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Elemental profiling of fungal species as a phenotyping tool is an understudied topic and is typically performed to examine plant tissue or non-biological materials. Traditional analytical techniques such as inductively coupled plasma-optical emission spectroscopy (ICP-OES) and inductively coupled plasma-mass spectrometry (ICP-MS) have been used to identify elemental profiles of fungi; however, these techniques can be cumbersome due to the difficulty of preparing samples. Additionally, the instruments used for these techniques can be expensive to procure and operate. Laser-induced breakdown spectroscopy (LIBS) is an alternative elemental analytical technique-one that is sensitive across the periodic table, easy to use on various sample types, and is cost-effective in both procurement and operation. LIBS has not been used on axenic filamentous fungal isolates grown in substrate media. In this work, as a proof of concept, we used LIBS on two genetically distinct fungal species grown on a nutrient-rich and nutrient-poor substrate media to determine whether robust elemental profiles can be detected and whether differences between the fungal isolates can be identified. Our results demonstrate a distinct correlation between fungal species and their elemental profile, regardless of the substrate media, as the same strains shared a similar uptake of carbon, zinc, phosphorus, manganese, and magnesium, which could play a vital role in their survival and propagation. Independently, each fungal species exhibited a unique elemental profile. This work demonstrates a unique and valuable approach to rapidly phenotype fungi through optical spectroscopy, and this approach can be critical in understanding these fungi's behavior and interactions with the environment. IMPORTANCE: Historically, ionomics, the elemental profiling of an organism or materials, has been used to understand the elemental composition in waste materials to identify and recycle heavy metals or rare earth elements, identify the soil composition in space exploration on the moon or Mars, or understand human disorders or disease. To our knowledge, ionomic profiling of microbes, particularly fungi, has not been investigated to answer applied and fundamental biological questions. The reason is that current ionomic analytical techniques can be laborious in sample preparation, fail to measure all potential elements accurately, are cost-prohibitive, or provide inconsistent results across replications. In our previous efforts, we explored whether laser-induced breakdown spectroscopy (LIBS) could be used in determining the elemental profiles of poplar tissue, which was successful. In this proof-of-concept endeavor, we undertook a transdisciplinary effort between applied and fundamental mycology and elemental analytical techniques to address the biological question of how LIBS can used for fungi grown axenically in a nutrient-rich and nutrient-poor environment.

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We identified two poplar (Populus sp.)-associated microbes, the fungus, Mortierella elongata strain AG77, and the bacterium, Burkholderia strain BT03, that mutually promote each other's growth. Using culture assays in concert with a novel microfluidic device to generate time-lapse videos, we found growth specific media differing in pH and pre-conditioned by microbial growth led to increased fungal and bacterial growth rates. Coupling microfluidics and comparative metabolomics data results indicated that observed microbial growth stimulation involves metabolic exchange during two ordered events. The first is an emission of fungal metabolites, including organic acids used or modified by bacteria. A second signal of unknown nature is produced by bacteria which increases fungal growth rates. We find this symbiosis is initiated in part by metabolic exchange involving fungal organic acids.

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Many plant-associated fungi host endosymbiotic endobacteria with reduced genomes. While endobacteria play important roles in these tri-partite plant-fungal-endobacterial systems, the active physiology of fungal endobacteria has not been characterized extensively by systems biology approaches. Here, we use integrated proteomics and metabolomics to characterize the relationship between the endobacterium Mycoavidus sp. and the root-associated fungus Mortierella elongata. In nitrogen-poor media, M. elongata had decreased growth but hosted a large and growing endobacterial population. The active endobacterium likely extracted malate from the fungal host as the primary carbon substrate for energy production and biosynthesis of phospho-sugars, nucleobases, peptidoglycan and some amino acids. The endobacterium obtained nitrogen by importing a variety of nitrogen-containing compounds. Further, nitrogen limitation significantly perturbed the carbon and nitrogen flows in the fungal metabolic network. M. elongata regulated many pathways by concordant changes on enzyme abundances, post-translational modifications, reactant concentrations and allosteric effectors. Such multimodal regulations may be a general mechanism for metabolic modulation.

