Arbuscular mycorrhizal (AM) fungi can sequester different potentially toxic elements, such as trace elements (TEs), within their structures to alleviate the toxicity for its host plant and themselves. To elucidate the role of AM fungi in TEs immobilization in the rhizosphere of host plants, it is important to know the TEs distribution in AM fungal structures. In the present study, we investigated the distribution and concentration of TEs within extraradical spores and mycelium of the AM fungus Rhizophagus intraradices, collected from the rhizosphere of Senecio bonariensis plants grown in a soil polluted with multiple TEs, by using Particle-Induced X-ray Emission with a micro-focused beam (micro PIXE). This technique enabled the simultaneous micrometric mapping of elements in a sample. The calculated values were compared with those in the polluted substrate, measured by the Wavelength Dispersive X-ray Fluorescence technique. The highest concentrations of Fe, P, Ti, Mn, Cr, Cu and Zn were found in AM fungal spores, where they were accumulated, while extraradical mycelium was enriched in Cu. Finally, we demonstrated that AM fungi can simultaneously accumulate high amounts of different TEs in their structures, thus reducing the toxicity of these elements to its host plant.

Bacillus spp. produce numerous antimicrobial metabolites. Among these metabolites, cyclic lipopeptides (CLP) including fengycins, iturins, and surfactins are known to have varying antifungal activity against phytopathogenic fungi. The differential activities of CLP have been attributed to diverse mechanisms of action on fungal membranes. However, the precise biochemical determinants driving their antifungal modes of action have not been conclusively identified. In this study, three plant pathogenic fungi of varying lipopeptide sensitivities, Alternaria solani, Cladosporium cucumerinum, and Fusarium sambucinum, were studied to determine how their cell membrane lipid compositions may confer sensitivity and/or tolerance to fengycin, iturin, and surfactin. Results indicated that sensitivity to all three lipopeptides correlated with lower ergosterol content and elevated phospholipid fatty acid unsaturation. Fungal sensitivity to surfactin was also notably different than fengycin and iturin, as surfactin was influenced more by lower phosphatidylethanolamine amounts, higher levels of phosphatidylinositol, and less by phospholipid fatty acyl chain length. Results from this study provide insight into the fungal membrane composition of A. solani, F. sambucinum, and C. cucumerinum and the specific membrane characteristics influencing the antifungal effectiveness of fengycin, iturin, and surfactin. Understanding of these determinants should enable more accurate prediction of sensitivity-tolerance outcomes for other fungal species exposed to these important CLP.

Cordyceps chanhua, an important cordycipitoid medical mushroom with wide use in Asia, has gained attention for its bioactive component beauvericin (BEA), which is of medicinal value as a drug lead, but also of food safety risk. Recent observations by our group revealed a significant decrease of BEA content in C. chanhua when exposed to light, but the underlying regulatory mechanisms remain elusive. In this study, a comprehensive approach combining metabolomics and transcriptomics was employed to investigate the effects of white light on the secondary metabolism of C. chanhua for elucidation of the influence of light on BEA biosynthesis in this fungus. The result showed that the genes and metabolites involved in the synthesis of D-hydroxyisovaleric acid, a precursor of BEA synthesis, were down-regulated under light exposure, while those associated with the synthesis of phenylalanine, another precursor of BEA synthesis, were up-regulated leading to elevated phenylalanine levels. It suggested that the suppressive effect of light on BEA synthesis in C. chanhua occurred primarily through the inhibition of D-hydroxyisovaleric acid synthesis, while the enhanced phenylalanine biosynthesis likely directed towards other metabolic pathway such as pigment synthesis. These results contributed to a better understanding on how light modulates the secondary metabolism of C. chanhua and provided valuable guidance for optimizing BEA production in cultivation practices.

