Evaluation of semiochemical based push-pull strategy for population suppression of ambrosia beetle vectors of laurel wilt disease in avocado.

Ambrosia beetles (Coleoptera: Curculionidae: Scolytinae and Platypodinae) bore into tree xylem to complete their life cycle, feeding on symbiotic fungi. Ambrosia beetles are a threat to avocado where they have been found to vector a symbiotic fungus, Raffaelea lauricola, the causal agent of the laurel wilt disease. We assessed the repellency of methyl salicylate and verbenone to two putative laurel wilt vectors in avocado, Xyleborus volvulus (Fabricius) and Xyleborus bispinatus (Eichhoff), under laboratory conditions. Then, we tested the same two chemicals released from SPLAT flowable matrix with and without low-dose ethanol dispensers for manipulation of ambrosia beetle populations occurring in commercial avocado. The potential active space of repellents was assessed by quantifying beetle catch on traps placed 'close' (~5-10 cm) and 'far' (~1-1.5 m) away from repellent dispensers. Ambrosia beetles collected on traps associated with all in-field treatments were identified to species to assess beetle diversity and community variation. Xyleborus volvulus was not repelled by methyl salicylate (MeSA) or verbenone in laboratory assays, while X. bispinatus was repelled by MeSA but not verbenone. Ambrosia beetle trap catches were reduced in the field more when plots were treated with verbenone dispensers (SPLAT) co-deployed with low-dose ethanol dispensers than when treated with verbenone alone. Beetle diversity was highest on traps deployed with low-dose ethanol lures. The repellent treatments and ethanol lures significantly altered the species composition of beetles captured in experiment plots. Our results indicate that verbenone co-deployed with ethanol lures holds potential for manipulating ambrosia beetle vectors via push-pull management in avocado. This tactic could discourage immigration and/or population establishment of ambrosia beetles in commercial avocado and function as an additional tool for management programs of laurel wilt.

Genomic comparisons of the laurel wilt pathogen, Raffaelea lauricola, and related tree pathogens highlight an arsenal of pathogenicity related genes.

Raffaelea lauricola is an invasive fungal pathogen and symbiont of the redbay ambrosia beetle (Xyleborus glabratus) that has caused widespread mortality to redbay (Persea borbonia) and other Lauraceae species in the southeastern USA. We compare two genomes of R. lauricola (C2646 and RL570) to seven other related Ophiostomatales species including R. aguacate (nonpathogenic close relative of R. lauricola), R. quercus-mongolicae (associated with mortality of oaks in Korea), R. quercivora (associated with mortality of oaks in Japan), Grosmannia clavigera (cause of blue stain in conifers), Ophiostoma novo-ulmi (extremely virulent causal agent of Dutch elm disease), O. ulmi (moderately virulent pathogen that cause of Dutch elm disease), and O. piceae (blue-stain saprophyte of conifer logs and lumber). Structural and functional annotations were performed to determine genes that are potentially associated with disease development. Raffaelea lauricola and R. aguacate had the largest genomes, along with the largest number of protein-coding genes, genes encoding secreted proteins, small-secreted proteins, ABC transporters, cytochrome P450 enzymes, CAZYmes, and proteases. Our results indicate that this large genome size was not related to pathogenicity but was likely lineage specific, as the other pathogens in Raffaelea (R. quercus-mongolicae and R. quercivora) had similar genome characteristics to the Ophiostoma species. A diverse repertoire of wood-decaying enzymes were identified in each of the genomes, likely used for toxin neutralization rather than wood degradation. Lastly, a larger number of species-specific, secondary metabolite, synthesis clusters were identified in R. lauricola suggesting that it is well equipped as a pathogen, which could explain its success as a pathogen of a wide range of lauraceous hosts.

Comparative analysis of the immune system of an invasive bark beetle, Dendroctonus valens, infected by an entomopathogenic fungus.

Dendroctonus valens LeConte is one of the most economically important forest pest in China. Leptographium procerum, a mutualistic fungus can assist the host beetle in overcoming the pine's chemical defenses, and Beauveria bassiana, an entomopathogenic fungus has shown high beetle killing efficiency. Considering that the D. valens immune system remains unknown at the genomic level, a mutualistic and antagonistic fungus associated with the beetle provides an ideal model for studying immune interactions between the insect and associated fungi. Here, B. bassiana killed most tested larvae more effectively than L. procerum and Tween. The entomopathogenic fungus provoked stronger responses than the symbiotic fungus at the transcriptome level. We identified 185 immunity-related genes, including pattern recognition receptors, signal modulators, members of immune pathways (Toll, IMD, and JAK/STAT), and immune effectors. Quantitative real-time PCR analysis confirmed that several recognition receptors and effector genes were activated at 1 or 2 days post infection, while the effector genes were suppressed at 4 days post infection by B. bassiana, respectively. In contrast, effector genes were upregulated in response to L. procerum. Together, this study provides a comprehensive sequence resource and insight into the D. valens immune system and lays a basis for understanding the molecular aspects of the interaction between the host and associated fungi.

