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Fungal life-styles and ecosystem dynamics: Biological aspects of plant pathogens, plant endophytes and saprophytes

This chapter discusses various biochemical, genetic, ecological, and evolutionary aspects of fungi that express either symbiotic or saprophytic life-styles. An enormous pool of potential pathogens exists in both agricultural and natural ecosystems, and virtually all plant species are susceptible to one or more fungal pathogens. Fungal pathogens have the potential to impact on the genetic structure of populations of individual plant species, the composition of plant communities and the process of plant succession. Endophytic fungi exist for at least part of their life cycles within the tissues of a plant host. This group of fungi is distinguished from plant pathogens because they do not elicit significant disease symptoms. However, endophytes do maintain the genetic and biochemical mechanisms required for infection and colonization of plant hosts. Fungi that obtain chemical nutrients from dead organic matter are known as saprophytes and are critical to the dynamics and resilience of ecosystems. There are two modes of saprophytic growth: one in which biomolecules that are amenable to transport across cell walls and membranes are directly absorbed, and another in which fungi must actively convert complex biopolymers into subunit forms amenable to transportation into cells. Regardless of life-style, fungi employ similar biochemical mechanisms for the acquisition and conversion of nutrients into complex biomolecules that are necessary for vegetative growth, production and dissemination of progeny, organismal competition, and survival during periods of nutrient deprivation or environmental inclemency.

Advances in Botanical Research

Fungal biology and agriculture: revisiting the field

Plant pathology has made significant progress over the years, a process that involved overcoming a variety of conceptual and technological hurdles. Descriptive mycology and the advent of chemical plant-disease management have been followed by biochemical and physiological studies of fungi and their hosts. The later establishment of biochemical genetics along with the introduction of DNA-mediated transformation have set the stage for dissection of gene function and advances in our understanding of fungal cell biology and plant-fungus interactions. Currently, with the advent of high-throughput technologies, we have the capacity to acquire vast data sets that have direct relevance to the numerous subdisciplines within fungal biology and pathology. These data provide unique opportunities for basic research and for engineering solutions to important agricultural problems. However, we also are faced with the challenge of data organization and mining to analyze the relationships between fungal and plant genomes and to elucidate the physiological function of pertinent DNA sequences. We present our perspective of fungal biology and agriculture, including administrative and political challenges to plant protection research.

Molecular Plant-Microbe Interactions

Genetic variation in the midcontinental population of sandhill cranes, Grus canadensis

Three subspecies of sandhill crane (Grus canadensis) are recognized in the Midcontinental population, the lesser (Grus c. canadensis), Canadian (G. c. rowani), and greater (G. c. tabida). Blood samples collected on the population's primary spring staging area in Nebraska, U.S.A., were used to resolve the genetic relationship among these subspecies. Phylogenetic analysis of 27 G. canadensis, by DNA sequencing of a 675 bp region of the mtDNA, supports the subspecies designations of G. c. canadensis and G. c. tabida. G. c. rowani individuals were intermediate with each of the other two subspecies. Genetic divergence ranged from 6.5 to 14.5% between G. c. canadensis and G. c. tabida, 0.5 to 6.6% within G. c. canadensis, and 0.1 to 6.0% within G. c. tabida. Sufficient DNA for analysis was obtained from shed feathers indicating a source of genetic material that does not require the capture or sacrifice of the birds. Other genetic markers and methods, including satellite telemetry, are required for obtaining detailed information on crane distributions as needed to establish effective management units for the MCP.

