Ambrosia Fungi Research Articles

Searching for laurel wilt resistance in avocados of Mexican and Mexican-Guatemalan ancestry.

Laurel wilt (LW), a lethal vascular disease caused by the ambrosia fungus Harringtonia lauricola, has severely reduced avocado (Persea americana Mill.) production in Florida and decimated populations of native lauraceous trees across twelve U.S. states. All commercial avocado cultivars evaluated to date succumb to the disease, but the speed at which the tree declines varies. Cultivars with West Indian (WI) genetic background develop severe symptoms faster than those with Mexican (M) and Guatemalan (G) pedigree. Genetic resistance to LW is urgently needed, as management relies on costly cultural practices. We screened non-commercial open-pollinated progenies from 19 Mexican and 6 Mexican x Guatemalan accessions, and 2 Guatemalan x West Indian cultivars recognized as tolerant by growers. From the five disease response parameters evaluated, the final disease intensity index and disease severity on the last evaluation day were used to classify genotypes. A wide variability of responses was observed within and among families. Symptomatic plants were present in all families, while some individuals within 15 families remained asymptomatic. 'Colín V-33' (M×G) family was identified as tolerant, and Libres 3 (M), Bladimiro M-06 (M) 'Colinmex' (M×G), 'Collinson' (G×WI), Libres 5 (M) and Rag-13 (M) families were classified as moderately tolerant. This is the first-time tolerance to laurel wilt in avocado is formally reported, though surviving material needs to be propagated for response validation and field testing. Identifying tolerant accessions can help understand the underlying mechanisms and provide breeders with genetic resources for the future incorporation of resistance genes into commercial cultivars.

A taxonomic review of Sueus Murayama, 1951 ambrosia beetles (Coleoptera: Curculionidae: Scolytinae: Hyorrhynchini) aided by molecular phylogenetic analyses

Four Sueus Murayama, 1951 species occur in Southeast Asia and Oceania. They all likely have a female-biased haplodiploid inbreeding mating system and feed on symbiotic ambrosia fungi. These life history traits increase the potential of adventive events. Indeed, Sueus has been recently discovered on the Caribbean island of Martinique. Morphological variation has been observed among some populations of Sueus niisimai (Eggers, 1926), which questioned species boundaries. Given the beetle’s potential economic importance, we provide a molecular phylogeny as a foundation for systematic study and review the status of the known species. We sequenced a total of 1117 nucleotides from mitochondrial COI and nuclear CAD genes for 25 specimens. Parsimony and Bayesian phylogenies were similar in topology and demonstrated the sister placement of S. granulatus (Eggers, 1936) to the other Sueus species, reciprocal monophyly of S. niisimai and S. pilosus (Eggers, 1936) status restored, the monophyly of S. obesus Browne, 1977 and elevated levels of nucleotide divergence (interspecific = 16–22%). Sueus chatterjeei Smith & Cognato sp. nov. (India) and Sueus insulanus Schiffer, Smith & Cognato sp. nov. (Papua New Guinea) are described. Hyorrhynchus granulatus Eggers, 1936 is removed from synonymy with Hyorrhynchus lewisi Blandford, 1894 and reinstated as a valid species, Sueus granulatus (Eggers, 1936) status restored, comb. nov. A key to the eight recognized species is given. In addition, the identity of the Martinique species is revised as S. pilosus. Geographic distribution of species and the potential existence of cryptic species are discussed.

Distribution of the invasive ambrosia beetle Anisandrus maiche (Coleoptera, Scolytinae) in Switzerland and first record in Europe of its ambrosia fungus Ambrosiella cleistominuta