Esteya Vermicola, a Nematophagous Fungus Attacking the Pine Wood Nematode, Harbors a Bacterial Endosymbiont Affiliated with Gammaproteobacteria.

Symbioses have played pivotal roles in biological, ecological, and evolutionary diversification. Symbiotic bacteria affect the biology of hosts in a number of ways. Esteya vermicola, an endoparasitic nematophagous fungus, has high infectivity in the pine wood nematode (PWN), which causes devastating ecological damage and economic losses in Asia and Europe. An integration of molecular, phylogenetic, and morphological analyses revealed that surface-sterilized E. vermicola with septate hyphae from different geographic locations harbor bacterial endosymbionts. 16S rRNA gene sequences from four fungal strains all clustered in a well-supported monophyletic clade that was the most closely related to Pseudomonas stutzeri and affiliated with Gammaproteobacteria. The existence and intracellular location of endobacteria was revealed by fluorescent in situ hybridization (FISH). Our results showed that endobacteria were coccoid, vertically inherited, as yet uncultured, and essential symbionts. Ultrastructural observations indicated that young and old endobacteria differed in cell size, cell wall thickness, and the degree of reproduction. The results of the present study provide a fundamental understanding of the endobacteria inside E. vermicola and raise questions regarding the impact of endobacteria on the biology, ecology, and evolution of their fungal host.

A genomic comparison of putative pathogenicity-related gene families in five members of the Ophiostomatales with different lifestyles.

Ophiostomatoid fungi are vectored by their bark-beetle associates and colonize different host tree species. To survive and proliferate in the host, they have evolved mechanisms for detoxification and elimination of host defence compounds, efficient nutrient sequestration, and, in pathogenic species, virulence towards plants. Here, we assembled a draft genome of the spruce pathogen Ophiostoma bicolor. For our comparative and phylogenetic analyses, we mined the genomes of closely related species (Ophiostoma piceae, Ophiostoma ulmi, Ophiostoma novo-ulmi, and Grosmannia clavigera). Our aim was to acquire a genomic and evolutionary perspective of gene families important in host colonization. Genome comparisons showed that both the nuclear and mitochondrial genomes in our assembly were largely complete. Our O. bicolor 25.3 Mbp draft genome had 10 018 predicted genes, 6041 proteins with gene ontology (GO) annotation, 269 carbohydrate-active enzymes (CAZymes), 559 peptidases and inhibitors, and 1373 genes likely involved in pathogen-host interactions. Phylogenetic analyses of selected protein families revealed core sets of cytochrome P450 genes, ABC transporters and backbone genes involved in secondary metabolite (SM) biosynthesis (polyketide synthases (PKS) and non-ribosomal synthases), and species-specific gene losses and duplications. Phylogenetic analyses of protein families of interest provided insight into evolutionary adaptations to host biochemistry in ophiostomatoid fungi.

Comparing Avocado, Swamp Bay, and Camphortree as Hosts of Raffaelea lauricola Using a Green Fluorescent Protein (GFP)-Labeled Strain of the Pathogen.

Raffaelea lauricola, a fungal symbiont of the ambrosia beetle Xyleborus glabratus, causes laurel wilt in members of the Lauraceae plant family. North American species in the family, such as avocado (Persea americana) and swamp bay (P. palustris), are particularly susceptible to laurel wilt, whereas the Asian camphortree (Cinnamomum camphora) is relatively tolerant. To determine whether susceptibility is related to pathogen colonization, a green fluorescent protein-labeled strain of R. lauricola was generated and used to inoculate avocado, swamp bay, and camphortree. Trees were harvested 3, 10, and 30 days after inoculation (DAI), and disease severity was rated on a 1-to-10 scale. By 30 DAI, avocado and swamp bay developed significantly more severe disease than camphortree (mean severities of 6.8 and 5.5 versus 1.6, P < 0.003). The extent of xylem colonization was recorded as the percentage of lumena that were colonized by the pathogen. More xylem was colonized in avocado than camphortree (0.9% versus 0.1%, P < 0.03) but colonization in swamp bay (0.4%) did not differ significantly from either host. Although there were significant correlations between xylem colonization and laurel wilt severity in avocado (r = 0.74), swamp bay (r = 0.82), and camphortree (r = 0.87), even severely affected trees of all species were scarcely colonized by the pathogen.

