematodes Meloidogyne spp. In this study, 70 Fusarium isolates were collected from both roots of healthy coffee plants and with corky-root disease symptoms. A phylogenetic analysis, and the detection of pathogenicity SIX genes and toxigenicity Fum genes was performed for 59 F. oxysporum and 11 F. solani isolates. Based on the molecular characterization, seven F. oxysporum and three F. solani isolates were assessed for their pathogenicity on coffee seedlings under optimal watering and water stress miming root-knot nematode effect on plants. Our results revealed that a drastic increment of plant colonization capacity and pathogenicity on coffee plants of some Fusarium isolates was caused by water stress. The pathogenicity on coffee of F. solani linked to coffee corky-root disease and the presence of SIX genes in this species were demonstrated for the first time. Our study provides evidence for understanding the pathogenic basis of F. oxysporum and F. solani isolates on coffee and revealed the presence of SIX and Fum genes as one of their pathogenicity-related mechanisms. We also highlight the relevance of chlorophyll, a fluorescence as an early and high-throughput phenotyping tool in Fusarium pathogenicity studies on coffee.

Diversity and Composition of the Gut Microbiota in the Developmental Stages of the Dung Beetle Copris incertus Say (Coleoptera, Scarabaeidae).

Dung beetles are holometabolous insects that feed on herbivorous mammal dung and provide services to the ecosystem including nutrient cycling and soil fertilization. It has been suggested that organisms developing on incomplete diets such as dungs require the association with microorganisms for the synthesis and utilization of nutrients. We describe the diversity and composition of the gut-microbiota during the life cycle of the dung beetle Copris incertus using 16S rRNA gene sequencing. We found that C. incertus gut contained a broad diversity of bacterial groups (1,699 OTUs and 302 genera). The taxonomic composition varied during the beetle life cycle, with the predominance of some bacterial genera in a specific developmental stage (Mothers: Enterobacter and Serratia; Eggs: Nocardioides and Hydrogenophaga; Larval and pupal stages: Dysgonomonas and Parabacteroides; offspring: Ochrobactrum). The beta diversity evidenced similarities among developmental stages, clustering (i) the adult stages (mother, male and female offsprings), (ii) intermediate developmental (larvae and pupa), and (iii) initial stage (egg). Microbiota differences could be attributed to dietary specialization or/and morpho-physiological factors involved in the transition from a developmental stage to the next. The predicted functional profile (PICRUSt2 analysis) for the development bacterial core of the level 3 categories, indicated grouping by developmental stage. Only 36 categories were significant in the SIMPER analysis, including the metabolic categories of amino acids and antibiotic synthesis, which were enriched in the larval and pupal stages; both categories are involved in the metamorphosis process. At the gene level, we found significant differences only in the KOs encoding functions related to nitrogen fixation, uric acid metabolism, and plant cell wall degradation for all developmental stages. Nitrogen fixation and plant cell wall degradation were enriched in the intermediate stages and uric acid metabolism was enriched in mothers. The data reported here suggested the influence of the maternal microbiota in the composition and diversity of the gut microbiota of the offspring.

Characterization of plant growth-promoting bacteria associated with avocado trees (Persea americana Miller) and their potential use in the biocontrol of Scirtothrips perseae (avocado thrips).

Plants interact with a great variety of microorganisms that inhabit the rhizosphere or the epiphytic and endophytic phyllosphere and that play critical roles in plant growth as well as the biocontrol of phytopathogens and insect pests. Avocado fruit damage caused by the thrips species Scirtothrips perseae leads to economic losses of 12-51% in many countries. In this study, a screening of bacteria associated with the rhizosphere or endophytic phyllosphere of avocado roots was performed to identify bacterial isolates with plant growth-promoting activity in vitro assays with Arabidopsis seedlings and to assess the biocontrol activity of the isolates against Scirtothrips perseae. The isolates with beneficial, pathogenic and/or neutral effects on Arabidopsis seedlings were identified. The plant growth-promoting bacteria were clustered in two different groups (G1 and G3B) based on their effects on root architecture and auxin responses, particularly bacteria of the Pseudomonas genus (MRf4-2, MRf4-4 and TRf2-7) and one Serratia sp. (TS3-6). Twenty strains were selected based on their plant growth promotion characteristics to evaluate their potential as thrips biocontrol agents. Analyzing the biocontrol activity of S. perseae, it was identified that Chryseobacterium sp. shows an entomopathogenic effect on avocado thrips survival. Through the metabolic profiling of compounds produced by bacteria with plant growth promotion activity, bioactive cyclodipeptides (CDPs) that could be responsible for the plant growth-promoting activity in Arabidopsis were identified in Pseudomonas, Serratia and Stenotrophomonas. This study unravels the diversity of bacteria from the avocado rhizosphere and highlights the potential of a unique isolate to achieve the biocontrol of S. perseae.

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.

Phytophthora Root Rot Modifies the Composition of the Avocado Rhizosphere Microbiome and Increases the Abundance of Opportunistic Fungal Pathogens.

