GENETIC CONTROL OF CONIDIATION IN ASPERGILLUS RUGULOSUS

The potential for the production of abundant conidia was suggested when an essentially non-conidial isolate of Aspergillus rugulosus, obtained from dune sand, was grown at 25 C rather than at the usual 37 C. Ultraviolet-light-induced conidial strains were obtained from the original non-conidial isolate. Evidence is presented which suggests that the production of conidia is under the control of a single nuclear gene. The data, with a single exception, suggest that induced conidial mutations represent alterations at the same locus. Crosses with isolates from various localities indicate that the genes controlling conidiation in these strains may be linked to or independent of the genes studied in the original dune-sand isolate. No evidence has been obtained which would suggest involvement of a diffusible substance in the control of conidiation.     

Malonate Metabolism, Stimulation of Conidiation and Succinate Dehydrogenase Activity in Neurospora crassa

Malonate was studied for its effect on succinate dehydrogenase activity and conidiation. It Was found to stimulate the succinate dehydrogenase activity and also conidiation of Neurospora crassa. The efficiency of sucrose metabolization for cellular synthesis was improved in malonate supplemented cultures. High Concentration (0.5 M) had a distinct toxic effect on conidiation and economic efficiency. Teratological structures were observed at 0.5 M malonate concentration. A stimulation of the glyoxylate cycle is considered to be one of the factors responsible for the conidiogenic effect.     

Submerged culture conidial germination and conidiation of the bioherbicideColletotrichum truncatum are influenced by the amino acid composition of the medium

Summary Submerged culture experiments were conducted to determine the optimal nitrogen source for rapidly producing conidia of the bioherbicide,Colletotrichum truncatum. Germination ofC. truncatum conidial inocula in submerged culture occurred most rapidly (>95% in 6 h) in media provided with a complete complement of amino acids. When (NH₄)₂SO₄, urea, or individual amino acids were provided as the sole nitrogen source, conidial germination was less than 20% after 6 h incubation. Conidia production was delayed inC. truncatum cultures grown in media with urea or individual amino acids as nitrogen sources compared to cultures supplied with Casamino acids or complete synthetic amino acid nitrogen sources. The use of methionine, lysine, tryptophan, isoleucine, leucine or cysteine as a sole nitrogen source severely inhibitedC. truncatum conidia production. Media with synthetic amino acid mixtures less these inhibitory amino acids produced significantly higher conidia yields compared to media with amino acid mixtures containing these amino acids. When various amounts of each individual inhibitory amino acid were added to media which contained amino acid mixtures, cysteine and methionine were shown to be most effective in reducing conidiation. An optimal nitrogen source forC. truncatum conidiation in submerged culture should contain a complete mixture of amino acids with low levels of cysteine, methionine, leucine, isoleucine, lysine and tryptophan for rapid conidiation and optimal conidia yield.The mention of firm names or trade products does not imply that they are endorsed or recommended by the US Department of Agriculture over other firms or similar products not mentioned.     

Development of a submerged-liquid sporulation medium for the johnsongrass bioherbicide<Emphasis Type="Italic"> Gloeocercospora sorghi</Emphasis>

Submerged culture experiments were conducted in three phases to determine the optimal medium for rapidly producing conidia of the fungal bioherbicide Gloeocercospora sorghi. In phase I, 18 crude carbon sources were evaluated to determine which would support sporulation. Under the conditions tested, butter bean and lima bean brines (1.5–4.6 mS/cm) provided best conidiation. In phase II, a fractional-factorial design was utilized to screen 76 different medium adjuncts in combination with butter bean brine for improved sporulation. d-Mannitol and carboxymethylcellulose (CMC) were the only acceptable factors that resulted in a significant improvement. In phase III, a central composite design with response surface methodology was used to optimize concentrations of these critical factors. The model predicted optimal sporulation in a medium composed of 2.69 mS/cm butter bean brine +0.043 M d-mannitol +0.37% w/v CMC with an expected titer of 1.51×10⁷ conidia/ml. Actual mean titer attained with the model-derived medium was 1.91×10⁷ conidia/ml. Optimal sporulation occurred at 25.5°C in this medium and conidia remained viable up to 2.71 days when stored at 12°C. No significant difference was observed in virulence of conidia produced on agar vs washed conidia produced in the model-derived (liquid) medium.     