Ambrosia beetles are highly successful as invaders because they are often transported internationally with wood packaging and other wood products and because their inbreeding mating systems facilitates establishment of invading populations. In 2022, two independent insect surveys in canton Ticino (southern Switzerland) revealed the widespread occurrence of the invasive ambrosia beetle Anisandrus maiche (Kurentzov, 1941) from southern to central-upper Ticino. This species is native to east Asia and has previously been found as a non-native invasive species in the United States, Canada, western Russia, Ukraine and, in 2021, in northern Italy. Here, we present the results of several trapping studies using different trap types (bottle traps, funnel traps and Polytrap intercept traps) and attractants and a map of the distribution of the species. In total, 715 specimens of A. maiche, all female, were trapped, and the identity of selected individuals was confirmed by morphological and molecular identification based on three mitochondrial and nuclear markers (COI, 28S and CAD). Trap samples from early April to early September 2022 in intervals of two to four weeks showed that flights of A. maiche occurred mainly from June to mid-August. Isolation of fungal associates of A. maiche from beetles trapped alive revealed the presence of four fungal species, including the ambrosia fungus Ambrosiella cleistominuta, the known mutualist of A. maiche. The identity of A. cleistominuta was confirmed by comparing DNA sequences of its nuclear, internal transcribed spacer (ITS) gene with reference sequences in NCBI and BOLDSYSTEMS. This represents the first record of A. cleistominuta in Europe. Of the other fungal associates isolated from A. maiche in Ticino, Fusarium lateritium is of note as there is a possibility that A. maiche could act as a vector of this plant pathogen. We highlight several research needs that should be addressed to gain insight into the potential impact of these non-native species and to overcome problems with heteroplasmy in COI sequences in studies of invasion and population genetics of ambrosia beetles.

Volatile organic compounds emitted by Megaplatypusmutatus associated fungi: chemical identification and temperature-modulated responses by the ambrosial beetle

BackgroundIn ambrosia and bark beetles–fungi interaction, volatile organic compounds (VOCs) play a central role in mediating various aspects of community dynamics of beetles and/or fungi. These functions include facilitating beetle habitat location, mate identification, and fungal partner differentiation. However, the understanding on this context remains limited, especially in the globally distributed subfamily Platypodinae, which comprises predominantly ambrosia beetles. There is a lack of chemical data on ambrosia fungi from native South American species. This study addresses this gap by characterizing VOCs from twelve fungal species associated with Megaplatypusmutatus and assessing species-specific behavioral responses during dispersal.MethodsFungal VOCs were collected by gas chromatography–mass spectrometry combined with solid-phase microextraction and Y-olfactometry assays of males and females were performed at dispersal stage. Statistical analyses involved: non-metric multidimensional scaling multivariate plot and PERMANOVA test, a cluster analysis through unweighted pair group method with Jaccard index, and finally, a chi-square goodness-of-fit test for beetle behavioral assays.ResultsWe identified 72 VOCs from the fungal species isolated from M.mutatus galleries, exocuticle, and gut. The olfactory behavior of M.mutatus demonstrated its capacity to discriminate between volatile profiles, showing a preference for either the fungus or the control source. Our results also enhance the understanding in a chemotaxonomic context and in the behavioral responses of M.mutatus revealing the beetle's remarkable low temperature tolerance and its capability to maintain mobility and orientation toward volatile sources even after zero-degree Celsius exposure.ConclusionThis study presents a comprehensive insight into fungal VOC profiles, emphasizing the sources of isolation within pest associated fungi, as well as its symbiotic species from the Raffaelea genus. In conclusion, our findings suggest that Megaplatypusmutatus exhibits a general aversion to its fungal VOCs symbiont. However, a notable exception arises when the beetles are pre-exposed for 48 h to freezing conditions, highlighting the beetles' ability to withstand freezing conditions as adults and to exhibit altered responses to their fungal associates under these circumstances.

Distribution of the invasive ambrosia beetle Xyleborinus attenuatus Blandford, 1894 (Coleoptera: Curculionidae: Scolytinae) in the Czech Republic (Central Europe)

Abstract In Europe, Xyleborinus attenuatus Blandford is considered an invasive bark beetle native to East Asia. We used the results of many local surveys and data from private and museum collections to get information on the distribution and abundance of X. attenuatus Blandford in the Czech Republic. The ambrosia beetle X. attenuatus Blandford is probably widespread throughout the territory of the Czech Republic, from the lowlands to the mountains, and has one generation per year in Central Europe. The flight activity lasts from mid-March to May, F1 adults emerge in August and September and overwinter in wood or in the ground under an infested tree. X. attenuatus Blandford predominantly occupies soft deciduous trees and so far there economic damage have not been reported yet. However, X. attenuatus Blandford is a vector of ambrosia fungi Ambrosiella spp. and Raffaelea spp., and therefore this ambrosia beetle should be monitored.

