Volatile Emissions and Relative Attraction of the Fungal Symbionts of Tea Shot Hole Borer (Coleoptera: Curculionidae)

Euwallacea perbrevis is an ambrosia beetle that vectors fungal pathogens causing Fusarium dieback in Florida avocado trees. Current monitoring lures contain quercivorol, a fungus-produced volatile, but the exact attractant is unknown since lures contain a mixture of p-menth-2-en-1-ol isomers and both α- and β-phellandrene. This study used pure cultures of six symbiotic fungi isolated from E. perbrevis to document volatile emissions and determine the relative attraction of symbionts in binary choice assays. In a comparative test, headspace solid-phase microextraction followed by gas chromatography–mass spectroscopy was used to identify and quantify emissions from 3-week-old cultures. In a temporal study, Super-Q collection followed by gas chromatography–flame ionization detection was used to measure cis- and trans-p-menth-2-en-1-ol emissions for three months. A total of 15 compounds were detected, with monoterpene hydrocarbons and oxygenated monoterpenoids predominating. Only trans-p-menth-2-en-1-ol was common to all six symbionts. Peak levels of both isomers were observed at day 7, then gradually declined over a 90 day period. In choice tests, avocado sawdust disks inoculated with Fusarium sp. nov. were the most attractive. This symbiont produced only two volatiles, trans-p-menth-2-en-1-ol and limonene. The combined results indicate that trans-p-menth-2-en-1-ol is the primary female attractant emitted from symbiotic fungi, but limonene may be a secondary attractant of E. perbrevis.

The Sugar Transporter MST1 Is Involved in Colonization of Rhizosphere and Rhizoplane by Metarhizium robertsii

Over 90% of all vascular plant species develop an intimate symbiosis with fungi, which has an enormous impact on terrestrial ecosystems. It is widely recognized that plant-symbiotic fungi are supported by photosynthates, but little is known about the mechanisms for fungi to utilize plant-derived carbon sources.

The effects of endophytic fungi of NZ terrestrial orchids: developing methods for conservation

<p>Nearly 40% of New Zealand (NZ) orchid species are of conservation concern, some critically endangered, largely due to habitat loss. In NZ, there are currently no propagation programs for terrestrial orchids all of which rely on symbiotic fungi to provide the nutrients required for germination, and little is known about the specific fungal species that might make this possible. To develop an understanding of the fungal interactions affecting recruitment in the field, a survey of endophytic fungal diversity from the roots of Chiloglottis valida, Microtis unifolia, Pterostylis banksii, Spiranthes novae-zelandiae and Thelymitra longifolia was carried out. The identification of fungi was assisted by obtaining sequences of the ITS rDNA gene marker. Seeds of M. unifolia, P. banksii, S. novae-zelandiae and T. longifolia were inoculated with cultured endophytes that were recovered from the roots of conspecific orchids, and their effect on seed germination evaluated. Seed viability using fluorescein diacetate was assayed on all species prior to all experiments and showed moderate to high viability scores for all species. Recovered endophytes belonged to the phyla Ascomycota, Basidiomycota, and Zygomycota. The effect of the different endophytes on seed germination was variable, with five inoculants exhibiting a positive response. Three inoculants had a consistent negative effect on seed germination. The distribution of orchid symbiotic mycorrhizae in situ was investigated at Otari-Wilton’s Bush, Wellington, NZ. Mesh seed packets containing seed of M. unifolia and T. longifolia were interred for 150 days, along transects (≤ 1 metre) that originated at adult orchids at three sites, and an additional site with no adult orchids was used as a control. No small-scale patterns were detected; however, germination rates were higher at undisturbed sites. Seed viability was considerably reduced to <2% after five months under the soil suggesting M. unifolia and T. longifolia seeds do not persist in the seed bank beyond one growing season. Sequences of ITS rDNA indicate Tulasnella calospora assists in the germination of M. unifolia at this site. Similarly, Tulasnella calospora promoted germination of the Nationally Vulnerable wetland species S. novae-zelandiae. Pelotons were isolated from the roots of S. novae-zelandiae plants from a wild population from the lower north island and cultured in Petri dishes. Germination of this orchid began after 30 days from inoculation when the pelotons are already observed inside the embryo. Chlorophyllus tissue was observed after c. 80 days of inoculation. The phylogenetic relationship of Asian-Pacific Spiranthes species with New Zealand Spiranthes was also investigated using nuclear (ITS) and chloroplast (trnL-trnF) DNA sequences. Phylogenetic analyses supported the recognition of Spiranthes novae-zelandiae ‘Motutangi’ as a distinct taxonomic unit. It was also found that the Asian-Pacific Spiranthes species are in need of taxonomic revision. Methods used and developed in this thesis study could be used to identify potential orchid symbionts and pathogens, assess suitable potential relocation sites, and propagation of NZ orchids using symbiotic fungi for restoration and conservation purposes.</p>