Studies of the functional activity of mitochondria and cell ultrastructure of the coleoptile in wheat,Agropyron and their hybrids (incomplete amphidiploids) during decreasing temperature

Summary The structure of the mitochondria and endoplasmic reticulum in the coleoptile of plants (Triticum aestivum var. Lutescens 329, Agropyron glaucum, Triticum × Agropyron 56-chromosome hybrids, incomplete amphidiploids, TAH, containing 42 wheat chromosomes and 14 chromosomes of genomes D or X of Agropyron), which differ in winterhardiness, was studied after exposure to 0 ° and -4 °C for periods varying from 10 min to 4 days. The functional activity of mitochondria isolated from 3 day old seedlings was also investigated in these cereals. The cells of Triticum and Agropyron seedlings grown at 23 °C were shown to differ in mitochondrial structure. In the cells of TAH both kinds of mitochondria were found.On day 4 of exposure to -4 °C, the mitochondria of Agropyron cells were not changed; the endoplasmic reticulum formed complex closed cavities. Under similar conditions most wheat mitochondria were destroyed and in the rest no cristae were observed.Morphometric analysis indicated that the volume of such mitochondria increases by two times, while the surface area of the internal membranes and cristae decreases by 1.54 times. In such cells, the endoplasmic reticulum is represented only by membranes of the smooth type. The structure of the mitochondria and endoplasmic reticulum in the seedling cells of TAH 829, which is more like Agropyron in winterhardiness, is similar to that of Agropyron cells; in hybrid 822 (more like wheat containing the D genome), changes arise resembling those observed in wheat. The existence of different types of mitochondria in seedling cells of TAH is especially distinct at low temperatures.The mitochondria of the cereals studied differ in biochemical activity after low temperature treatment (0 ° and — 4 °C). Phosphorylative and oxidative activity of mitochondria of the winterhardy forms (Agropyron glaucum, TAH 829) decreases just after the beginning of low temperature treatment. At the same time, the morphology of the mitochondria undergoes reversible changes. The mitochondria of cold-susceptible forms of wintering plants (Triticum aestivum, TAH 822) do not conform to this pattern. Under long-term low temperature treatment they display irreversibly damaged mitochondria. It is suggested that the winterhardy forms have high adaptability connected with a rapid protective response of the cell mitochondria and endoplasmic reticulum. This adaptability is regulated by nuclear genes: TAH have different mitochondria in the coleoptile cells; if genome X of Agropyron is present, which TAH derives from the male parent, the related mitochondria become more resistant to low temperature treatment.     

Heat-induced changes in respiratory pathways and mitochondrial structure during microcycle conidiation of Neurospora crassa

Changes in both respiratory pathways and mitochondrial structure of Neurospora crassa occurred under conditions of microcycle conidiation. Upon heat-treatment at 46°C, conidia developed a highly cyanide-insensitive, hydroxamate-sensitive respiration associated with morphological alterations in mitochondrial membranes; such changes were time-dependent. When heat-treated conidia were shifted down to 25°C, the alternate, hydroxamate-sensitive respiration decreased significantly, paralleling the recovery of well-cristated mitochondria with an electron-dense matrix in the germ tubes. The decrease in hydroxamate-sensitivity was associated with two periods of increase in cyanide sensitivity corresponding to the events of germination and precocious proconidial budding.     

Hepatic organelle interaction. IV. Mechanism of succinate enhancement of formaldehyde accumulation from endoplasmic reticulum N-dealkylations

Further evidence for organelle interaction during drug metabolism by the liver is presented. The apparent stimulation by succinate of formaldehyde accumulation in the medium, which was reported to occur with liver slices and homogenates as well as with mitochondria plus microsomes, has been shown to be the result of succinate inhibition of mitochondrial aldehyde dehydrogenase. The mechanism of succinate inhibition is shown to be by reverse electron transport, and an increase in the NADH to NAD+ ratio in the mitochondria; the aldehyde dehydrogenase requires the oxidized form of the pyridine nucleotide as its cofactor. Studies on in vitro N-demethylation by liver microsomes and endoplasmic reticulum segments which cosediment with the mitochondria indicate that formaldehyde produced by the mixed function oxidase is handled differently from formaldehyde added to the medium. The latter is mainly retained in the medium containing 5 mM semicarbazide, while the generated formaldehyde is more than 50% consumed by the mitochondria. Electron microscopy has indicated that the microsomes and the endoplasmic reticulum fragments have a tendency to align themselves close to the mitochondria when present in the same medium. Consequently, it is possible that formaldehyde released to the medium adjacent to the mitochondria, as by N-demethylation, would be exposed to semicarbazide for shorter periods than that added directly to the medium. In agreement with this suggestion, complexing of formaldehyde with semicarbazide was observed spectroscopically not to be an extremely rapid reaction even at 37 degrees C. This is believed to be the reason for the greater extent of consumption of formaldehyde generated by the endoplasmic reticulum.     