Two sympatric lineages of Australian Cnestus solidus share Ambrosiella symbionts but not Wolbachia

Sympatric lineages of inbreeding species provide an excellent opportunity to investigate species divergence patterns and processes. Many ambrosia beetle lineages (Curculionidae: Scolytinae) reproduce by predominant inbreeding through sib mating in nests excavated in woody plant parts wherein they cultivate symbiotic ambrosia fungi as their sole source of nutrition. The Xyleborini ambrosia beetle species Cnestus solidus and Cnestus pseudosolidus are sympatrically distributed across eastern Australia and have overlapping morphological variation. Using multilocus sequencing analysis of individuals collected from 19 sites spanning their sympatric distribution, we assessed their phylogenetic relationships, taxonomic status and microbial symbionts. We found no genetic differentiation between individuals morphologically identified as C. solidus and C. pseudosolidus confirming previous suggestions that C. pseudosolidus is synonymous to C. solidus. However, within C. solidus we unexpectedly discovered the sympatric coexistence of two morphologically indistinguishable but genetically distinct lineages with small nuclear yet large mitochondrial divergence. At all sites except one, individuals of both lineages carried the same primary fungal symbiont, a new Ambrosiella species, indicating that fungal symbiont differentiation may not be involved in lineage divergence. One strain of the maternally inherited bacterial endosymbiont Wolbachia was found at high prevalence in individuals of the more common lineage but not in the other, suggesting that it may influence host fitness. Our data suggest that the two Australian Cnestus lineages diverged allopatrically, and one lineage then acquired Wolbachia. Predominant inbreeding and Wolbachia infection may have reinforced reproductive barriers between these two lineages after their secondary contact contributing to their current sympatric distribution.

Infestation of pinhole borer [Euplatypus parallelus (F.)] on Dalbergia latifolia Roxb.

Dalbergia latifolia Roxb. (Indian rosewood) is India's one of the main timber tree. This is the first report of infestation of D. latifolia by Euplatypus parallelus (Fabricius, 1801) (Coleoptera: Curculionidae: Platypodinae), beetle in Karnataka, India. Pinhead-sized holes with reddish resinous exudation oozing from them and blackened areas around the holes caused by the ambrosia fungi are the characteristic feature of infestation. Fusarium oxysporum Schlecht. was isolated from infected portion of sapwood. Infested trees wilt, lose leaves and die. The infection may seriously threaten Indian rosewood.

Flexibility in the ambrosia symbiosis of Xyleborus bispinatus.

Ambrosia beetles maintain strict associations with specific lineages of fungi. However, anthropogenic introductions of ambrosia beetles into new ecosystems can result in the lateral transfer of their symbionts to other ambrosia beetles. The ability of a Florida endemic ambrosia beetle, Xyleborus bispinatus, to feed and establish persistent associations with two of its known symbionts (Raffaelea subfusca and Raffaelea arxii) and two other fungi (Harringtonia lauricola and Fusarium sp. nov.), which are primary symbionts of invasive ambrosia beetles, was investigated. The stability of these mutualisms and their effect on the beetle's fitness were monitored over five consecutive generations. Surface-disinfested pupae with non-developed mycangia were reared separately on one of the four fungal symbionts. Non-treated beetles (i.e., lab colony) with previously colonized mycangia were used as a control group. Xyleborus bispinatus could exchange its fungal symbionts, survive, and reproduce on different fungal diets, including known fungal associates and phylogenetically distant fungi, which are plant pathogens and primary symbionts of other invasive ambrosia beetles. These changes in fungal diets resulted in persistent mutualisms, and some symbionts even increased the beetle's reproduction. Females that developed on Fusarium sp. nov. had a significantly greater number of female offspring than non-treated beetles. Females that fed solely on Harringtonia or Raffaelea symbionts produced fewer female offspring. Even though some ambrosia beetles like X. bispinatus can partner with different ambrosia fungi, their symbiosis under natural conditions is modulated by their mycangium and possibly other environmental factors. However, exposure to symbionts of invasive beetles can result in stable partnerships with these fungi and affect the population dynamics of ambrosia beetles and their symbionts.