Heavy metal Cd can easily be accumulated by fungi, causing significant stress, with the fungal cell membrane being one of the primary targets. However, the understanding of the mechanisms behind this stress remains limited. This study investigated the changes in membrane lipid molecules of Pleurotus ostreatus mycelia under Cd stress and the antagonistic effect of Ca on this stress. Cd in the growth media significantly inhibited mycelial growth, with increasing intensity at higher concentrations. The addition of Ca mitigated this Cd-induced growth inhibition. Lipidomic analysis showed that Cd reduced membrane lipid content and altered lipid composition, while Ca counteracted these changes. The effects of both Cd and Ca on lipids are dose dependent and phosphatidylethanolamine appeared most affected. Cd also caused a phosphatidylcholine/phosphatidylethanolamine ratio increase at high concentrations, but Ca helped maintain normal levels. The acyl chain length and unsaturation of lipids remained unaffected, suggesting Cd doesn't alter acyl chain structure of lipids. These findings suggest that Cd may affect the growth of mycelia by inhibiting the synthesis of membrane lipids, particular the synthesis of phosphatidylethanolamine, providing novel insights into the mechanisms of Cd stress in fungi and the role of Ca in mitigating the stress.

The coconut rhinoceros beetle (CRB; Oryctes rhinoceros) is one of the most destructive insect pests of coconut and oil palms in tropical Asia and the Pacific islands. Members of a new variant, known as CRB-G (clade I), have recently spread into the Pacific islands, causing significant damage. Biopesticides containing Metarhizium spp. are the strongest candidates for inundative biological control against the emerging CRB threat. Selection of the most virulent and robust isolate may determine the impact of this control option on the pest. In this work, CRB specimens with natural fungal infection were collected in Papua New Guinea (PNG) and Solomon Islands (SI). Putative entomopathogenic fungi were isolated and identified. These new isolates and some previously obtained from other Pacific countries were molecularly identified, characterized, and tested for virulence against CRB larval populations in PNG and SI in laboratory bioassays. Of the new isolates collected, four obtained from SI were identified as Metarhizium majus (conidia length ⁓11-15 μm), and four from PNG were identified as Metarhizium pingshaense (conidia length ⁓4-6 μm). The most virulent isolate was M. majus AgR-F717, which caused 100 % mortality in 20-23 days against a CRB variant from the CRB-S grouping (clade II) in laboratory bioassays carried out in PNG. Isolates of M. pingshaense did not show pathogenicity against CRB larvae. M. majus AgR-F717 was also the most virulent in laboratory bioassays using the mixed SI population (from both CRB-S and CRB-G groupings) and was selected for further evaluation using artificial breeding sites. Under field conditions, this isolate demonstrated its ability to infect CRB, dispersal up to 100 m from treated artificial breeding sites, and persistence in soil for at least four months. The new isolate AgR-F717 of M. majus has demonstrated potential as an augmentative biological control agent for CRB in PNG and SI.

An intense black pigmented halotolerant yeast GUBPC1, was obtained from the solar salterns of Nerul, Goa-India. The isolate could tolerate 0 to 20 % NaCl. FE-SEM analysis revealed its polymorphic nature, exhibiting oval cells at higher salt concentrations and filamentous spindle like shapes at lower concentrations. Initially, the cells appear oval, yeast like in shape but gradually after 15 days of incubation, it becomes elongated and undergoes budding, exhibiting various budding patterns, from single polar bud to bipolar buds with annellidic ring, to lateral buds and eventually forming filamentous hyphae. The intracellular black pigment was identified as melanin based on ultraviolet-visible spectroscopy analysis. The molecular identification of the culture showed closest similarity with Hortaea werneckii. Plant polymer-degrading enzymatic activities such as cellulase, laccase, chitinase, xylanase, pectinase, amylase and protease were exhibited by the isolate GUBPC1. To further understand and explore its biotechnological potential, we performed whole-genome sequencing and analysis. The obtained genome size was 26.93 Mb with 686 contigs and a GC content of 53.24 %. We identified 9383 protein-coding genes, and their functional annotation revealed the presence of 435 CAZyme genes and 16 functional genes involved in secondary metabolite synthesis, thus providing a basis for its potential value in various biotechnological applications.