Biochemical Genetics

Polyphasic characterization of Aeromonas salmonicida isolates recovered from salmonid and non-salmonid fish

Michigan's fisheries rely primarily upon the hatchery propagation of salmonid fish for release in public waters. One limitation on the success of these efforts is the presence of bacterial pathogens, including Aeromonas salmonicida, the causative agent of furunculosis. This study was undertaken to determine the prevalence of A. salmonicida in Michigan fish, as well as to determine whether biochemical or gene sequence variability exists among Michigan isolates. A total of 2202 wild, feral and hatchery-propagated fish from Michigan were examined for the presence of A. salmonicida. The examined fish included Chinook salmon, Oncorhynchus tshawytscha (Walbaum), coho salmon, O. kisutcha (Walbaum), steelhead trout, O. mykiss (Walbaum), Atlantic salmon, Salmo salar L., brook trout, Salvelinus fontinalis (Mitchill), and yellow perch, Perca flavescens (Mitchill). Among these, 234 fish yielded a brown pigment-producing bacterium that was presumptively identified as A. salmonicida. Further phenotypic and phylogenetic analyses identified representative isolates as Aeromonas salmonicida subsp. salmonicida and revealed some genetic and biochemical variability. Logistic regression analyses showed that infection prevalence varied according to fish species/strain, year and gender, whereby Chinook salmon and females had the highest infection prevalence. Moreover, this pathogen was found in six fish species from eight sites, demonstrating its widespread nature within Michigan.

Journal of Fish Diseases

Inheritance of muscle and liver types of supernatant NADP-dependent isocitrate dehydrogenase in rainbow trout (Salmo gairdneri)

The genetics of allelic variation for NADP-dependent isocitrate dehydrogenase (IDH-s) found in the supernatant of liver and white muscle extracts of rainbow trout (Salmo gairdneri) was examined. Twenty progeny from each of 50 controlled matings were examined for IDH phenotypes. Progeny data clearly indicated that the IDH-s variation in the muscle is controlled by two loci—one fixed and one with two alleles producing molecules of different electrophoretic mobilities. IDH-s variation in the liver is controlled by two disomic loci which code for four alleles. No linkage between the loci controlling IDH-s in the liver and the loci controlling it in the muscle was detected.

Biochemical Genetics

On the origin of brucellosis in bison of Yellowstone National Park: a review

Brucellosis caused by Brucella abortus occurs in the free-ranging bison ( Bison bison ) of Yellowstone and Wood Buffalo National Parks and in elk ( Cervus elaphus ) of the Greater Yellowstone Area. As a result of nationwide bovine brucellosis eradication programs, states and provinces proximate to the national parks are considered free of bovine brucellosis. Thus, increased attention has been focused on the wildlife within these areas as potential reservoirs for transmission to cattle. Because the national parks are mandated as natural areas, the question has been raised as to whether Brucella abortus is endogenous or exogenous to bison, particularly for Yellowstone National Park. We synthesized diverse lines of inquiry, including the evolutionary history of both bison and Brucella , wild animals as Brucella hosts, biochemical and genetic information, behavioral characteristics of host and organism, and area history to develop an evaluation of the question for the National Park Service. All lines of inquiry indicated that the organism was introduced to North America with cattle, and that the introduction into the Yellowstone bison probably was directly from cattle shortly before 1917. Fistulous withers of horses was a less likely possibility. Elk on winter feedgrounds south of Yellowstone National Park apparently acquired the disease directly from cattle. Bison presently using Grand Teton National Park probably acquired brucellosis from feedground elk.

Idaho, Montana, Wyoming

Nonlethal evaluation of the physiological health of unionid mussels: Method for biopsy and glycogen analysis

In long-lived unionid mussels, many short-term measures of growth are of limited value. Changes in physiological condition may be an early indication of stress, because the increased energy demand associated with stress often results in a depletion of glycogen reserves, the principal storage form of carbohydrates in unionid mussels. Our goal was to nonlethally extract tissue from freshwater mussels and then to develop a rapid and dependable method for the analysis of glycogen in the tissue extracts. A biopsy technique was developed to remove between 5 and 10 mg of foot tissue in Amblema plicata plicata. The survival rate did not differ between biopsied and non-biopsied mussels during a 581-d observation period, demonstrating that the biopsy technique will allow nonlethal evaluation of the physiological condition of individual mussels through measurement of changes in contaminant, genetic, and biochemical indicators in tissue. We also modified the standard alkaline digestion and phenol-sulfuric acid analysis of glycogen for use on the small samples of biopsied tissue and to reduce analysis time and cost. We present quality control data, including method detection limits and estimates of precision and bias. The modified analytical method is rapid and accurate and has a method detection limit of 0.014 mg glycogen. Glycogen content in the biopsied samples was well above the method detection limit; it ranged from 0.09 to 0.36 mg, indicating that the method should be applicable to native mussels.