Symbiotic Fungi Associated With Xyleborine Ambrosia Beetles (Coleoptera: Curculionidae: Scolytinae) and the Imperative of Global Collaboration

AbstractAmbrosia beetles from the tribe Xyleborini are part of nearly all forest ecosystems. Because of their small size, haplodiploid mating structure, and protected lives inside the sapwood of woody plants, they have a unique ability to expand into new regions via inadvertent human transport. A small number of invasive xyleborines cause significant damage to forests, lumber concerns, and agricultural systems. Most ambrosia pests damage or kill trees by the accumulation of beetle attacks, one is known to cause tree death through the introduction of pathogenic fungus into susceptible Lauraceae trees. The relationships between ambrosia fungi and their beetle vectors range from mutualistic symbiosis to facultative association, but most remain unstudied. Unresolved taxonomies, convergent morphologies, and the difficulty of sampling ambrosia fungi over their entire global ranges make comprehensive surveys of ambrosia fungi difficult to achieve. Ambrosia fungi from Europe and North America are moderately well documented, however, we have yet to sufficiently document those from Africa, Asia, Australia, and South America. Worldwide cooperation to improve and standardize scientific study of the ambrosia symbioses is needed to better understand these impactful organisms.

Partitioning of Ambrosia Beetle Diversity on Teak Plantations in Java, Sumbawa, and Sulawesi Islands

Ambrosia beetles are the largest group of beetles living mutualistically with ambrosia fungi. Increased global shipments of forest and agricultural products have expanded the distribution of some species of ambrosia beetle. We investigated the partitioning diversity of the ambrosia beetle community on teak plantations in Indonesia’s Java, Sumbawa, and Sulawesi Islands. The ambrosia beetles were collected on the twelve sites of teak plantations with different managements (un-thinned and thinned) in Java, Sulawesi, and Sumbawa Islands. Ambrosia beetles were collected ten times at 7-day intervals. The diversity of ambrosia beetles recorded in teak plantations across twelve sites in Java, Sumbawa, and Sulawesi Islands were 17 species and 6154 individuals. Xyleborus affinis (47.17%), Xylosandrus crassiusculus (27.64%), and Hypothenemus sp. (12.33%) were the three dominant species. The highest and lowest species richness were found in the teak plantations in Java and Sumbawa Islands, respectively. The highest and lowest populations of ambrosia beetles were in Sulawesi and Sumbawa islands, respectively. Three factors contribute to the species richness of ambrosia beetles, i.e., temperature, rainfall, and altitude. Stand age, temperature, rainfall, altitude, and teak management contribute to ambrosia beetle abundance. Ambrosia beetle communities among islands show differences between each group, as confirmed by analysis of variance based on homogeneity of multivariate dispersion (sig. 0.001) and permutation test for homogeneity of multivariate dispersions (sign. 0.001). For the group of teak managements, there are differences between both teak managements, as confirmed by analysis of variance based on homogeneity of multivariate dispersion (sig. 0.001) and permutation test for homogeneity of multivariate dispersions (sign. 0.01). Based on the eigenvalues for PCoA axes by the Bray–Curtis method, Sulawesi Island is separate from both Java, and Sumbawa islands. However, Java and Sumbawa islands overlap each other. For groups of teak managements (thinning and non-thinning), there are overlap with each other based on the eigenvalues for PCoA axes by the Bray–Curtis method. The β-1 (Within bottle trap/local scale) contributes the highest to γ-diversity (42.46%). The relative contribution of species replacement (β-sim) in multiple sites across Java, Sumbawa, and Sulawesi Islands (regional scale) provides a high contribution (85%) to overall beta diversity, and the relative contribution of β-nes to the β-sor among sites is 14.03%.

The Role of Mycangial Fungi Associated with Ambrosia Beetles (Euwallacea interjectus) in Fig Wilt Disease: Dual Inoculation of Fusarium kuroshium and Ceratocystis ficicola Can Bring Fig Saplings to Early Symptom Development.