Macrophages play critical protective roles as sentinels of the innate immune system against fungal infection. It is therefore important to understand the dynamics of the interaction between these phagocytes and their fungal prey. We show here that many of the hyphal apices formed by Candida albicans within the macrophage ceased elongating, and apical and sub-apical hyphal compartments became swollen. Swollen hyphal cell compartments assimilated less Lysotracker-Red than non-swollen compartments, suggesting they had enhanced viability. Staining with florescent dyes suggested that there were higher levels of β-glucan and chitin in internalized fungal filaments compared to non-internalized hyphae, suggesting active cell wall remodelling within macrophages. These observations suggest that the stresses imposed by macrophages upon the fungus lead to changes in cell wall composition, inhibition of polarised growth and the induction of swelling in hyphal compartments, and that this can prevent or delay loss of viability of hyphal cells within the phagocyte.

Boeremia was established to accommodate phoma-resembling fungi. Its species occur in terrestrial ecosystems as endophytes, saprobes and pathogens, except one species reported from a marine ecosystem. Boeremia species are characterized by hyaline, thin-walled, and aseptate (occasionally 1(-2)-septate) conidia that are variable in shape, and hyaline, straight or slightly curved, thick-walled, and 1-septate ascospores that are usually constricted at the septum. In the past, host associations were used to delimit Boeremia species. However, since Boeremia taxa have overlapping morphological characters and are cryptic, it renders taxonomic identification arduous. Therefore, the use of other approaches including multi-gene phylogenetic analyses are imperative. Recommended DNA markers for species delineation are the internal transcribed spacer (ITS, nuclear rDNA consisting of ITS1-5.8S-ITS2) and large subunit (28S, D1-D2 domains of nuclear 28S rDNA) loci, and the genes for actin (ACT1), beta-tubulin (TBB1), RNA polymerase 2 (RPB2) and translation elongation factor 1α (TEF1). Here, we applied morphological and molecular phylogenetic analyses to establish a new taxon (B. albae), and a new host and geographical record for B. maritima associated with leaf spots of Morus alba (Moraceae) in northern Thailand. By providing sequence data for three additional gene regions, our phylogenetic analyses impart a stable phylogenetic placement of the ex-type strain of B. maritima, as illustrated. This is the first study that reports Boeremia species from M. alba, and B. maritima from a terrestrial habitat.

Biotic factors in fungal exudates impact plant-fungal symbioses establishment. Mutualistic ectomycorrhizal fungi play various ecological roles in forest soils by interacting with trees. Despite progress in understanding secreted fungal signals, dynamics of signal production in situ before or during direct host root contact remain unclear. We need to better understand how variability in intra-species fungal signaling at these stages impacts symbiosis with host tissues. Using the ECM model Pisolithus microcarpus, we selected two isolates (Si9 and Si14) with different abilities to colonize Eucalyptus grandis roots. Hypothesizing that distinct early signalling and metabolite profiles between these isolates would influence colonization and symbiosis, we used microdialysis to non-destructively collect secreted metabolites from either the fungus, host, or both, capturing the dynamic interplay of pre-symbiotic signalling over 48 hours. Our findings revealed significant differences in metabolite profiles between Si9 and Si14, grown alone or with a host root. Si9, with lower colonization efficiency than Si14, secreted a more diverse range of compounds, including lipids, oligopeptides, and carboxylic acids. In contrast, Si14's secretions, similar to the host's, included more aminoglycosides. This study emphasizes the importance of intra-specific metabolomic diversity in ectomycorrhizal fungi, suggesting that early metabolite secretion is crucial for establishing successful mutualistic relationships.

