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Transition Metal Ion Uptake in Yeasts and Filamentous Fungi * Hydroxamates and Polycarboxylates as Iron Transport Agents (Siderophores) in Fungi * Enzymology of Siderophore Biosynthesis in Fungi * Molecular Biology of Iron Transport in Fungi * Reductive Iron Assimilation in Saccharomyces cerevisiae * Iron Storage in Fungi * Manganese: Function and Transport in Fungi * Uptake of Zinc by Fungi * Accumulation of Radionuclides in Fungi * Metal Ion Resistance and the Role of Metallothionein in Yeast * Cadystin (Phytochelatin) in Fungi * Molecular Genetic Analysis of Cadmium Tolerance in Schizosaccharomyces pombe * Superoxide Dismutases in Saccharomyces Cerevisiae * Evolution and Biological Roles of Fungal Superoxide Dismutases Ferrochelatase of Saccharomyces cerevisiae * Heme-Mediated Gene Regulation in Saccharomyces cerevisiae.

Detailed crown condition information, including numbers of broken branches ≥ 5 cm diameter, broken tops, and healthy branches, were recorded for 5434 living trees > 9 cm dbh from 603 ten-basal-area-factor prism plots (three per forest stand) at 201 random points (stands) throughout the ice damage region of northern New York State. Twenty five percent of the sample stands had ≥ 20% branch breakage. Bigtooth aspen, red oak, red maple, and white pine had the most breakage. Comparison of potential mortality of trees associated with ≥ 75% ice damage (severe damage) to baseline (predicted) mortality to maintain the existing forest structure suggests that ice damage may alter the health of 18% of the forest stands but this is not sufficient to alter the health (sustainability) of the larger forest system. Key words: ice storm, forest health, sustainability, growth, mortality, dbh classes

Three aspen (Populus tremuloides Michx.) clones were grown hydroponically in growth chambers and subjected daily to 6 h of drought stress by exposing the roots to air. Relative to that of unstressed control plants, growth of the drought-stressed plants was severely reduced, but the leaves remained green and healthy. Amino acid concentrations varied among clones and organs. Drought stress caused both organ-specific and clone-specific changes in amino acid concentrations. Among organs, succulent stems usually had the highest concentrations of amino acids in both control and drought-stressed plants. Glutamate, glutamine, asparagine, arginine, and an unidentified amino acid were usually the major amino acids in all organs, except root tips where y-aminobutyrate replaced arginine as one of the most abundant amino acids. Proline was a minor constituent in both control and drought-stressed plants. Comparison of the results of the present study with those of earlier studies with the same aspen clones indicates that the method of inducing drought stress affects the responses observed.

We have developed an automated microprocessor controlled system for subjecting hydroponically grown plants to drought. Pumps and valves were used to move nutrient solutions into and out of a system of culture vessels in a growth chamber to provide periods of drought. Drought conditions were obtained by exposing the roots of hydroponically grown clones of aspen, Populus tremuloides Michx., to air in culture vessels temporarily emptied of nutrient medium. Over a 3-week period, the daily duration of drought was increased from 0 to 6 h. During this period, the plants became increasingly tolerant to drought, as shown by a decreasing propensity to wilt. All three clones sustained diurnal drought periods of 6 h for up to 5 weeks without detectable deterioration of health. Typical drought stress symptoms were observed including inhibition of growth, increased tissue amino acid content, and decreased water, solute, and turgor potentials in young leaves. In all clones, control plants had leaf water potentials between -1.0 and -1.6 MPa, whereas leaf water potentials of drought-treated plants were significantly lower, ranging from -1.7 to -3.0 MPa. Only one of the clones showed a significant decrease in leaf solute potential in response to drought. The decrease in leaf solute potential paralleled the decrease in water potential resulting in no significant difference in turgor potential. The other two clones had nonsignificant decreases to more negative leaf solute potentials under drought conditions resulting in significantly lowered turgor potentials. Leaf water potentials, solute potentials, and turgor potentials of the drought-treated plants returned to control values within two hours after rewatering. The growth inhibitions observed could not have been the consequence of loss of turgor. These results demonstrate genetic differences among aspen clones in water relations responses to drought.