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BACKGROUND: The soil fungus Rhizoctonia solani anastomosis group 3 (AG-3) is an important pathogen of cultivated plants in the family Solanaceae. Isolates of R. solani AG-3 are taxonomically related based on the composition of cellular fatty acids, phylogenetic analysis of nuclear ribosomal DNA (rDNA) and beta-tubulin gene sequences, and somatic hyphal interactions. Despite the close genetic relationship among isolates of R. solani AG-3, field populations from potato and tobacco exhibit comparative differences in their disease biology, dispersal ecology, host specialization, genetic diversity and population structure. However, little information is available on how field populations of R. solani AG-3 on potato and tobacco are shaped by population genetic processes. In this study, two field populations of R. solani AG-3 from potato in North Carolina (NC) and the Northern USA; and two field populations from tobacco in NC and Southern Brazil were examined using sequence analysis of two cloned regions of nuclear DNA (pP42F and pP89). RESULTS: Populations of R. solani AG-3 from potato were genetically diverse with a high frequency of heterozygosity, while limited or no genetic diversity was observed within the highly homozygous tobacco populations from NC and Brazil. Except for one isolate (TBR24), all NC and Brazilian isolates from tobacco shared the same alleles. No alleles were shared between potato and tobacco populations of R. solani AG-3, indicating no gene flow between them. To infer historical events that influenced current geographical patterns observed for populations of R. solani AG-3 from potato, we performed an analysis of molecular variance (AMOVA) and a nested clade analysis (NCA). Population differentiation was detected for locus pP89 (Phi ST = 0.257, significant at P < 0.05) but not for locus pP42F (Phi ST = 0.034, not significant). Results based on NCA of the pP89 locus suggest that historical restricted gene flow is a plausible explanation for the geographical association of clades. Coalescent-based simulations of genealogical relationships between populations of R. solani AG-3 from potato and tobacco were used to estimate the amount and directionality of historical migration patterns in time, and the ages of mutations of populations. Low rates of historical movement of genes were observed between the potato and tobacco populations of R. solani AG-3. CONCLUSION: The two sisters populations of the basidiomycete fungus R. solani AG-3 from potato and tobacco represent two genetically distinct and historically divergent lineages that have probably evolved within the range of their particular related Solanaceae hosts as sympatric species.

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Hemiascomycetes are species of yeasts within the order Saccharomycetales. The order encompasses disparate genera with a variety of life styles, including opportunistic human pathogens (e.g., Candida albicans), plant pathogens (e.g., Eremothecium gossypii), and cosmopolitan yeasts associated with water and decaying vegetation. To analyze the phylogeny of medically important species of yeasts, we selected 38 human pathogenic and related strains in the order Saccharomycetales. The DNA sequences of six nuclear genes were analyzed by maximum likelihood and Bayesian phylogenetic methods. The maximum likelihood analysis of the combined data for all six genes resolved three major lineages with significant support according to Bayesian posterior probability. One clade was mostly comprised of pathogenic species of Candida. Another major group contained members of the family Metschnikowiaceae as a monophyletic group, three species of Debaryomyces, and strains of Candida guilliermondii. The third clade consisted exclusively of species of the family Saccharomycetaceae. Analysis of the evolution of key characters indicated that both codon reassignment and coenzyme Q(9) likely had single origins with multiple losses. Tests of correlated character evolution revealed that these two traits evolved independently.

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ABSTRACT The relative contribution of migration of Rhizoctonia solani anastomosis group 3 (AG-3) on infested potato seed tubers originating from production areas in Canada, Maine, and Wisconsin (source population) to the genetic diversity and structure of populations of R. solani AG-3 in North Carolina (NC) soil (recipient population) was examined. The frequency of alleles detected by multilocus polymerase chain reaction-restriction fragment length polymorphisms, heterozygosity at individual loci, and gametic phase disequilibrium between all pairs of loci were determined for subpopulations of R. solani AG-3 from eight sources of potato seed tubers and from five soils in NC. Analysis of molecular variation revealed little variation between seed source and NC recipient soil populations or between subpopulations within each region. Analysis of population data with a Bayesian-based statistical method previously developed for detecting migration in human populations suggested that six multilocus genotypes from the NC soil population had a statistically significant probability of being migrants from the northern source population. The one-way (unidirectional) migration of genotypes of R. solani AG-3 into NC on infested potato seed tubers from Canada, Maine, and Wisconsin provides a plausible explanation for the lack of genetic subdivision (differentiation) between populations of the pathogen in NC soils or between the northern source and the NC recipient soil populations.