Leaf blotch, caused by Zymoseptoria tritici, is a fungal disease that poses a severe threat to wheat production worldwide. Knowledge of virulence variability is crucial in choosing effective control measures. However, there have only been a few studies of the pathogenic variability and pathotypes within Ethiopian isolates. Hence, the objective of this study was to assess the virulence spectrum and variability of Z. tritici isolates. Forty-three isolates were tested for their virulence and pathotype against 7 wheat differential lines that have different resistance genes. A pathogenicity assay detected 41 differential line-specific virulent isolates among 301 interactions between a host and pathogen based on the percentage coverage of the leaf area by pycnidia. Some isolates were virulent against 50 %-60 % of the resistant genes, but most of them were virulent against some differential lines. Isolates such as EtA-11, EtSh-1, EtSh-2, EtSh-4, and EtA-19 expressed broad-spectrum virulence, highlighting that such isolates are useful for germplasm screening. The isolates were classified into 25 pathotypes, defined by their differential virulence responses. They were also assigned to two clusters according to their mean pycnidia percent. Pathotypes and principal component analysis detected 58.1 % and 62.2 % pathogenic diversity in Ethiopian isolates, respectively. The current findings provide information that breeders can use to identify and select more resistant varieties for farmers.

Some fungi have demonstrated the ability to adapt rapidly to changing environments by exhibiting morphological plasticity, a trait influenced by species and environmental factors. Here, an anamorphic yeast strain IOJ-3 exhibited unique sectorization characteristics, naturally producing diverse filamentous sectors when cultivated on potato dextrose agar (PDA) medium or natural culture medium for durations exceeding 13 days. The strain IOJ-3 and its filamentous sectors were identified as Dothiora sorbi. The morphology of the sectors was consistent and heritable. The life cycle of strain IOJ-3 was investigated through microscopic observation, emphasizing the development of conidiogenous cells as a crucial stage, from which filamentous sectors originate. Some physiological characteristics of IOJ-3 and filamentous sectors are compared, and strain IOJ-3 has a higher antibiotic tolerance than two filamentous sectors, IOJ-3a expands faster on the culture medium, and IOJ-3b can penetrate cellophane. A transcriptomic analysis was conducted to investigate the differentially expressed genes between the yeast form IOJ-3 and its two filamentous sectors, revealing a total of 594 genes that exhibited consistent differential expression relative to IOJ-3, including 44 silencing genes in IOJ-3 that were activated. Gene Ontology analysis indicated that these differentially expressed genes were primarily associated with the cellular component category. Furthermore, adding 5-Azacytidine accelerated filamentous sectorization and increased the proportion of filamentous cells of strain IOJ-3 in PD liquid media, suggesting that the filamentous sectorization observed in strain IOJ-3 is linked to processes of DNA demethylation. In conclusion, this study sheds light on the biological characteristics of D. sorbi regarding morphological transitions and provides substantial direction for exploring genes related to fungal filamentous development.

Mutations have underpinned research into gene characterization across all domains of life. This includes the discovery of the genes involved in the development of asexual spores in filamentous fungi. Mutants in the ascomycete Paecilomyces variotii were isolated with impaired biosynthesis of the characteristic yellow pigment produced by this filamentous fungus. The affected genes were identified as pvpP, encoding the polyketide synthase that is required for synthesis of the pigment YWA1, and abaA and wetA that are two genes that encode components of the AbaA-BrlA-WetA module required for the development of asexual spores in species in the Eurotiales order. WetA was further characterized. A strain expressing a functional WetA-GFP fusion was created and used to find that WetA is expressed primarily in spores and concentrated in their nuclei, providing evidence that this conserved protein likely functions as a regulator of transcription in conidia. Analysis of the phenotypes of the P. variotii wetA mutant suggests that how this three-protein module impacts fungal biology will vary from species-to-species, despite being conserved amongst filamentous Ascomycete species.

Fusarium verticillioides is both an endophyte and pathogen of maize. During growth on maize, the fungus often synthesizes the mycotoxins fumonisins, which have been linked to a variety of diseases, including cancer in some animals. How F. verticillioides responds to other fungi, such as Fusarium proliferatum, Aspergillus flavus, Aspergillus niger, and Penicillium oxalicum, that coinfect maize, has potential to impact mycotoxin synthesis and disease. We hypothesize that low molecular weight acids produced by these fungi play a role in communication between the fungi in planta/nature. To address this hypothesis, we exposed 48-h maize kernel cultures of F. verticillioides to oxalic acid, citric acid, fusaric acid, or kojic acid and then compared transcriptomes after 30 min and 6 h. Transcription of some genes were affected by multiple chemicals and others were affected by only one chemical. The most significant positive response was observed after exposure to fusaric acid which resulted in >2-fold upregulation of 225 genes, including genes involved in fusaric acid synthesis. Exposure of cultures to the other three chemicals increased expression of only 3-15 genes. The predicted function and frequent co-localization of three sets of genes support a role in protecting the fungus from the chemical or a role in catabolism. These unique transcriptional responses support our hypothesis that these chemicals can act as signaling molecules. Studies with gene deletion mutants will further indicate if the initial transcriptional response to the chemicals benefit F. verticillioides.