Comparative behavior of root pathogens in stems and roots of southeastern Pinus species.

Root diseases are expected to become a greater threat to trees in the future due to accidental pathogen introductions and predicted climate changes, thus there is a need for accurate and efficient pathogenicity tests. For many root pathogens, these tests have been conducted in stems instead of roots. It, however, remains unclear whether stem and root inoculations are comparable for most fungal species. In this study we compared the growth and damage caused by five root pathogens (Grosmannia huntii, Grosmannia alacris, Leptographium procerum, Leptographium terebrantis, and Heterobasidion irregulare) in root and stem tissue of two Pinus species by inoculating mature trees and tissue amended agar in the laboratory. Most fungal species tested caused greater damage in roots of both pine hosts following inoculation. The relationship between root and stem damage was, however, similar when most combinations of pathogens were compared. These results suggest that although stem inoculations are not suitable for determining the actual damage potential of a given species, they may be viewed as a useful surrogate for root inoculations when comparing the relative pathogenicity of multiple species. When grown on amended agar, fungal species generally had greater growth in stem tissue, contrasting with the findings from tree inoculations.

Cloning and characterization of chitinases from interior spruce and lodgepole pine.

Chitinases have been implicated in the defence of conifers against insects and pathogens. cDNA for six chitinases were cloned from interior spruce (Picea glauca x engelmannii) and four from lodgepole pine (Pinus contorta). The cloned interior spruce chitinases were annotated class I PgeChia1-1 and PgeChia1-2, class II PgeChia2-1, class IV PgeChia4-1, and class VII PgeChia7-1 and PgeChia7-2; lodgepole pine chitinases were annotated class I PcChia1-1, class IV PcChia4-1, and class VII PcChia7-1 and PcChia7-2. Chitinases were expressed in Escherichia coli with maltose-binding-protein tags and soluble proteins purified. Functional characterization demonstrated chitinolytic activity for the three class I chitinases PgeChia1-1, PgeChia1-2 and PcChia1-1. Transcript analysis established strong induction of most of the tested chitinases, including all three class I chitinases, in interior spruce and lodgepole pine in response to inoculation with bark beetle associated fungi (Leptographium abietinum and Grosmannia clavigera) and in interior spruce in response to weevil (Pissodes strobi) feeding. Evidence of chitinolytic activity and inducibility by fungal and insect attack support the involvement of these chitinases in conifer defense.

Population structure and migration pattern of a conifer pathogen, Grosmannia clavigera, as influenced by its symbiont, the mountain pine beetle.

We investigated the population structure of Grosmannia clavigera (Gc), a fungal symbiont of the mountain pine beetle (MPB) that plays a crucial role in the establishment and reproductive success of this pathogen. This insect-fungal complex has destroyed over 16 million ha of lodgepole pine forests in Canada, the largest MPB epidemic in recorded history. During this current epidemic, MPB has expanded its range beyond historically recorded boundaries, both northward and eastward, and has now reached the jack pine of Alberta, potentially threatening the Canadian boreal forest. To better understand the dynamics between the beetle and its fungal symbiont, we sampled 19 populations in western North America and genotyped individuals from these populations with eight microsatellite markers. The fungus displayed high haplotype diversity, with over 250 unique haplotypes observed in 335 single spore isolates. Linkage equilibria in 13 of the 19 populations suggested that the fungus reproduces sexually. Bayesian clustering and distance analyses identified four genetic clusters that corresponded to four major geographical regions, which suggested that the epidemic arose from multiple geographical sources. A genetic cluster north of the Rocky Mountains, where the MPB has recently become established, experienced a population bottleneck, probably as a result of the recent range expansion. The two genetic clusters located north and west of the Rocky Mountains contained many fungal isolates admixed from all populations, possibly due to the massive movement of MPB during the epidemic. The general agreement in north-south differentiation of MPB and G. clavigera populations points to the fungal pathogen's dependence on the movement of its insect vector. In addition, the patterns of diversity and the individual assignment tests of the fungal associate suggest that migration across the Rocky Mountains occurred via a northeastern corridor, in accordance with meteorological patterns and observation of MPB movement data. Our results highlight the potential of this pathogen for both expansion and sexual reproduction, and also identify some possible barriers to gene flow. Understanding the ecological and evolutionary dynamics of this fungus-beetle association is important for the modelling and prediction of MPB epidemics.

Growth of Esteya vermicola in media amended with nitrogen sources yields conidia with increased predacity and resistance to environmental stress.