The ambrosia beetle, Euwallacea interjectus, is a wood-boring pest and a vector of Ceratocystis ficicola, a pathogenic fungus causing fig (Ficus carica) wilt disease (FWD) in Japan. The ambrosia fungi, Fusarium kuroshium and Neocosmospora metavorans, have been frequently isolated from heads (including mycangia) of wild and reared adult female E. interjectus, respectively. However, the exact mechanisms driving FWD as well as the interactions between F. kuroshium and C. ficicola in fig orchard remain unclear. To verify the role of the mycangial fungi in the FWD progression, fig saplings were subjected to inoculation treatments (T1, F. kuroshium; T2, N. metavorans, reference positive control; T3, C. ficicola; T4, F. kuroshium + C. ficicola, realistic on-site combination). T3 and T4 saplings began wilting approximately 12 days after inoculation, leading to eventual death. Median duration from inoculation to death of the T4 saplings was approximately four days significantly faster than that of the T3 saplings. Xylem sap-conduction test indicated that dysfunction and necrosis area were considerably wider in the T4 saplings than in T3 saplings. These results demonstrate that the synergistic action of F. kuroshium and C. ficicola contributed to accelerated wilting in the saplings. Based on these discoveries, we proposed a model for system changes in the symbiosis between E. interjectus and its associated fungi in FWD in Japan.

Ecuadorian Coptoborus beetles harbor Fusarium and Graphium fungi previously associated with Euwallacea ambrosia beetles

ABSTRACT Ambrosia beetles from the scolytine tribe Xyleborini (Curculionidae) are important to the decomposition of woody plant material on every continent except Antarctica. These insects farm fungi on the walls of tunnels they build inside recently dead trees and rely on the fungi for nutrition during all stages of their lives. Such ambrosia fungi rely on the beetles to provide appropriate substrates and environmental conditions for growth. A small minority of xyleborine ambrosia beetle–fungal partnerships cause significant damage to healthy trees. The xyleborine beetle Coptoborus ochromactonus vectors a Fusarium (Hypocreales) fungus that is lethal to balsa (Ochroma pyramidale (Malvaceae)) trees in Ecuador. Although this pathogenic fungus and its associated beetle are not known to be established in the United States, several other non-native ambrosia beetle species are vectors of destructive plant diseases in this country. This fact and the acceleration of trade between South America and the United States demonstrate the importance of understanding fungal plant pathogens before they escape their native ranges. Here we identify the fungi accompanying Coptoborus ambrosia beetles collected in Ecuador. Classification based ribosomal internal transcribed spacer 1 (ITS) sequences revealed the most prevalent fungi associated with Coptoborus are Fusarium sp. and Graphium sp. (Microascales: Microascaceae), which have been confirmed as ambrosia fungi for xyleborine ambrosia beetles, and Clonostsachys sp. (Hypocreales), which is a diverse genus found abundantly in soils and associated with plants. Phylogenetic analyses of the Fusarium strains based on ITS, translation elongation factor (EF1-α), and two subunits of the DNA-directed RNA polymerase II (RPB1 and RPB2) identified them as Fusarium sp. AF-9 in the Ambrosia Fusarium Clade (AFC). This Fusarium species was previously associated with a few xyleborine ambrosia beetles, most notably the species complex Euwallacea fornicatus (Eichhoff 1868) (Curculionidae: Scolytinae: Xyleborini). Examination of ITS and EF1-α sequences showed a close affinity between the Graphium isolated from Coptoborus spp. and other xyleborine-associated Graphium as well as the soil fungus Graphium basitruncatum. This characterization of ambrosia fungi through DNA sequencing confirms the identity of a putative plant pathogen spread by Coptoborus beetles and expands the documented range of Fusarium and Graphium ambrosia fungi.