The enzymatic conversion of xanthoxin to abscisic acid by cell-free extracts of Phaseolus vulgaris L. leaves has been found to be a two-step reaction catalyzed by two different enzymes. Xanthoxin was first converted to abscisic aldehyde followed by conversion of the latter to abscisic acid. The enzyme activity catalyzing the synthesis of abscisic aldehyde from xanthoxin (xanthoxin oxidase) was present in cell-free leaf extracts from both wild type and the abscisic acid-deficient molybdopterin cofactor mutant, Az34 (nar2a) of Hordeum vulgare L. However, the enzyme activity catalyzing the synthesis of abscisic acid from abscisic aldehyde (abscisic aldehyde oxidase) was present only in extracts of the wild type and no activity could be detected in either turgid or water stressed leaf extracts of the Az34 mutant. Furthermore, the wilty tomato mutants, sitiens and flacca, which do not accumulate abscisic acid in response to water stress, have been shown to lack abscisic aldehyde oxidase activity. When this enzyme fraction was isolated from leaf extracts of P. vulgaris L. and added to extracts prepared from sitiens and flacca, xanthoxin was converted to abscisic acid. Abscisic aldehyde oxidase has been purified about 145-fold from P. vulgaris L. leaves. It exhibited optimum catalytic activity at pH 7.25 in potassium phosphate buffer.

Plantlets from five clones of Populus tremuloides were cultured in media at three different osmotic potentials to assess the effects of water stress on amino acid content. Differences in amino acid content were then related to differences in susceptibility (determined in a previous study) of clones to Hypoxylon mammatum. Changes in amino acid concentrations varied among the amino acids. Alanine, arginine, asparagine, glutamine, glutamate, and proline increased generally but not consistently among all clones. Comparisons among the clones for these six amino acids showed significant variations. Proline, which has been shown to accelerate radial growth of H. mammatum more than other amino acids, increased progressively with increased water stress in all clones. Levels of proline among the clones were correlated (r = 0.89) with susceptibility to infection and colonization of the clones by H. mammatum. Alanine and glutamine were also similarly correlated with infection rating. However, one clone, which grew poorly in culture, did not show this relationship. In comparison with the other clones, it had low levels of all amino acids, which did not increase significantly in response to water stress. Yet in the inoculation study, it had the highest infection rating at all stress levels, suggesting that other stress-induced factors are also important. These results suggest that nitrogen status of some aspen clones under water stress may increase their susceptibility to hypoxylon canker.

The firm of KPMG, LLP (KPMG) and the Defense Contract Audit Agency (DCAA) performed the single audit for the Illinois Institute of Technology Research Institute (IITRI), a non-profit research organization located in Chicago, Illinois. The Office of Management and Budget (0MB) Circular No. A-133, "Audits of States, Local Governments, and Non-Profit Organizations," (0MB Circular A-133) requires the audit. IITRI expended approximately $97.1 million of Federal awards, of which $75.5 million was for DoD) and $21.6 million for other Federal agencies, during the fiscal year that ended September 30, 1997. Quality Control Review Objective. As the Federal cognizant agency for IITRI, the Office of the Inspector General, DoD, performed a quality control review to determine whether the FY 1997 report that IITRI submitted to the Single Audit Clearinghouse met the applicable reporting standards and whether KPMG and DCAA conducted the audit as applicable standards and 0MB Circular A-133 require. See Appendix A for a discussion of the scope of this review and the requirements for an 0MB Circular A-133 single audit.

Hypoxylon canker of aspen has many of the features of an ideal system for studying woody plant defense mechanisms against fungi. The primary hosts, trembling and European aspens (Populus tremuloides and P. tremula) occur over broad geographic areas of the northern hemisphere, providing an immense array of genetic and environmental diversity for the development of this system. Less commonly affected hosts such as P. grandidentata, P. trichocarpa, P. balsamifera, P. alba, and others provide a foundation of plant materials with different levels of resistance and susceptibility. The capacity to generate hybrids among these and other nonhost poplars further expands the foundations of test plant materials.

Two lines of evidence, population structure and conidial germination properties, support the hypothesis that ascospores and not conidia disperse the fungus in the disease cycle of hypoxylon canker of aspen. Mycelial interactions, identified by the formation of a conidial interface between genetically different isolates, provided phenotypic markers for determining population structure in Hypoxylon mammatum. Single ascospore isolates from 14 asci from six perithecia found on one canker were tested for interactions (...)