Journal of the North American Benthological Societ

Biochemical and molecular typing of Streptococcus iniae isolated from fish and human cases

Streptococcus iniae is an important bacterial pathogen of fish, causing up to 50% mortality in stocks, which has recently been associated with human infections. To determine whether S. iniae isolates from humans and fish are similar, the present authors examined the biochemical profiles and genetic relatedness of these isolates by random amplified polymorphic DNA (RAPD) analysis and repetitive primer polymerase chain reaction(REP PCR). The biochemical profiles differentiated between the human and fish isolates of S. iniae using pyrrolidonyl arylamidase, arginine dehydrogenase, ribose, β-glucoronidase and glycogen as markers. These biochemical results suggest that the fish and human S. iniae isolates are genetically different. However, RAPD and REP PCR do not have the discriminatory power to differentiate between these streptococcus isolates using five different RAPD primers and BoxA primer.

Journal of Fish Diseases

Variation in glycogen concentrations within mantle and foot tissue in Amblema plicata plicata: Implications for tissue biopsy sampling

With the development of techniques to non-lethally biopsy tissue from unionids, a new method is available to measure changes in biochemical, contaminant, and genetic constituents in this imperiled faunal group. However, before its widespread application, information on the variability of biochemical components within and among tissues needs to be evaluated. We measured glycogen concentrations in foot and mantle tissue in Amblema plicata plicata (Say, 1817) to determine if glycogen was evenly distributed within and between tissues and to determine which tissue might be more responsive to the stress associated with relocating mussels. Glycogen was measured in two groups of mussels: those sampled from their native environment (undisturbed mussels) and quickly frozen for analysis and those relocated into an artificial pond (relocated mussels) for 24 months before analysis. In both undisturbed and relocated mussels, glycogen concentrations were evenly distributed within foot, but not within mantle tissue. In mantle tissue, concentrations of glycogen varied about 2-fold among sections. In addition, glycogen varied significantly between tissues in undisturbed mussels, but not in relocated mussels. Twenty-four months after relocation, glycogen concentrations had declined by 80% in mantle tissue and by 56% in foot tissue relative to the undisturbed mussels. These data indicate that representative biopsy samples can be obtained from foot tissue, but not mantle tissue. We hypothesize that mantle tissue could be more responsive to the stress of relocation due to its high metabolic activity associated with shell formation.

American Malacological Bulletin

Population genetic studies of the sea otter ( Enhydra lutris ): A review and interpretation of available data

C urrent information about the utility of genetic markers for estimating population structuring in sea otters ( Enhydra lutris ) is reviewed. Analyses of spatial population structuring with biochemical and molecular genetic markers are discussed in the context of the species' ecology and history of exploitation. Studies that have used a diversity of genetic markers including allozymes, mitochondrial DNA (mtDNA), and multilocus minisatellites revealed that geographically spearated populations of sea otters are highly differentiated, though little evidence for phylogeographic structuring was suggested. Analyses of population relationships based on mtDNA haplotype frequency distribution suggested that populations can be separated into four major groups: (1) California; (2) Prince William Sound, Alaska; (3) Kodiak Island, Alaska, and islands of the Aleutian archipelago, including the Commander Islands; and (4) the Kuril Islands. Populations from locales separated by large geographic distances often shared haplotypes, suggesting recent common ancestry and some degree of historical gene flow. THe large differences among populations in nuclear and mtDNA gene frequency suggested strong constrains on contemporary gene flow and/or considerable drift in gene frequencies due to population bottlenecks. No evidence for microgeographic structuring was noted. Levels of genetic diversity within populations varied greatly across the species range but were not related to contemporary estimates of population size.