Successful induction and recognition of conidiation,conidial germination and chlamydospore formation in pure culture of Morchella

Morels, fungi from the genus Morchella, are popular edible mushrooms. However, little knowledge of their asexual reproduction and inaccessible pure mitospores hamper illumination of their life cycle. Herein, we successfully induced conidiation, conidial germination and chlamydospore formation in pure culture of Morchella sextelata. Conidiation proceeded via four morphologically distinct stages: development of the conidiophore stalk, stalk branching, phialide differentiation, and conidium production. Terminal and intercalary chlamydospores were formed on conidial hyphae. The development of conidiophores occurred earlier, with more conidia produced, in cross-mating cultures than in single-spore cultures. Mature conidia were spherical and 2.5–8 μm in diameter, with a vast majority (nearly 99%) 2.5–5 μm in diameter. Each conidium contained one to three nuclei (80.2% conidia contained one nucleus, 19.1% contained two nuclei, and 0.7% contained three nuclei). The conidial nucleus diameter was 1–2 μm. The nuclear mitosis in detached conidia that was observed may benefit their colony initiation. Additionally, morel conidia formed conidial anastomosis tubes. Conidia (mitospores) likely not only function as spermatia, but also as reproductive propagules in Morchella. Further research is imperative to elucidate the relationship between the conidia and chlamydospores, and their unique function in the morel life cycle.     

Some required events in conidial germination of Neurospora crassa.

A temperature-sensitive mutant of Neurospora crassa, with reduced levels of protein synthesis at 37°C, was used to identify some essential events in conidial germination. Conidia of mutant strain psi-1 were incubated for 2 hr at 37°C and then shifted to 20°C. Germination was inhibited at 37°C, but commenced after 1.5 hr at 20°C. Increases in aspartate transcarbamylase activity, cell wall synthesis, and nuclear number preceded germination. However, increases in glutamate dehydrogenase activity, amino acid uptake, and DNA synthesis were inhibited prior to germination. Although all of these events were correlated with germination in control cultures of the mutant at 20°C and of its parent strain at 20 and 37°C, some events were apparently not essential for germination. The requirement for aspartate transcarbamylase activity was demonstrated independently by the failure of strain pyr-3d (lacking the activity) to germinate in the absence of uridine. The dispensability of glutamate dehydrogenase activity and DNA synthesis for the germination of some conidia was verified by the germination of strain am-1 (lacking glutamate dehydrogenase activity) in the absence of glutamate and by the germination of the parent strain in the presence of hydroxyurea (an inhibitor of DNA synthesis). These findings identify some landmarks in germination which may be useful in further studies of the regulation of a developmental program. They also provide preliminary evidence that the resting conidia may contain nuclei arrested at different stages of their division cycle.     

Oxidations in kidney mitochondria of heat-exposed rats: Regulation by cytochromec

Exposure of rats to higher environmental temperature (36–37°C) decreased the capacity of their kidney mitochondria to oxidize succinate. The decrease was corrected on the addition of exogenous cytochromec. Kidney mitochondria of heat-exposed animals showed decreased rates of H₂O₂ generation when -glycerophosphate, but not succinate, was used as electron donor. These mitochondria also showed decreased activity of -glycerophosphate dehydrogenase but not of succinate dehydrogenase. The content of cytochromec in kidney mitochondria of heat-exposed animals was low even though the concentration of the pigment in the whole tissue did not decrease. Starvation as well as administration of an antithyroid agent like propylthiouracil simulated some of the effects of heat exposure on kidney mitochondria, but the cytochromec-dependent reversal of inhibition of oxidation was obtained only in heat exposure.     