Variation among isolates ofHypoxylon mammatum caused by amino acids added as sole nitrogen sources demonstrated genetic heterogeneity among the population in the mechanisms regulating hyphal growth rates. Isolates were obtained by a random sample consisting of 22 single ascospore isolates from a local population of hypoxylon cankers onP. tremuloides. Hyphal growth rates were determined from colony diameter measurements on defined media containing either alanine, asparagine, glycine, leucine, lysine, or proline. All isolates but two grew faster on proline than the other amino acids tested. The mean growth rate of the population sample was 3.9 mm day−1 on proline compared to 1.96 mm day−1 on asparagine, the second fastest mean growth rate. Growth rates of the 22 isolates on these six amino acids were largely uncorrelated, indicating independent mechanisms of regulation. Only asparagine gave significant correlations with more than two other amino acids. A more detailed examination of selected isolates representing a range of growth rates on proline showed that the rapid growth rates were also dependent on other materials present in Difco Bacto-Agar. Growth of the proline stimulated isolates was considerably slower on media gelled with Noble agar. Other nitrogen sources, especially glutamate, added with proline stimulated the growth of these isolates. Isolate dependent variation in the stimulatory effect of the N source added with proline was demonstrated. Stimulation of growth rate by proline may be related to drought stress-enhanced canker elongationin vivo, since drought stress caused proline accumulation in the host,Populus tremuloides.

Inhibitors of protein synthesis (cycloheximide, dl -p- fluorophenylalanine , puromycin, and blasticidin-S) were tested for their effect on the in vivo synthesis of both whole cell and ribosomal RNA by the water mold Achlya bisexualis. Whereas none of the drugs affected the uptake of [5-3H]uridine, all resulted in a decrease in the rate of accumulation of this precursor into acidinsoluble polynucleotides. Analysis of uridine incorporation into rRNA showed that, while dl -p- fluorophenylalanine , puromycin and blasticidin-S inhibited synthesis gradually over a period of 30–40 min, cycloheximide resulted in complete cessation of synthesis within 2.5 min. In the presence of each of the inhibitors except cycloheximide the capacity of the cells to synthesize RNA, as measured by incorporation of uridine into either acid-insoluble nucleic acid or into ribosomes, decays rapidly following pseudo-first-order kinetics, with a half-life of between 10 and 23 min. In the presence of cycloheximide, synthetic capacity decays with a half-life of 1.4 min. These results are interpreted to mean that in Achlya (1) there is a rapidly turning over protein requisite to rRNA synthesis, and (2) the effect on rRNA synthesis elicited by cycloheximide is not exclusively the result of inhibition of protein synthesis.

A B S T R A C T Parental crosses, backcrosses, repeated backcrosses, and sib matings with several strains of Achlya bisexualis Coker and Couch have been made. Progeny were tested for sexual reaction with the parental strains. The distribution of sex observed could not be explained on the basis of any simple gene or chromosomal mechanism. Two factors were suggested which may affect the distribution of sex. They are selfing in the female and the complexity of the hormonal system which controls the expression of sex. The inheritance of a cycloheximideresistant mutant argues against selfing; however, further studies with other genetic markers

Analogs of the glutarimide antibiotic cycloheximide were tested for their effect on growth and incorporation of proline and uridine into acid-insoluble material in Achlya bisexualis. Each of the compounds tested had reduced antibiotic activity as compared to cycloheximide. The effects of the antibiotics on protein and RNA synthesis were varied. While cycloheximide inhibited both protein and RNA synthesis immediately, two of the analogs inhibited proline incorporation without effect on uridine incorporation, while three, each representing a modification of the hydroxyl of cycloheximide, stimulated uridine incorporation and either had no effect on or inhibited protein synthesis. These results indicate that the control of RNA synthesis by protein synthesis in Achlya can be released by glutarimide antibiotics.