The effects of endophytic fungi of NZ terrestrial orchids: developing methods for conservation

<p>Nearly 40% of New Zealand (NZ) orchid species are of conservation concern, some critically endangered, largely due to habitat loss. In NZ, there are currently no propagation programs for terrestrial orchids all of which rely on symbiotic fungi to provide the nutrients required for germination, and little is known about the specific fungal species that might make this possible. To develop an understanding of the fungal interactions affecting recruitment in the field, a survey of endophytic fungal diversity from the roots of Chiloglottis valida, Microtis unifolia, Pterostylis banksii, Spiranthes novae-zelandiae and Thelymitra longifolia was carried out. The identification of fungi was assisted by obtaining sequences of the ITS rDNA gene marker. Seeds of M. unifolia, P. banksii, S. novae-zelandiae and T. longifolia were inoculated with cultured endophytes that were recovered from the roots of conspecific orchids, and their effect on seed germination evaluated. Seed viability using fluorescein diacetate was assayed on all species prior to all experiments and showed moderate to high viability scores for all species. Recovered endophytes belonged to the phyla Ascomycota, Basidiomycota, and Zygomycota. The effect of the different endophytes on seed germination was variable, with five inoculants exhibiting a positive response. Three inoculants had a consistent negative effect on seed germination. The distribution of orchid symbiotic mycorrhizae in situ was investigated at Otari-Wilton’s Bush, Wellington, NZ. Mesh seed packets containing seed of M. unifolia and T. longifolia were interred for 150 days, along transects (≤ 1 metre) that originated at adult orchids at three sites, and an additional site with no adult orchids was used as a control. No small-scale patterns were detected; however, germination rates were higher at undisturbed sites. Seed viability was considerably reduced to <2% after five months under the soil suggesting M. unifolia and T. longifolia seeds do not persist in the seed bank beyond one growing season. Sequences of ITS rDNA indicate Tulasnella calospora assists in the germination of M. unifolia at this site. Similarly, Tulasnella calospora promoted germination of the Nationally Vulnerable wetland species S. novae-zelandiae. Pelotons were isolated from the roots of S. novae-zelandiae plants from a wild population from the lower north island and cultured in Petri dishes. Germination of this orchid began after 30 days from inoculation when the pelotons are already observed inside the embryo. Chlorophyllus tissue was observed after c. 80 days of inoculation. The phylogenetic relationship of Asian-Pacific Spiranthes species with New Zealand Spiranthes was also investigated using nuclear (ITS) and chloroplast (trnL-trnF) DNA sequences. Phylogenetic analyses supported the recognition of Spiranthes novae-zelandiae ‘Motutangi’ as a distinct taxonomic unit. It was also found that the Asian-Pacific Spiranthes species are in need of taxonomic revision. Methods used and developed in this thesis study could be used to identify potential orchid symbionts and pathogens, assess suitable potential relocation sites, and propagation of NZ orchids using symbiotic fungi for restoration and conservation purposes.</p>

Nutritional resources of the yeast symbiont cultivated by the lizard beetle Doubledaya bucculenta in bamboos