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Cryptococcus neoformans is a major pathogen of humans throughout the world. Using commercial mAbs to capsular epitopes, strains of C. neoformans manifest five distinct serotypes--A, B, C, D and AD. Previous studies demonstrated significant divergence among serotypes A, B, C and D, which are thought to be haploid. In this study the origins and evolution of strains of serotype AD were investigated. A portion (537 bp) of the laccase gene was cloned and sequenced from 14 strains of serotype AD. Each strain contained two different alleles and sequences for both alleles were obtained. These sequences were compared to those from serotypes A, B, C and D. This analysis indicated that each of the 14 serotype AD strains contained two phylogenetically distinct haplotypes: one haplotype was highly similar to the serotype A group and the other to the serotype D group. To explain the origins of these serotype AD strains, genealogical analysis is consistent with at least three recent and independent hybridization events. The results demonstrate that the evolution of C. neoformans is continuing and dynamic.

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A polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method was developed to identify and differentiate genotypes of Rhizoctonia solani anastomosis group 3 subgroup PT (AG-3 PT), a fungal pathogen of potato. Polymorphic co-dominant single-locus PCR-RFLP markers were identified after sequencing of clones from a genomic library and digestion with restriction enzymes. Multilocus genotypes were determined by a combination of PCR product and digestion with a specific restriction enzyme for each of seven loci. A sample of 104 isolates from one commercial field in each of five counties in eastern North Carolina was analyzed, and evidence for high levels of gene flow between populations was revealed. When data were clone-corrected and samples pooled into one single North Carolina population, random associations of alleles were found for all loci or pairs of loci, indicating random mating. However, when all genotypes were analyzed, the observed genotypic diversity deviated from panmixia and alleles within and between loci were not randomly associated. These findings support a model of population structure for R. solani AG-3 PT on potato that includes both recombination and clonality.

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Anastomosis group 3 (AG-3) of Rhizoctonia solani (teleomorph = Thanatephorus cucumeris) is frequently associated with diseases of potato (AG-3 PT) and tobacco (AG-3 TB). Although isolates of R. solani AG-3 from these two Solanaceous hosts are somatically related based on anastomosis reaction and taxonomically related based on fatty acid, isozyme and DNA characters, considerable differences are evident in their biology, ecology, and epidemiology. However, genetic diversity among field populations of R. solani AG-3 PT and TB has not been documented. In this study, the genetic diversity of field populations of R. solani AG-3 PT and AG-3 TB in North Carolina was examined using somatic compatibility and amplified fragment length polymorphism (AFLP) criteria. A sample of 32 isolates from potato and 36 isolates from tobacco were paired in all possible combinations on PDA plus activated charcoal and examined for their resulting somatic interactions. Twenty-eight and eight distinct somatic compatibility groups (SCG) were identified in the AG-3 PT and AG-3 TB samples, respectively. AFLP analyses indicated that each of the 32 AG-3 PT isolates had a distinct AFLP phenotype, whereas 28 AFLP phenotypes were found among the 36 isolates of AG-3 TB. None of the AG-3 PT isolates were somatically compatible or shared a common AFLP phenotype with any AG-3 TB isolate. Clones (i.e., cases where two or more isolates were somatically compatible and shared the same AFLP phenotype) were identified only in the AG-3 TB population. Four clones from tobacco represented 22% of the total population. All eight SCG from tobacco were associated with more than one AFLP phenotype. Compatible somatic interactions between AG-3 PT isolates occurred only between certain isolates from the same field (two isolates in each of four different fields), and when this occurred AFLP phenotypes were similar but not identical.