Microbial mutualism suppression by Trichoderma and Bacillus species for controlling the invasive ambrosia beetle Xylosandrus compactus

Xylosandrus compactus (Eichhoff) (Coleoptera: Curculionidae: Scolytinae) is among the most invasive ambrosia beetles causing severe damage to host plants belonging to various ecosystems. Foundress females excavate galleries into the plant xylem introducing mutualistic ambrosia fungi which are the exclusive food source for their offspring. The cryptic habits of this fungus-farming insect and its wide host range facilitated its rapid spreading worldwide. Despite several advances in deepening the bio-ecology of this beetle, there is still a general lack of information about effective control strategies. In this context, microbials, such as antagonistic bacteria and mycoparasitic fungi, can have the potential to act as alternative management tools. Here, we evaluated, for the first time, the potential of commercially available microbial agents for suppressing the growth of the main beetle mutualist, Ambrosiella xylebori, and consequently to hamper the beetle progeny development. The antagonistic activity of five Trichoderma and two Bacillus-based biofungicides was first evaluated in vitro by means of dual-culture experiments in Petri dishes. Then, beetle bioassays were conducted by exposing carob twigs, previously treated with different concentrations of each biofungicide, to beetle foundresses. Tested microbial antagonists significantly suppressed the growth of A. xylebori on Petri dishes. A significant reduction of the beetle progeny was observed in galleries of treated twigs compared to controls. The higher reduction in mutualist growth and brood production was obtained within galleries of T. atroviride treated twigs. Our results show the potential of the tested microbials to suppress the beetle mutualist growth within galleries and consequently to affect the development of its progeny.

A Diagnostic Guide for Laurel Wilt Disease in Avocado

Laurel wilt disease (LWD), caused by the ambrosia fungus Raffaelea lauricola, is a deadly vascular disease affecting numerous hosts in the Lauraceae family, including important forest species and avocado trees. The fungal pathogen is spread by beetle-vectors and through root grafts. Symptoms include leaf wilting, foliar desiccation, stem and limb dieback, and depending on the host, localized or systemic vascular damage. R. lauricola and its main vector, Xyleborus glabratus (Redbay Ambrosia Beetle), are invasive species that were introduced into the United States in 2002. This guide describes methods used to diagnose LWD in avocado. R. lauricola is isolated from infected sapwood or ambrosia beetles using the semi-selective medium CSMA (cycloheximide–streptomycin malt agar). Molecular identification is based on the amplification of the LSU (Large Subunit of the ribosomal cassette) or of the species-specific microsatellites IFW and CHK by conventional PCR. More current detection methods use DNA extracted directly from infected material and include the amplification of specific DNA regions through conventional PCR, qPCR, or LAMP. However, to date, there is no method that can detect the pathogen early in the infection process (<3 days) when the vascular tissue remains asymptomatic. Besides detailed protocols on how to diagnose the disease and confirm the presence of the causal agent, we include protocols on pathogen preservation and inoculation. Although this guide focuses on avocado, the techniques included here can be implemented to diagnose LWD in other susceptible species.

Ambrosia Beetles Prefer Closed Canopies: A Case Study in Oak Forests in Central Europe

Research Highlights: The percentage of canopy closure was found to be the main factor associated with ambrosia beetle abundance and species richness. The latter two variables increased as canopy closure increased, probably because a high percentage of canopy closure provides a stable and humid environment suitable for the growth of ambrosia fungi. Objectives: Oak is a common host tree for ambrosia beetles (Coleoptera: Curculionidae: Scolytinae), which have independently evolved a nutritional mutualism with fungi. We suspected that ambrosia beetles might have specific habitat preferences that are different from those of other saproxylic beetles and that reflect the specific habitat preferences of their food, i.e., ambrosia fungi. Methods: We assessed ambrosia beetle abundance with ethanol-lured traps in five old-growth oak dominated forests and five managed oak dominated forests (one trap per forest) during the vegetation period in 2020. We determined whether ambrosia beetle abundance and species richness depend on forest type (managed vs. unmanaged), degree of canopy closure, abundance of oak trees, abundance of coarse deadwood, and abundance of dead oak branches. Results: In total, 4137 individuals of six species of ambrosia beetles associated with oaks were captured. The native ambrosia beetle Anisandrus dispar represented the majority of trapped ambrosia bark beetles. A. dispar along with another ambrosia beetle, Xyleborinus saxesenii, represented 99% of all captured beetles. Conclusions: In addition to canopy closure, the abundance of oak trees and the abundance of dead oak branches were significantly associated with ambrosia beetle abundance and species richness. The abundance of A. dispar was mainly correlated with dead oak branch abundance and the degree of canopy closure, whereas the abundances of X. saxesenii and of the invasive species Xyleborinus attenuatus and Cyclorhipidion bodoanum were mainly correlated with the net area occupied by oak trees.