Symbiotic fungi of wood-inhabiting insects are often considered to aid wood digestion of host insects when the associated fungi can assimilate wood-associated indigestible materials. In most cases, however, the components of wood that are utilized by fungal symbionts remain poorly understood. The lizard beetle Doubledaya bucculenta (Coleoptera, Erotylidae, Languriinae) farms the symbiotic yeast Wickerhamomyces anomalus inside the cavity of host bamboo internodes, which serves as food for larvae. To determine the carbon sources of the internodes serving as nutritional substrates for W. anomalus, we used ion exchange chromatography measurements to analyze free and structural sugar compositions in fresh pith (FP), yeast-cultured pith (YP), and larva-reared pith (LP) of internodes. Glucose and fructose were the major free sugars in FP and markedly decreased in YP and LP. For structural sugars, no sugar significantly decreased in YP or LP compared with FP. Carbon assimilation tests showed that W. anomalus assimilated glucose, mannose, fructose, and sucrose strongly, xylose and cellobiose moderately, and xylan weakly. Elemental analysis revealed that the compositions of carbon, hydrogen, and nitrogen were not significantly different among tissue types. These results suggest that W. anomalus does not consume bamboo-associated indigestible sugars but most free sugars, mainly glucose and fructose, in the pith. Our findings suggest that a symbiont’s abilities may not always benefit its host in nature.

Influence of Dosages of Biofertilizers Composed of Mycorrhizae and Diazotrophs on the Corn Productivity

Corn (Zea mays L.) is a crop of world importance, however, one of the problems present during its development is the lack of nutrients or the presence of these in a form not available to the plant. Because of this, the objective of this review article is to gather information on the influence of different dosages of mycorrhiza and diazotroph biofertilizers on the corn crop. A meticulous bibliographic research was carried out, detailing the methods used by various authors; Among the most repetitive and significant factors are the use of mycorrhizae and nitrogen-fixing bacteria plus fertilization with P, the following treatment stands out: 200 gr ha−1 (using mycorrhizae and nitrogen-fixing bacteria); the common response variable of the authors mentioned in methodology was grain yield. The results obtained by the majority of authors show that grain yield improves significantly in the interaction of mycorrhizae and diazotrophs plus chemical fertilization; As a result of the aforementioned treatment, the yield of corn kg ha−1 was increased by 37%, by virtue of these results it is concluded that the most appropriate thing would be to take it into account to apply it on the corn crop. Keywords: Inoculants, Nitrogen Fixing Bacteria, Symbiotic Fungi. Resumen El maíz (Zea mays L.) es un cultivo de importancia mundial, sin embargo, uno de los problemas presentes durante su desarrollo, es la falta de nutrientes o la presencia de estos en forma no disponible para la planta. Debido a esto, el objetivo de este artículo de revisión es recopilar información sobre la influencia de las diferentes dosificaciones de biofertilizantes a base de micorriza y diazótrofos sobre el cultivo de maíz. Se realizó una minuciosa investigación bibliográfica, detallando métodos utilizados por varios autores; entre los factores más reiterativos y significativos están el empleo de micorrizas y bacterias fijadoras de nitrógeno más la fertilización con P, sobresaliendo el siguiente tratamiento: 200 gr ha−1 (usando micorrizas y bacterias fijadoras de nitrógeno); la variable respuesta en común de los autores mencionados en metodología fue el rendimiento del grano. Los resultados obtenidos por la mayoría de autores muestran que el rendimiento del grano mejora significativamente en la interacción de micorrizas y diazótrofos más la fertilización química; producto del tratamiento mencionado anteriormente, se aumentó un 37% el rendimiento del maíz kg ha−1, en virtud a estos resultados se concluye que lo más adecuado sería tomar en cuenta para aplicarla sobre el cultivo de maíz. Palabras Clave: Inoculantes, bacterias fijadoras de nitrógeno, hongos simbiontes.

Taxonomy and Molecular Phylogeny of Phellodon (Thelephorales) with Descriptions of Four New Species from Southwest China