Development of Media and Automated Liquid Fermentation Methods to Produce Desiccation-Tolerant Propagules ofTrichoderma harzianum

A suitable medium was developed from modified Richard's medium plus V8 juice (RM8) to produce high levels of desiccation-tolerant conidia ofTrichoderma harzianumstrain 1295-22. The addition of 9% (v/v) glycerol to RM8 improved both biomass production and desiccation tolerance of the conidia ofT. harzianum.This medium was then used in a laboratory scale fermenter (1.5 liter) to determine optimal operating conditions. The optimal temperature for conidial production and desiccation tolerance improvement in the fermenter was 32°C when dissolved oxygen was maintained at 50% saturation of air, and the stirring rate was 1000 revolutions per minute. The initial water potential of the medium (with 9% glycerol) was −3.7 MPa, the pH was 6, and neither was controlled during fermentation. Changes in medium pH and dissolved oxygen were associated with the stages of morphological development and conidiation. The pH of the medium decreased concurrently with germ-tube elongation and mycelium development and then increased to 6.0–6.2 at phialide formation. Intensive conidiation occurred at pH 6.3–6.5 and reached its maximal level at 6.9–7.1. Changes in pH values could be used as indicators to monitor the morphological development and conidiation ofT. harzianumduring fermentation. The use of a 48-h-old culture inoculum, rather than conidial inoculum, to start fermentation reduced the time required to complete the shift from vegetative growth to phialide formation. Intensive conidiation occurred immediately after the addition of culture inoculum and reached maximum levels within 68 h of fermentation. Dry weight of biomass increased with the duration of fermentation and was greatest at 96 h. However, no improvements in conidia/gram and CFU/gram were achieved after 72 h of fermentation. The desiccation tolerance of conidia harvested at 72 or 96 h was significantly (P = 0.05) greater than that of conidia harvested at 48 h of fermentation. Results obtained from this study could be used for further scale-up of the fermentation process.     

Ultrastructure ofAspergillus nidulans conidia and conidial lomasomes

Summary Lomasomes in the conidia ofAspergillus nidulans can be divided into at least two distinct structures. The first is a twice double membrane bound core of cytoplasmic origin. The outermost membrane of the lomasome becomes incorporated into the plasmalemma as it migrates to rest next to the cell wall. The second lomasome structure appears to be a triangle shaped series of tubules arranged in a parallel fashion. The wide end next to the cell wall connected to the plasmalemma and the opposite end to an element of the endoplasmic reticulum. The term membranosome has been coined to designate this lomasome structure with its function of plasmalemma extension. Various structures of the conidium such as wall, endoplasmic reticulum and the cytoplasmic matrix undergo changes from the conidial chain stage to the free or resting conidial stage. This suggests that after conidiation and before the resting stage, the conidium continues to mature.     

Microcycle conidiation and the conidial properties in the entomopathogenic fungus Metarhizium acridum on agar medium

Conidiation of the entomopathogenic fungus Metarhizium acridum on agar media was investigated. M. acridum CQMa102 exhibits two different conidiation patterns on agar media: normal conidiation in which conidia are formed on extended hyphae and microcycle conidiation in which conidiation occurs directly after conidia germination. Microcycle conidiation resulted in a mass of conidia produced via budding by accelerated development at the inoculation site. The mean total conidial yield (conidiation at day 10) was 4–5-fold greater after microcycle conidiation than during normal conidiation. Insect pathology assays indicated that microcycle conidia produced on SYA agar were as effective as normal aerial conidia against the locust. Ultraviolet (UV)-resistance tests showed no significant differences between the two types of cell propagules. However, microcycle conidia were more heat resistant than normal aerial conidia, and accumulated higher levels of trehalose in response to heat induction compared to normal aerial conidia.     

Activités de quelques enzymes associés à la conidiogenèse du Neurospora crassa

Summary Enzyme activities have been measured and compared at several stages of the development of Neurospora crassa i.e. from free conidia (inoculum) to conidiated mycelia grown on sucrose versus acetate (poor versus highly conidiogenous) media.Glucose-6-phosphate dehydrogenase (G-6-PDH) and NADP nucleotidase (NADPase) show inverse activity-time curves, both on sucrose and acetate media. G-6-PDH has its higher activity at the preconidiating stage while NADPase progressively increases to reach its maximal value in the mature conidia.Malate dehydrogenase (MDH) has a higher activity in extracts from acetate compared to sucrose cultures; in both conditions, maximal MDH activity corresponds with the initiation of conidiation. Malate synthetase (MS) has a delayed activity which is much higher in acetate extracts.Succinate dehydrogenase (SDH) is more active in sucrose than acetate extract with a sharp activity peak just preceding conidiation proper.Isozymes of G-6-PDH and MDH, as well as total soluble proteins from extracts of sucrose versus acetate cultures have been compared after their separation on polyacrylamide columns.     