Microbial Biocontrol Strategies for Ambrosia Beetles and Their Associated Phytopathogenic Fungi

Ambrosia beetles and their symbiotic fungi are causing severe damage in natural and agro-ecosystems worldwide, threatening the productivity of several important tree crops such as avocado. Strategies aiming at mitigating their impact include the application of broad-spectrum agrochemicals and the incineration of diseased trees, but the increasing demand for environment-friendly strategies call for exploring biological control for the management of ambrosia beetles and their phytopathogenic fungal symbionts. The aim of this review is to examine the existing knowledge on biocontrol approaches using beneficial microorganisms and microbial natural products with entomopathogenic and antifungal activity against ambrosia beetles and fungi. We show that biocontrol has been mainly focused on the insect, using entomopathogenic fungi (EPF) such as Beauveria spp. or Metarhizium spp. However, recent studies have been integrating EPF with mycoparasitic fungi such as Trichoderma spp. to simultaneously challenge the vector and its fungal symbionts. Novel approaches also include the use of microbial natural products as insect lures or antifungal agents. Contrastingly, the potential of bacteria, including actinobacteria (actinomycetes), as biocontrol agents of ambrosia fungi has been little investigated. We thus suggest that future research should further examine the antifungal activity of bacterial strains, with an emphasis on harsh environments. We also suggest pursuing the isolation of more effective microbial strains with dual biocontrol effect, i.e., exhibiting fungicidal/insecticidal activities. Moreover, additional efforts should aim at determining the best application methods of biocontrol agents in the field to ensure that the positive effects detected in vitro are sustained. Finally, we propose the integration of microbiome studies in pest and disease management strategies as they could provide us with tools to steer the beneficial host plant microbiome and to manipulate the beetle microbiome in order to reduce insect fitness.

Evaluating Sexual Dimorphism in the Ambrosia Beetle Xyleborus affinis (Coleoptera: Curculionidae) Using Geometric Morphometrics

Species within genus Xyleborus Eichhoff (Curculionidae: Scolytinae) live in a feeding symbiosis with ambrosia fungi and present inbreeding polygyny with highly biased sex ratios and sexual dimorphism in size. Here we evaluate the expression of sexual dimorphism, the allometric effect on shape variation, and the morphological integration of 2 body structures (elytra and pronotum) in Xyleborus affinis (Eichhoff) (Curculionidae: Scolytinae) under controlled growth conditions (26 °C, 60% RH, and continuous darkness), using geometric morphometrics. Differences between sexes from statistical multivariate tests indicate the presence of sexual size and shape dimorphism in both structures, with the largest differences and shape variability presented in the pronotum. We found significant developmental integration of both structures (morphological covariation) and no significant differences in allometric trends between sexes. The results suggest a specific allometric pattern in X. affinis that could be constrained by genetic or phylogenetic factors. However, quantification of shape and size variation could help to determine the differential effects of environmental stress between sexes during growth in this species. This approach could be valuable for monitoring populations of other Xyleborus species living in contrasting environmental conditions, given their wide geographical distributions and the vast amount of host plant species with which they interact.

Origin and evolution of fungus farming in wood-boring Coleoptera - a palaeontological perspective.

Insect-fungus mutualism is one of the better-studied symbiotic interactions in nature. Ambrosia fungi are an ecological assemblage of unrelated fungi that are cultivated by ambrosia beetles in their galleries as obligate food for larvae. Despite recently increased research interest, it remains unclear which ecological factors facilitated the origin of fungus farming, and how it transformed into a symbiotic relationship with obligate dependency. It is clear from phylogenetic analyses that this symbiosis evolved independently many times in several beetle and fungus lineages. However, there is a mismatch between palaeontological and phylogenetic data. Herein we review, for the first time, the ambrosia system from a palaeontological perspective. Although largely ignored, families such as Lymexylidae and Bostrichidae should be included in the list of ambrosia beetles because some of their species cultivate ambrosia fungi. The estimated origin for some groups of ambrosia fungi during the Cretaceous concurs with a known high diversity of Lymexylidae and Bostrichidae at that time. Although potentially older, the greatest radiation of various ambrosia beetle lineages occurred in the weevil subfamilies Scolytinae and Platypodinae during the Eocene. In this review we explore the evolutionary relationship between ambrosia beetles, fungi and their host trees, which is likely to have persisted for longer than previously supposed.