Phellodon is a genus of ectomycorrhizal fungi belonging to the group known as the stipitate hydnoids. It is associated with coniferous trees in forest ecosystems and is widely distributed in the northern hemisphere. Phellodon, together with Hydnellum, and Sarcodon, is classified in the Bankeraceae, members of which are generally considered as symbiotic fungi. Ectomycorrhizal fungi can help plant roots fix nitrogen and improve the absorption capacity of soil nutrients by trees, so they play an important role in ecosystem protection. Taxonomic and phylogenetic studies of Chinese Phellodon collections were carried out. Four new Phellodon species were discovered from southwestern China based on a combination of morphological characters and molecular data. Phellodon atroardesiacus is characterized by the blackish blue to dark grey pileus, dark grey to ash grey spines, and presence of clamp connections in spines. Phellodon cinereofuscus is distinguished by a cottony tomentose pileal margin, long spines which become clay-buff when dry, and echinulate basidiospores. Phellodon stramineus is characterized by a depressed and tomentose pileus, straw buff-colored pileal surface, and dark grey to ash grey spines. Phellodon yunnanensis is distinguished by a clay-pink to brown pileus, pale brown to white spines, and the presence of clamp connections in the outer layer of stipe. Detailed descriptions, illustrations, and ecological traits for the new taxa are provided. Phylogenetic analyses inferred from the internal transcribed spacer (ITS) regions confirmed that the four new species are distinct within Phellodon.

COPHYLOGENETIC ANALYSES OF TRACHYMYRMEX ANT-FUNGAL SPECIFICITY: ‘ONE TO ONE WITH SOME EXCEPTIONS’

Over the past few decades, large-scale phylogenetic analyses of fungus-gardening ants and their symbiotic fungi have depicted strong concordance among major clades of ants and their symbiotic fungi, yet within clades, fungus sharing is somewhat widespread among unrelated ant lineages. These symbioses are thought to be explained by a diffuse coevolution model within major clades. Understanding horizontal exchange within clades has been limited by conventional genetic markers that lack both interspecific and geographic variation. To examine whether reports of horizontal exchange was indeed symbiont sharing or an issue of employing relatively uninformative molecular markers, samples of Trachymyrmex arizonensis and Trachymyrmex pomonae and their fungi were collected from native populations in Arizona and genotyped using conventional marker genes and genome-wide single nucleotide polymorphisms (SNPs). Conventional markers of the fungal symbionts generally exhibited cophylogenetic patterns that were consistent with some symbiont sharing, but most fungal clades had low support. SNP analysis, in contrast, indicated that each ant species exhibited fidelity to its own fungal subclade with only one instance of a colony growing a fungus that was otherwise associated with a different ant species. This evidence supports a pattern of codivergence between Trachymyrmex species and their fungi, and thus a diffuse coevolutionary model may not accurately predict symbiont exchange. These results suggest that fungal sharing across host species in these symbioses may be less extensive than previously thought.

Context Dependency in Bark Beetle-Fungus Mutualisms Revisited: Assessing Potential Shifts in Interaction Outcomes Against Varied Genetic, Ecological, and Evolutionary Backgrounds

Context dependency occurs when biological interactions shift in sign or magnitude depending upon genetic, abiotic, and biotic context. Most models of mutualism address systems where interaction outcomes slide along a mutualism-antagonism continuum as environmental conditions vary altering cost-benefit relationships. However, these models do not apply to the many mutualisms that involve by-product benefits and others that do not have antagonistic alternate states. The ubiquity of such mutualisms indicates a need for different approaches and models to understand how environmental variability influences their strength, stability, and ecological roles. In this paper, we apply the concept of context dependency to mutualisms among bark beetles and fungi that span a variety of life strategies and exposures to environmental variability. Bark beetles and their mutualist fungi co-construct a niche based on by-product benefits that allows them to exist in a resource that is otherwise intractable or inaccessible. For the closest of these partnerships, this has resulted in some of the most influential agents of forest mortality in conifer forests worldwide. Understanding these symbioses is key to understanding their influence on forest structure and dynamics and responses to change. We found no evidence that bark beetle mutualisms change in sign as conditions vary, only in magnitude, and that the “closest” (and most environmentally influential) of these partnerships have evolved behaviors and mechanisms to reduce context-dependency and stabilize benefit delivery. The bark beetle-fungus symbioses most likely to slide along a mutualism-antagonism continuum are those involving loosely associated facultative symbionts that may provide benefits under some circumstances and that are horizontally transmitted by the beetle host. Additionally, some symbiotic fungi are never mutualists – these “third party” fungi are exploiters and may shift from commensalism to antagonism depending on environmental context. Our assessment indicates that a careful differentiation between bark beetle-fungus partnerships is crucial to understanding how they influence forests and respond to environmental variability.