Esteya vermicola , an endoparasitic fungus of pinewood nematode, exhibits great potential as a biological agent against nematodes. In this study to enhance the sporulation, predacity, and environmental resistance of E. vermicola, various nitrogen sources, such as glycine, L-leucine, and ammonium nitrate, were tested. The supplement of glycine and L-leucine had a significant influence on the growth rate of the colony, enhancing colony dry mass by 5-fold more than did ammonium nitrate or the control. Of the nitrogen sources tested, ammonium nitrate and L-leucine promoted sporulation, yielding more than 6 × 10(6) CFU/g, while glycine enhanced the proportion of lunate spores. Meanwhile, the supplement of nitrogen sources had a significant influence on adhesive rate and mortality rate against Bursaphelenchus xylophilus . Moreover, the supplement of glycine enhanced the survival rate against heat stress by more than 3-fold that of L-leucine, ammonium nitrate, and control. The spores produced in media amended with glycine, L-leucine, and ammonium nitrate had slightly but not significantly higher UV resistance and drought resistance than spores produced without nitrogen sources. These results suggested that the addition of glycine resulted in the production of E. vermicola conidia with increased predacity and resistance to environmental stress that may be more suitable for control of pine wilt disease.

Limited response of ponderosa pine bole defenses to wounding and fungi.

Tree defense against bark beetles (Curculionidae: Scolytinae) and their associated fungi generally comprises some combination of constitutive (primary) and induced (secondary) defenses. In pines, the primary constitutive defense against bark beetles consists of preformed resin stored in resin ducts. Induced defenses at the wound site (point of beetle entry) in pines may consist of an increase in resin flow and necrotic lesion formation. The quantity and quality of both induced and constitutive defenses can vary by species and season. The inducible defense response in ponderosa pine is not well understood. Our study examined the inducible defense response in ponderosa pine using traumatic mechanical wounding, and wounding with and without fungal inoculations with two different bark beetle-associated fungi (Ophiostoma minus and Grosmannia clavigera). Resin flow did not significantly increase in response to any treatment. In addition, necrotic lesion formation on the bole after fungal inoculation was minimal. Stand thinning, which has been shown to increase water availability, had no, or inconsistent, effects on inducible tree defense. Our results suggest that ponderosa pine bole defense against bark beetles and their associated fungi is primarily constitutive and not induced.

Diversity and pathogenicity of ophiostomatoid fungi associated with Tetropium species colonizing Picea abies in Poland.

The ophiostomatoid fungi associated with cerambycid beetles Tetropium spp. (their symbiotic vectors) colonizing Norway spruce in Poland (six species collected) were isolated. The virulence of representative isolates was evaluated through inoculations using 2-year-old Norway spruce seedlings. A total of 1325 isolates (Ophiostoma piceae, O. tetropii, O. minus, Grosmannia piceiperda, G. cucullata, and five other less frequent taxa) were obtained. Tetropium castaneum and T. fuscum were vectors of similar spectra of ophiostomatoid fungi although some differences in fungal frequency between these Tetropium spp. were found. Among the fungal associates of the Tetropium spp. collected only G. piceiperda was pathogenic, which suggests that it can play a role in the death of spruce trees following attack by Tetropium spp.

Ophiostomatoid fungi (Ascomycota) associated with Pinus tabuliformis infested by Dendroctonus valens (Coleoptera) in northern China and an assessment of their pathogenicity on mature trees.

Dendroctonus valens is an invasive pest in coniferous forests of northern China. It was suspected of being responsible for the death of more than three million Pinus tabuliformis trees. The present study sought to identify the ophiostomatoid fungi associated with D. valens in northern China and understand the possible role of these fungi in the pine decline. On the basis of morphology, physiology, mating compatibility and phylogenetic analyses of multiple DNA sequences, seven species of ophiostomatoid fungi were isolated from and around D. valens galleries: Leptographium alethinum, Grosmannia koreana (teleomorph of L. koreanum), L. procerum, L. sinoprocerum, L. truncatum, Pesotum aureum and P. pini. All have been recorded for the first time in China. Among them, the occurrence of the dominant species L. procerum is positively linked to attack intensities of D. valens. The pathogenicity of four species (L. koreanum, L. procerum, L. sinoprocerum and L. truncatum) was tested on mature P. tabuliformis trees by stem inoculation. All inoculated strains caused significant necrotic lesions on the inner bark. However, L. koreanum and L. truncatum induced more extensive lesions than L. procerum and L. sinoprocerum. Their association with D. valens and the P. tabuliformis decline is discussed.