Metarhizium robertsii in combination with Trichoderma asperellum reduce the malathion doses used to control ambrosia beetles: the case of Xyleborus affinis

ABSTRACT Invasive ambrosia beetles and their symbiotic fungi are responsible for destructive diseases like Laurel Wilt and Fusarium Dieback. Some biological treatments and chemical pesticides (e.g. malathion) have been proposed as alternatives to control these pests, however, to date there are no effective treatments. Here we evaluated the susceptibility of the ambrosia beetle Xyleborus affinis Eichhoff to the entomopathogenic fungus Metarhizium robertsii strain Xoch 8.1, and the efficacy of the mycopathogenic fungus Trichoderma asperellum strain Th-T4 (3) to inhibit the growth of the pathogenic ambrosia fungus Fusarium euwallaceae strain 744. Also, we compared the efficacy of a combination of M. robertsii Xoch 8.1 and T. asperellum Th-T4 (3) against X. affinis, and examined the feasibility of using this combination as an alternative to reduce the malathion doses that have been proposed to control ambrosia beetles. The treatment of X. affinis with M. robertsii (1 × 107 con/mL) alone or in combination with T. asperellum (1 × 106 con/mL) resulted in an average mortality of 76% (±7.5), which was statistically similar to that obtained with malathion at 600 ppm (96% ± 4.0). The combination of a lower concentration of malathion (60 ppm or 200 ppm) and both fungi was as efficient as malathion at 600 ppm. Mortality in negative controls (without fungal conidia or malathion) resulted in an average of 16% (±7.5). The proposed combined treatment reduces by up to one order of magnitude the required amount of malathion, and thus represents a potential alternative to control ambrosia beetle pests.

Habitat Quality Determines Dispersal Decisions and Fitness in a Beetle – Fungus Mutualism

Delayed dispersal of sexually mature offspring is a fundamental component of cooperative breeding. In ambrosia beetles, female offspring temporarily remain in their natal nest and refrain from reproduction, instead investing in alloparental care. Previous work has demonstrated a link between helping behaviour and the increased need for pathogen defence, arising from their close association with fungal cultivars. In the ambrosia beetle Xyleborinus saxesenii, mature female offspring can effectively fight pathogen infections and manage the microbial composition within the nest by adjusting the frequency of different hygienic and nest maintenance behaviours. This suggests a potential to respond flexibly to the ecology of their nest, which calls for a better understanding of the connection between behaviour and the microbial community thriving within their nests. Here, we studied the significance of the mutualistic fungus garden composition for the beetles’ nest ecology and fitness by experimentally varying substrate quality. We found that the vertically transmitted ambrosia fungus garden is composed of at least two fungus mutualist species and a wide variety of other microbes varying in their relative abundance. This is strongly affected by the moisture content of the substrate, which in nature depends on the age and type of wood. We found that the mutualist fungi complement each other in terms of dryness-resistance, allowing the beetles to utilise a broad range of substrates over prolonged time during which the wood gradually desiccates. Under suboptimal humidity conditions, the interaction between host and multiple fungus species has important ramifications for the behaviour of philopatric helpers, including their alloparental investment, sibling cannibalism and the timing of dispersal. Rearing five generations of beetles consecutively in dry substrate resulted in transgenerational effects on philopatry and alloparental care, probably mediated through the dominance of a particular fungus species that was driven by the experimental habitat condition. Interestingly, the nests of these selection lines produced much more offspring after five generations than any first-generation nest, which may have reflected increased egg laying by non-dispersing daughters. Our study highlights the importance of considering the interactions between the microbial community and their insect hosts for understanding social evolution in cooperatively breeding beetles.