The structure and function of rhizosphere microbial communities are affected by the plant health status. In this study, we investigated the effect of root rot on the avocado rhizosphere microbiome, using 16S rDNA and ITS sequencing. Furthermore, we isolated potential fungal pathogens associated with root rot symptoms and assessed their pathogenic activity on avocado. We found that root rot did not affect species richness, diversity or community structure, but induced changes in the relative abundance of several microbial taxa. Root rot increased the proportion of Pseudomonadales and Burkholderiales in the rhizosphere but reduced that of Actinobacteria, Bacillus spp. and Rhizobiales. An increase in putative opportunistic fungal pathogens was also detected in the roots of symptomatic trees; the potential pathogenicity of Mortierella sp., Fusarium spp., Lasiodiplodia sp. and Scytalidium sp., is reported for the first time for the State of Veracruz, Mexico. Root rot also potentially modified the predicted functions carried out by rhizobacteria, reducing the proportion of categories linked with the lipid and amino-acid metabolisms whilst promoting those associated with quorum sensing, virulence, and antibiotic resistance. Altogether, our results could help identifying microbial taxa associated to the disease causal agents and direct the selection of plant growth-promoting bacteria for the development of biocontrol microbial consortia.

Functional Signatures of the Epiphytic Prokaryotic Microbiome of Agaves and Cacti.

Microbial symbionts account for survival, development, fitness and evolution of eukaryotic hosts. These microorganisms together with their host form a biological unit known as holobiont. Recent studies have revealed that the holobiont of agaves and cacti comprises a diverse and structured microbiome, which might be important for its adaptation to drylands. Here, we investigated the functional signatures of the prokaryotic communities of the soil and the episphere, that includes the rhizosphere and phyllosphere, associated with the cultivated Agave tequilana and the native and sympatric Agave salmiana, Opuntia robusta and Myrtillocactus geometrizans by mining shotgun metagenomic data. Consistent with previous phylogenetic profiling, we found that Proteobacteria, Actinobacteria and Firmicutes were the main represented phyla in the episphere of agaves and cacti, and that clustering of metagenomes correlated with the plant compartment. In native plants, genes related to aerobic anoxygenic phototrophy and photosynthesis were enriched in the phyllosphere and soil, while genes coding for biofilm formation and quorum sensing were enriched in both epiphytic communities. In the episphere of cultivated A. tequilana fewer genes were identified, but they belonged to similar pathways than those found in native plants. A. tequilana showed a depletion in several genes belonging to carbon metabolism, secondary metabolite biosynthesis and xenobiotic degradation suggesting that its lower microbial diversity might be linked to functional losses. However, this species also showed an enrichment in biofilm and quorum sensing in the epiphytic compartments, and evidence for nitrogen fixation in the rhizosphere. Aerobic anoxygenic phototrophic markers were represented by Rhizobiales (Methylobacterium) and Rhodospirillales (Belnapia) in the phyllosphere, while photosystem genes were widespread in Bacillales and Cyanobacteria. Nitrogen fixation and biofilm formation genes were mostly related to Proteobacteria. These analyses support the idea of niche differentiation in the rhizosphere and phyllosphere of agaves and cacti and shed light on the potential mechanisms by which epiphytic microbial communities survive and colonize plants of arid and semiarid ecosystems. This study establishes a guideline for testing the relevance of the identified functional traits on the microbial community and the plant fitness.

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.

Plant compartment and biogeography affect microbiome composition in cultivated and native Agave species.

Desert plants are hypothesized to survive the environmental stress inherent to these regions in part thanks to symbioses with microorganisms, and yet these microbial species, the communities they form, and the forces that influence them are poorly understood. Here we report the first comprehensive investigation of the microbial communities associated with species of Agave, which are native to semiarid and arid regions of Central and North America and are emerging as biofuel feedstocks. We examined prokaryotic and fungal communities in the rhizosphere, phyllosphere, leaf and root endosphere, as well as proximal and distal soil samples from cultivated and native agaves, through Illumina amplicon sequencing. Phylogenetic profiling revealed that the composition of prokaryotic communities was primarily determined by the plant compartment, whereas the composition of fungal communities was mainly influenced by the biogeography of the host species. Cultivated A. tequilana exhibited lower levels of prokaryotic diversity compared with native agaves, although no differences in microbial diversity were found in the endosphere. Agaves shared core prokaryotic and fungal taxa known to promote plant growth and confer tolerance to abiotic stress, which suggests common principles underpinning Agave-microbe interactions.

Diazotrophic potential among bacterial communities associated with wild and cultivated Agave species.

Agaves are major biotic resources in arid and semi-arid ecosystems. Despite their ecological, economical and cultural relevance, many aspects of the microbial communities associated with agaves are still unknown. Here, we investigated the bacterial communities associated with two Agave species by 16S rRNA- Denaturing gradient gel electrophoresis fingerprinting and sequencing. We also evaluated the effects of biotic and abiotic factors in the structure of the bacterial communities. In parallel, we isolated and characterized diazotrophic bacteria associated with agaves, as Agave soils are characterized by their low nitrogen content. Our results demonstrate that in Agave, the structure of prokaryotic assemblages was mostly influenced by the community group, where the soil, episphere, and endosphere were clearly distinct. Proteobacteria (γ and α), Actinobacteria, and Acidobacteria were the dominant phyla. Bacterial communities in the episphere of agaves were mainly influenced by the host species, whereas in the endosphere were affected by the season. Fifteen bacterial taxa were common and abundant in the endosphere of both Agave species during the dry season. Notably, some of the confirmed diazotrophic strains belonged to this group, suggesting a possible beneficial role in planta.