Conference Paper

Genetic management of endangered species at the Patuxent Wildlife Research Center

Summary: The Patuxent Wildlife Research Center conducts one of the world's largest and best-known research programs for captive propagation of endangered wildlife. In order to be effective and to ensure the long-term survival of species, researchers at Patuxent attempt to manage captive populations according to the principles of population genetics. This includes the use of estimated inbreeding levels for mate selections in Masked Bobwhites and biochemical analyses to measure extant genetic material and determine relationships among Whooping Cranes. As added insurance against catastrophic losses, or even random losses of key individuals representing unique lineages, cryopreservation of semen has been studied and used for some species. Artificial insemination, using either stored or fresh semen, is used to improve fertility rates, thereby increasing the chances for survival of unique genetic lines. Finally, a periodic influx of unrelated stock occurs, when feasible, in order to enhance the genetic base of captive populations. The application of these techniques will ensure that future releases utilize genetically viable animals, thereby improving the potential for successful reintroductions into the wild.

Book chapter

Mycoplasma agassizii sp., nov., isolated from the upper respiratory tract of the desert tortoise (Gopherus agassizii) and the gopher tortoise (Gopherus polyphemus).

Biochemical, serological and molecular genetic studies were performed on seven mycoplasma isolates that were recovered from the upper respiratory tract of clinically ill desert tortoises. The isolates were serologically related to each other but serologically distinct from previously described species. Unique mycoplasma species-specific 16S rRNA nucleotide sequences were found in the proposed type strain. The name Mycoplasma agassizii is proposed for these isolates. The type strain is PS6 T (=ATCC 700616 T ) which caused upper respiratory tract disease (URTD) in experimentally infected tortoises.

Nevada

Fish and wildlife species as sentinels of environmental endocrine disruption

This chapter provides an overview of the history and criteria for use of captive and free-ranging fish and wildlife (amphibians, reptiles, birds, and mammals) species as sentinels of potential environmental endocrine disruption. Biochemical, behavioral, physiological, immunological, genetic, reproductive, developmental, and ecological correlates of endocrine disruption in these sentinels are presented and reviewed. In addition, data needs to promote better use of sentinel species in the assessment of endocrine disruption are discussed.