Green mould contamination causes a significant challenge to mushroom growers in Malaysia leading to reduced yields and economic losses in the widely cultivated and marketed edible grey oyster mushroom, Pleurotus pulmanorius. This study aimed to identify the causal agents of green mould contaminants and determine the critical points in the cultivation process in the farm that contribute to green mould contamination. Samples of mushroom substrate (sawdust), spawn substrate (corn), environmental sources and tools were collected at different stages of mushroom cultivation. As results, the causal agents of green mould contamination were identified as Trichoderma pleuroti, T. harzianum and T. ghanese. Prior to steam pasteurisation and after steam pasteurisation, the spawn substrate and mushroom substrate were found to be free of Trichoderma. However, Trichoderma was detected in water, air within the production house and on cleaning tools. This findings suggests that water could serve as the source of green mould introduction in mushroom farms, while cultivation practices such as watering and scratching during the harvesting cycle may contribute to adverse green mould. Understanding these critical points and causal agents provides information to mitigate the green mould contamination throughout the grey oyster mushroom cultivation process.

Calonectria leaf blight (CLB) is one of the best-known diseases of Eucalyptus spp., particularly in Asia and South America. Recently, typical symptoms of leaf and shoot blight caused by Calonectria spp. Were observed in a Eucalyptus plantation in the YunNan Province of southwestern China. Isolations were made from diseased leaves and top soil collected below the diseased trees to determine the causal agent of the disease and to consider the distribution characteristics of the Calonectria species. This resulted in 417 isolates, of which 228 were from leaves and 189 were from soil. Based on comparisons of DNA sequences for the act (actin), cmdA (calmodulin), his3 (histone H3), rpb2 (the second largest subunit of RNA polymerase), tef1 (translation elongation factor 1-alpha) and tub2 (β-tubulin) gene regions, as well as morphological characteristics, 11 Calonectria species were identified. These included Calonectria aciculata (0.7 %), Ca. colhounii (1.2 %), Ca. eucalypti (10.6 %) and Ca. honghensis (43.2 %) in the Ca. colhounii species complex, and Ca. aconidialis (15.3 %), Ca. asiatica (9.8 %), Ca. hongkongensis (1.0 %), Ca. ilicicola (6.0 %), Ca. kyotensis (0.5 %), and Ca. yunnanensis (11.3 %) in the Ca. kyotensis species complex. In addition, a novel species, accounting for 0.5 % of the isolates, was discovered and is described here as Ca. dianii sp. nov. in the Ca colhounii species complex. Most (99.1 %) of the isolates collected from the leaves resided in the Ca. colhounii species complex and a majority (95.8 %) of those from the soils were in Ca. kyotensis species complex. These results suggest that Calonectria spp. in the Ca. colhounii species complex infecting leaves might be adapted to that niche and that those in the Ca. kyotensis species complex are better adapted to a soil habitat.