Resistant sporangia of Blastocladiella emerson?i were induced by the addition of bicarbonate, potassium chloride, sodium chloride, or ammonium chloride to the medium and by the exposure of the zoospores to ultraviolet irradiation. Mature resistant sporangia induced by all of these conditions exhibit similar areolate wall pitting. Under suitable conditions resistant sporangia in all cases examined germinated with the cracking of the outer sporangial wall, with the formation of exit tubes by the inner sporangial wall, and with the cleavage and release of zoospores through discharge papillae formed in the tips of the exit tubes. BLASTOCLADIELLA EMERSONII Cant. & Hyatt, a non-filamentous water mold in the order Blastocladiales, has been the object of many morphogenetic and biochemical studies (Cantino and Lovett, 1964). The zoospores grow into thalli, each producing a single sporangium of either of two types. The resistant sporangia (RS) have thick, pitted walls and are orange-brown in color. The ordinary colorless sporangia (OC) have thin, non-pitted walls and no pigmentation. Under suitable conditions both types of sporangia germinate, cleaving their protoplasts into many tiny zoospores which escape through discharge papillae formed in the sporangial wall. Cantino (1951) showed that the addition of bicarbonate to a medium which otherwise supports the development of OC causes RS to develop instead. Griffin (1965a) showed that potassium chloride in suitable concentrations would also induce the formation of resistant sporangia. In a preliminary report (Griffin, 1965b) it was further shown that sodium and ammonium chlorides are equally as effective as potassium chloride in the induction of RS formation. These data are presented here in full for the first time. Lovett (personal communication) discovered that if a suspension of zoospores is exposed to ultraviolet light and then allowed to develop on an agar medium, a large percentage of the first-generation thalli will form resistant sporangia. This study shows that the effect of potassium chloride in inducing the formation of resistant sporangia is not specific and that sodium and ammonium chlorides are equally effective. The effect of these salts is not purely an osmotic phenomenon, since sucrose added in concentra1 Received for publication 13 May 1968. Supported in part by National Science Foundation Grant GB4747 to the junior author. 2 Present address: Department of Forest Botany, State University College of Forestry, Syracuse, N. Y. 13210. tions up to the point w-here the spores can no. longer grow does not cause the formation of RS. Resistant sporangia formed under all known conditions of induction, bicarbonate, chlorides of potassium, sodium and ammonium, and ultraviolet irradiation of the zoospores are essentially alike in wall sculpturing and germination behavior. MATERIALS AND METHODS-Cultures of B. emersonii (Berkeley Culture Collection strain 49-1) were used throughout this study. This is a subculture of the original isolate (Cantino and Hyatt, 1953). Stock cultures were maintained on PYG agar containing peptone (Difco), 1.25 g;yeast extract (Difco), 1.25 g; glucose (Fisher), 3.0 g; and agar (Difco), 15 g per liter of water. Experiments to test the specificity of salts in inducing RS formation were performned usinga medium consisting of yeast extract (Difco), 1.25 g; glucose (Fisher), 3.0 g; and agar (Difco), 15 g per liter of water, and salts as specified in the text. RS grown for photography and germination were grown on medium PYG supplemented with either KC1 3.5 X 10-2M, NaCl 7.5 X 102-m, NH4Cl 7.5 X 10-2M, or KHCO3 1.0 X 10-2M before autoclaving. For obtaining ultraviolet-induced RS a suspension of zoospores, was spread evenly over the surface of a PYG agarplate and irradiated for 10 sec at a distance of 15 cm with a UVS-11 short wave ultraviolet lamp (Ultraviolet Products, Inc., San Gabriel, Calif.). Individual RS thalli were removed from theagar with a fine needle and water mounts weremade for observation and photography. To obtain Fig. 1-6.-Fig. 1-3. Mature RS showing areolate pitting. -Fig. 1. HCO3-grown.-Fig. 2. KCl-grown.-Fig. 3. U1traviolet-induced.-Fig. 4-6. Germinating RS showing cracked wall.-Fig. 4. HCO3-grown.-Fig. 5. KCI-grown. -Fig. 6. Ultraviolet-induced.--Magnification of all, X560.

Studies of cytochrome oxidase in isolated mitochondria of Blastocladiella emersonii Cant. and Hyatt show that the enzyme was present in zoospores and throughout the development of ordinary colorless sporangia and of resistant sporangia. The enzyme activity was present in KCl, NaCl, NH(4)Cl, and KHCO(3) induced resistant sporangia, and was shown to be as active or more active than the enzyme found in ordinary colorless sporangia and zoospores. Interfering substances causing difficulties in the measurement of cytochrome oxidase activity were found in whole cell homogenates of KHCO(3) grown resistant sporangia, but not in KCl, NaCl, or NH(4)Cl grown thalli. These substances could be removed by dialysis or by sedimentation of the mitochondria.

Some factors controlling the differentiation of sporangia and gemmae in an isolate of Achlya sp. were investigated by replacement culture technique. Factors in the replacement medium which affect differentiation were as follows: A) Calcium is required for the differentiation of both sporangia and gemmae. B) Calcium cannot be replaced by magnesium, potassium or sodium. C) Gemmae are induced when the replacement medium contains salt at 40 to 80 mm. D) The salt induction of gemmae is nonspecific, sodium, potassium, magnesium and calcium chlorides are equally effective.

Multiple forms of RNA polymerase have been reported from representatives of six classes of fungi. The number of forms reported varies from two to four. The general properties of these forms of RNA polymerase suggest that they are very similar to the multiple forms of RNA polymerase reported from higher plants and animals. All species studied have at least one polymerase sensitive to α-samanitin. All forms are more active in the presence of manganese than in the presence of magnesium. Where studied, the fungal enzymes have high molecular weight and multiple subunits.