Book chapter

Molt and aging criteria for four North American grassland passerines

Prairie and grassland habitats in central and western North America have declined substantially since settlement by Europeans (Knopf 1994) and many of the birds and other organisms that inhabit North American grasslands have experienced steep declines (Peterjohn and Sauer 1999; Johnson and Igl 1997; Sauer, Hines, and Fallon 2007). The species addressed here, Sprague’s Pipit (Anthus spragueii), Grasshopper (Ammodramus savannarum) and Baird’s (A. bairdii) sparrows, and Chestnut-collared Longspurs (Calcarius ornatus), are grassland birds that are of special conservation concern throughout their ranges due to declining populations and the loss of the specific grassland habitats required on both their breeding and wintering ranges (Knopf 1994, Davis and Sealy 1998, Davis 2003, Davis 2004, Jones and Dieni 2007). Population-trend data on grassland birds, while clearly showing declines, provides no information on the causes of population declines. Without demographic information (i.e., productivity and survivorship), there are no means to determine when in their life cycle the problems that are creating these declines are occurring, or to determine to what extent population trends are driven by factors that affect birth rates, death rates, or both (DeSante 1995). For migratory birds, population declines may be driven by factors on breeding grounds, during migration, and/or on wintering grounds. Lack of data on productivity and survivorship thus impedes the formulation of effective management and conservation strategies to reverse population declines (DeSante 1992). Furthermore, if deficiencies in survivorship are revealed, management strategies may need to address habitats on both breeding and non-breeding grounds, as well as along migratory pathways. One technique that helps inform management strategies is the biochemical analysis of isotopes and genetic markers, from the sampling of individual feathers from live birds (Smith et al. 2003, Pérez and Hobson 2006; Appendix). Determining demographic parameters and effectively sampling feathers to reveal connectivity between breeding and wintering grounds requires detailed knowledge of molt patterns and age determination criteria for the target species, in the hand. For example, productivity, survivorship, and territory acquisition may all be age-dependent, with first-year birds showing different patterns and responses than older birds. In many cases it may be possible to sample both a feather grown on the breeding grounds and one grown on the wintering grounds from a single individual, but knowledge of age-specific molt patterns, as well as an ability to recognize different feather generations, is needed to accomplish such a task. While some information on molt and aging criteria exists for grassland passerine species (Pyle 1997a), these species have been rarely captured during mark-recapture studies (Jones et al. 2007) and this information thus needs refining. There is a need for additional resources to assist field workers in determining molt patterns and age in captured individuals. Our objective is to describe molt and aging criteria for four grassland passerine species with the aid of digital photographs taken in the field. We hope that this document will be useful for researchers studying grassland species through capture and banding of live individuals on either the breeding or the wintering grounds. We present a general section on molt and aging techniques, followed by specific accounts for the four species treated: Sprague’s Pipits, Grasshopper and Baird sparrows, and Chestnut-collared Longspur. We also provide a brief protocol on collecting feather samples (Appendix).

Biological Technical Publication

Genetic analysis of paramyxovirus isolates from pacific salmon reveals two independently co-circulating lineages

Viruses with the morphological and biochemical characteristics of the family Paramyxoviridae (paramyxoviruses) have been isolated from adult salmon returning to rivers along the Pacific coast of North America since 1982. These Pacific salmon paramyxoviruses (PSPV), which have mainly been isolated from Chinook salmon Oncorhynchus tshawytscha, grow slowly in established fish cell lines and have not been associated with disease. Genetic analysis of a 505-base-pair region of the polymerase gene from 47 PsPV isolates produced 17 nucleotide sequence types that could be grouped into two major sublineages, designated A and B. The two independently co-circulating sublineages differed by 12.1-13.9% at the nucleotide level but by only 1.2% at the amino acid level. Isolates of PSPV from adult Pacific salmon returning to rivers from Alaska to California over a 25-year period showed little evidence of geographic or temporal grouping. Phylogenetic analyses revealed that these paramyxoviruses of Pacific salmon were most closely related to the Atlantic salmon paramyxovirus (ASPV) from Norway, having a maximum nucleotide diversity of 26.1 % and an amino acid diversity of 19.0%. When compared with homologous sequences of other paramyxoviruses, PSPV and ASPV were sufficiently distinct to suggest that they are not clearly members of any of the established genera in the family Paramyxoviridae. in the course of this study, a polymerase chain reaction assay was developed that can be used for confirmatory identification of PSPV. ?? Copyright by the American Fisheries Society 2008.

Journal of Aquatic Animal Health

Bioindicators used in aquatic and terrestrial monitoring

There are many different types of contaminants present in the environment ranging from synthetic chemicals, which would not be present in the environment without man's intervention, to trace metals that are required for life. Concerns range from possible harmful effects on flora and fauna to possible harm to humans consuming such organisms. The complete chemical analysis for all possible environmental contaminants in sediments, water, air, and every species and sample of interest would not only be excessively costly, but the facilities to handle do many samples do not exist. Such chemical analysis would only establish the presence of contaminants, without revealing how available or active they were within the organism. Bioindicators, sometimes called biomarkers , are responses in living organisms that may simply signify exposure to contaminants, may predict future harm, or may themselves be harmful effects. In this chapter, a bioindicator will be interpreted to mean the same as a biomarker (a biochemical, physiological, or morphological response), but not a population or ecosystem bioindicator such as a sentinel species, genetic variability, or species richness. thus, a bioindicator (biomarker) may be studied in a particular species to assess the status of that species, or in a sentinel species to assess habitat status. Individuals of the sentinel species might be those already residing at the site of interest or might be pristine organisms brought to the site and maintained in cages (fish) or released and recovered (birds).