The aim of this study is to develop safe biological methods for controlling fungal deterioration of historical manuscripts. Therefore, fifteen fungal isolates were obtained from paper sheets and leather skins of a deteriorated historical manuscript (dated back to the 13th century). Those isolates were identified using both traditional methods and ITS-sequencing analysis. Aspergillus niger accounted for seven strains, Penicillium citrinum for one strain, Aspergillus flavus for three, Aspergillus fumigatus for one, Aspergillus nidulans for one, and Penicillium chrysogenum for two of the fungal strains that were obtained. The ability of fungal strains for the secretion of cellulase, amylase, gelatinase, and pectinase as hydrolytic enzymes was evaluated. The capability of the probiotic-bacterial strain Lactobacillus plantarum DSM 20174 for inhibition of fungal strains that cause severe deterioration was studied using ethyl acetate-extract. The metabolic profile of the ethyl acetate-extract showed the presence of both high- and low-molecular-weight active compounds as revealed by GC-MS analysis. The safe dose to prevent fungal growth was determined by testing the ethyl acetate extract's biocompatibility against Wi38 and HFB4 as normal cell lines. The extract was found to have a concentration-dependent cytotoxic impact on Wi38 and HFB4, with IC values of 416 ± 4.5 and 349.7 ± 5.9 μg mL, respectively. It was suggested that 100 μg mL as a safe concentration could be used for paper preservation. Whatman filter paper treated with ethyl acetate extract was used to cultivate the fungal strain Penicillium citrinum AX2. According to data analysis, fungal inhibition measurement, SEM, ATR-FT-IR, XRD, color change measurement, and mechanical property assessment, the recommended concentration of ethyl acetate extract was adequate to protect paper inoculated with the highest enzymatic producer fungi, P. citrinum AX2.

Understanding species habitat preferences is essential for conservation and management efforts, as it enables the identification of areas with a higher likelihood of species presence. Lactarius deliciosus (L.) Gray, an economically important edible mushroom, is influenced by various environmental variables, yet information regarding its ecological niche remains elusive. Therefore, in this study, we aim to address this gap by modeling the fundamental niche of L. deliciosus. Specifically, we explore its distribution patterns in response to large-scale environmental factors, including long-term temperature averages and topography. We employed 242 presence-only georeferenced points in Europe obtained from the Global Biodiversity Information Facility (GBIF). Utilizing the Kuenm R package, we constructed 210 models incorporating five sets of environmental variables, 14 regularization multiplier values, and three feature class combinations. Evaluation metrics included statistical significance, predictive power, and model complexity. The final model was transferred to Turkiye, with careful consideration of extrapolation risk using MESS (multivariate similarity surface) and MoD (most dissimilar variable) metrics. In alignment with all three evaluation criteria, the algorithm implemented in Kuenm identified the best model as the linear-quadratic combination with a regularization multiplier of 0.2, based on variables selected by the contribution importance method. Results underscore temperature-related variables as critical determinants of L. deliciosus habitat preferences within the calibration area, with solar radiation also playing a significant role in the final model. These results underscored the effectiveness of ecological niche modeling (ENM) in understanding how climatic patterns may alter the distribution of species like L. deliciosus. The findings contribute to the development of informed conservation strategies and decision-making in dynamic environments. Emphasizing a comprehensive approach to ecological modeling is crucial for promoting sustainable forest management.

P. umbellatus sclerotium is a traditional Chinese medicine that is widely utilized in China, Korea, Japan, and other countries due to its diverse medicinal activities, such as diuretic, antitumor, anticancer, and immune system enhancement effects. Conidia, which are common asexual spores in various fungi, are not universally present in Polyporus species. In this study, the asexual life cycle of P. umbellatus was elucidated. Conidia, i.e. arthorconidia, were produced by both dikaryotic and monokaryotic strains. In the dikaryotic strain, binucleate, uninucleate, and nuclei-free conidia were identified with proportions of 67.9 %, 12.4 %, and 19.7 %, respectively. Conversely, the monokaryotic strain did not produce binucleate conidia. This discrepancy suggests that binucleate spores are heterokaryons, while uninucleate spores are homokaryons. Clamp connections were observed in dikaryotic hyphae, but were absent in monokaryotic hyphae. Monokaryotic strains were obtained from conidia of the dikaryotic strain. Additionally, mating types were determined through pairing tests, and successful crossbreeding occurred between monokaryotic strains derived from conidia and basidiospores from different strains. This study introduced the first crossbreeding strategy for P. umbellatus.