Two in vitro procedures are being tested for screening trembling aspen for resistance to Hypoxylon canker: (1) tissue culture response to fungal culture metabolites and (2) ascospore inoculation of moisture stressed plantlets. In the first procedure, the sensitivity of five clones to the acidic ethyl acetate (AEA) fraction of partially purified culture filtrate was determined by comparing callus growth on ACM medium containing the AEA fraction with that of a control containing non-inoculated culture filtrate. Treatments of 0.1x and 1.0x AEA concentrations were inhibitory compared with controls. The clones varied considerably in callus growth and callus inhibition, with the inhibition relative to growth of the control directly related to that growth. Correlations of the resistance responses of 10 year old ramets to artificial inoculations with mycelial-infested grain show a moderate positive correlation of both callus growth of the control and callus inhibition with canker length and with incidence of branch death.

During the synchronous differentiation of sporangia in the absence of added nutrients, the water mold Achlya bisexualis (Coker and Couch) actively synthesized protein. Inhibition of protein synthesis at any time during the sporulation process completely inhibited further differentiation. Large changes in the rate of radioactive amino acid uptake resulted in changes in the specific activity of the cellular amino acid pool. The rate of protein synthesis was calculated from the amino acid pool specific activity and the incorporation of isotope into protein. During the 1st h after induction of the sporulation process, the rate of protein synthesis increased to two times the initial value. The amino acid precursors for this synthesis were supplied by the degradation of preexisting protein. Proteolytic enzyme activity assayed in vitro increased in proportion to the in vivo rates of protein synthesis and degradation. Differentiation was accompanied by a slight decline in dry weight of the mycelium as well as by a decrease in the protein content, whereas the relative size of the amino acid pools remained constant.

Specific inhibitors of each of the three RNA polymerases of Blastocladiella emersonii have been found. Cycloheximide specifically inhibited the in vitro activity of the DEAE-fraction I enzyme, alpha-amanitin specifically inhibited the DEAE-fraction II enzyme, and rifampicin specifically inhibited the fraction III enzyme. DNA stimulation and dependency on the four riboside triphosphates were shown to be characteristic of each of the three fractions. Optimum concentrations of magnesium ions required were shown to differ among the three fractions and to be somewhat higher than optimum concentrations of manganese ions. The effect of pH on activity was essentially identical for each of the three fractions. Kinetic experiments and nuclease assays indicated the presence of some interfering substances in the partially purified RNA polymerase fractions.

MULTIPLE RNA polymerases have been shown to exist in a wide variety of eukaryotic organisms1–5. Two nuclear polymerases have been found in all the cells studied, each with a specific location and a specific function: the DEAE fraction I enzyme is located in the nucleolus and may be involved in the synthesis of ribosomal RNA1,2,5,6; the DEAE fraction II enzyme is located in the non-nucleolar nucleoplasm and functions in the synthesis of DNA-like RNA2–5,7. The DEAE fraction III enzyme was reported to exist in sea urchin1, the aquatic fungus B. emersonii5 and to be present sometimes in rat liver preparations1,8. Although there have been some reports that polymerase III is nuclear, Horgen and Griffin5 showed that the enzyme was sensitive to the prokaryotic RNA polymerase inhibitor rifampicin. They suggested that the fraction III enzyme may be mitochondrial, formed as the result of organelle contamination in their crude nuclear preparations. The results of this study show that the DEAE fraction III enzyme in B. emersonii is a mitochondrial enzyme, most likely functioning in the synthesis of mitochondrial RNA. The rifampicin sensitivity of the enzyme is further evidence of a prokaryotic origin of mitochondria9,10.

Cankers low on the bole were associated with mortality. Small stems had more cankers below the lowest living branch. The frequency of cankers above the lowest living branch increased with tree size, suggesting that the infection court occurs on branches. With increasing tree size, cankers on branches originate farther away from the bole and fewer cankers cause mortality. Considerable variation existed in disease incidence and mortality among the clones and four sample locations

The inhibition of indoleacetic acid oxidase activity by chlorogenic acid is markedly variable. Aqueous extracts of acetone precipitates from etiolated pea epicotyls contain heat-stable substances which decrease the inhibition produced by chlorogenic acid and also have a small activating effect. Low concentrations of pcoumaric acid (1 micro/ml) can stimulate this action. The possible role of activators and inhibitors in the in vivo regulation of indoleacetic acid oxidase activity is discussed.