Book chapter

Comparative analysis of Edwardsiella isolates from fish in the eastern United States identifies two distinct genetic taxa amongst organisms phenotypically classified as E. tarda

Edwardsiella tarda, a Gram-negative member of the family Enterobacteriaceae, has been implicated in significant losses in aquaculture facilities worldwide. Here, we assessed the intra-specific variability of E. tarda isolates from 4 different fish species in the eastern United States. Repetitive sequence mediated PCR (rep-PCR) using 4 different primer sets (ERIC I & II, ERIC II, BOX, and GTG 5 ) and multi-locus sequence analysis of 16S SSU rDNA, groEl, gyrA, gyrB, pho, pgi, pgm, and rpoA gene fragments identified two distinct genotypes of E. tarda (DNA group I; DNA group II). Isolates that fell into DNA group II demonstrated more similarity to E. ictaluri than DNA group I, which contained the reference E. tarda strain (ATCC #15947). Conventional PCR analysis using published E. tarda-specific primer sets yielded variable results, with several primer sets producing no observable amplification of target DNA from some isolates. Fluorometric determination of G + C content demonstrated 56.4% G + C content for DNA group I, 60.2% for DNA group II, and 58.4% for E. ictaluri. Surprisingly, these isolates were indistinguishable using conventional biochemical techniques, with all isolates demonstrating phenotypic characteristics consistent with E. tarda. Analysis using two commercial test kits identified multiple phenotypes, although no single metabolic characteristic could reliably discriminate between genetic groups. Additionally, anti-microbial susceptibility and fatty acid profiles did not demonstrate remarkable differences between groups. The significant genetic variation (<90% similarity at gyrA, gyrB, pho, phi and pgm; <40% similarity by rep-PCR) between these groups suggests organisms from DNA group II may represent an unrecognized, genetically distinct taxa of Edwardsiella that is phenotypically indistinguishable from E. tarda.

Veterinary Microbiology

Signatures of adaptive divergence among populations of an avian species of conservation

Understanding the genetic underpinning of adaptive divergence among populations is a key goal of evolutionary biology and conservation. Gunnison sage‐grouse ( Centrocercus minimus ) is a sagebrush obligate species with a constricted range consisting of seven discrete populations, each with distinctly different habitat and climatic conditions. Though geographically close, populations have low levels of natural gene flow resulting in relatively high levels of differentiation. Here, we use 15,033 SNP loci in genomic outlier analyses, genotype–environment association analyses, and gene ontology enrichment tests to examine patterns of putatively adaptive genetic differentiation in an avian species of conservation concern. We found 411 loci within 5 kbp of 289 putative genes associated with biological functions or pathways that were overrepresented in the assemblage of outlier SNPs. The identified gene set was enriched for cytochrome P450 gene family members (CYP4V2, CYP2R1, CYP2C23B, CYP4B1) and could impact metabolism of plant secondary metabolites, a critical challenge for sagebrush obligates. Additionally, the gene set was also enriched with members potentially involved in antiviral response (DEAD box helicase gene family and SETX). Our results provide a first look at local adaption for isolated populations of a single species and suggest adaptive divergence in multiple metabolic and biochemical pathways may be occurring. This information can be useful in managing this species of conservation concern, for example, to identify unique populations to conserve, avoid translocation or release of individuals that may swamp locally adapted genetic diversity, or guide habitat restoration efforts.

Arizona, Colorado, New Mexico, Utah