The Oomycetes fungus Phytophthora spp. which causes Abnormal leaf fall (ALF) disease poses a significant threat as one of the most devastating diseases affecting rubber trees in India. A total of 30 Phytophthora isolates were obtained from ALF-affected samples collected during the Southwest monsoon season of Kerala. The colony morphology of Phytophthora isolates revealed eight different types of growth patterns, with stellate, stellate striated, and petaloid patterns growing rapidly, whereas chrysanthemum pattern grew slowly. Sporangia were papillate to non-papillate in various shapes, and sporangiophores exhibited simple, simple sympodial, or irregularly branching patterns. Highly virulent isolates exhibited petaloid morphology and rapid growth rates. Regardless of their virulence, all isolates showed susceptibility to the fungicide metalaxyl. Under in vitro conditions, the highly virulent isolate (R17) from rubber caused severe infections in chili, brinjal, and tomato with brown water-soaked lesions. Sequence analysis and multi-locus phylogeny of Internal transcribed spacer (ITS), cCytochrome c oxidase 1 (COX 1), Heat shock protein 90 (HSP 90), and Ribosomal protein L10 (RPL 10) confirmed the pathogen as Phytophthora meadii. A comprehensive understanding of both morphological and molecular traits of P. meadii is crucial for precise identification and future genetic variability studies.

Ectomycorrhizal (ECM) fungi play a major role in forest ecosystems and managed tree plantations. Particularly, they facilitate mineral weathering and nutrient transfer towards colonized roots. Among nutrients provided by these fungi, potassium (K) has been understudied compared to phosphorus (P) or nitrogen (N). The ECM fungus Paxillus ammoniavirescens is a generalist species that interacts with the root of many trees and can directly transfer K to them, including loblolly pine. However, the forms of K that ECM fungi can store is still unknown. Here, we used synchrotron potassium X-ray fluorescence (XRF) and K-edge X-ray Absorption Near Edge Structure (XANES) spectroscopy on P. ammoniavirescens growing in axenic conditions to investigate the K chemistries accumulating in the center and the edge of the mycelium. We observed that various K forms accumulated in different part of the mycelium, including K-nitrate (KNO), K-C-O compounds (such as K-tartrate K(CHO) and K-oxalate (KCO)), K-S and K-P compounds. Saprotrophic fungi have been shown to excrete carboxylic acids, which in turn play a role in soil mineral weathering. Our finding of several K counter-ions to carboxylic acids may suggest that, besides their direct transfer to colonized roots, K ions can also be involved in the production of compounds necessary for sourcing nutrients from their surrounding environment by ECM fungi. Additionally, this work reveals that XANES spectroscopy can be used to identify the various forms of K accumulating in biological systems.

Eucalyptus spp. in plantations are negatively affected by canker and wilt diseases caused by several species of Ceratocystis, particularly those in the Latin American Clade (LAC). Ceratocystis eucalypticola and Ceratocystis manginecans are of particular concern where disease epidemics are reported globally, with recent outbreaks emerging in South African and Indonesian Eucalyptus plantations. Consequently, a rapid screening protocol is required for these pathogens. In this study, a high-resolution melting curve analysis (HRMA) was developed to detect C. eucalypticola and C. manginecans that bypasses time-consuming isolation and post-PCR procedures. Primers targeting a 172 bp region of the cerato-platanin (CP) gene were designed. Using these primers, the accuracy of HRMA to detect and distinguish between these two LAC species was assessed using pure fungal DNA, and DNA extracted directly from Eucalyptus samples naturally infected with C. eucalypticola. The assay accurately detected the presence of C. eucalypticola and C. manginecans and quantifies their DNA, both from cultures, and directly from wood samples. HRMA further differentiated these two species from all other tested LAC individuals. This assay was also able to detect the presence of all the tested LAC species and distinguish seven of these, including C. fimbriata, to species level. Ceratocystis polyconidia was the only non-LAC off-target species detected. Based on these results, the developed assay can be used to rapidly identify C. eucalypticola and C. manginecans directly from infected plant material or fungal cultures, with the potential to also screen for several other LAC species.