Effects of fluctuating moisture and temperature regimes on the persistence of quiescent conidia of Isaria fumosorosea

Conidia of Isaria fumosorosea were submitted to three regimes of temperature and moisture to simulate microclimatic conditions which prevail in temperate (43% RH and 28 °C to 98% RH and 15 °C), subtropical (75% RH and 35 °C to 98% RH and 25 °C), and arid areas (13% RH and 40 °C to 33% RH and 15 °C) with daily fluctuating cycles. Germination, conidial viability, and virulence to Spodoptera frugiperda larvae were less affected after 20 days exposure under temperate cycling conditions than under arid and subtropical conditions. Exposure of conidia for 20 days to constant nocturnal simulated conditions of any tested regime weakly affected conidial persistence, whereas diurnal conditions exerted the most detrimental effects of high temperatures. However, when tested at both 45 °C and 50 °C at 33% RH for 160 h, the persistence of I. fumosorosea conidia was relatively higher than expected. These results emphasize that climatic conditions prevailing in environments and ecological fitness of fungal isolates have to be taken into account for assessing microbial control strategies.     

Cell-free transfer and sorting of membrane lipids in spinach

Summary The donor and acceptor specificity of cell-free transfer of radiolabeled membrane constituents, chiefly lipids, was examined using purified fractions of endoplasmic reticulum, Golgi apparatus, nuclei, plasma membrane, tonoplast, mitochondria, and chloroplasts prepared from green leaves of spinach. Donor membranes were radiolabeled with [¹⁴C]acetate. Acceptor membranes were unlabeled and immobilized on nitrocellulose filters. The assay was designed to measure membrane transfer resulting from ATP-and temperature-dependent formation of transfer vesicles by the donor fraction in solution and subsequent attachment and/or fusion of the transfer vesicles with the immobilized acceptor. When applied to the analysis of spinach fractions, significant ATP-dependent transfer in the presence of cytosol was observed only with endoplasmic reticulum as donor and Golgi apparatus as acceptor. Transfer in the reverse direction, from Golgi apparatus to endoplasmic reticulum, was only 0.2 to 0.3 that from endoplasmic reticulum to Golgi apparatus. ATP-dependent transfers also were indicated between nuclei and Golgi apparatus from regression analysis of transfer kinetics. Specific transfer between Golgi apparatus and plasma membrane and, to a lesser extent, from plasma membrane to Golgi apparatus was observed at 25°C compared to 4°C but was not ATP plus cytosol-dependent. All other combinations of organelles and membranes exhibited no ATP plus cytosol-dependent transfer and only small increments of specific transfer comparing transfer at 37°C to transfer at 4°C. Thus, the only combinations of membranes capable of significant cell-free transfer in vitro were those observed by electron microscopy of cells and tissues to be involved in vesicular transport in vivo (endoplasmic reticulum, Golgi apparatus, plasma membrane, nuclear envelope). Of these, only with endoplasmic reticulum (or nuclear envelope) and Golgi apparatus, where transfer in situ is via 50 to 70 nm transition vesicles, was temperature-and ATP-dependent transfer of acetatelabeled membrane reproduced in vitro. Lipids transferred included phospholipids, mono-and diacylglycerols, and sterols but not triacylglycerols or steryl esters, raising the possibility of lipid sorting or processing to exclude transfer of triacylglycerols and steryl esters at the endoplasmic reticulum to Golgi apparatus step.     

Differential deoxyribonucleic acid synthesis during microcycle conidiation in Neurospora crassa

In conidia of Neurospora crassa germinating at 25°C, DNA synthesis measured by incorporation of tritiated adenosine reaches a maximum soon after the outgrowth of the germ tube (6–7h after inoculation). In conidia heat-treated at 46°C (for 15h), a maximum of incorporation of the DNA precursor occurs already 1h after inoculation, then the incorporation progressively declines until the end of the heat-shock. When such conidia are shifted to 25°C, a maximum of DNA synthesis occurs during the development of the presumptive conidiophore as at the outgrowth of normal germ tubes. This wave of DNA synthesis is followed by a second maximum of DNA synthesis, occurring only in the microcyclized cultures, when the premature differentiation of proconidia takes place. Prevention of this second wave of DNA synthesis with hydroxyurea or 5-fluorodeoxyuridine respectively reduces or fully inhibits such induced conidial differentiation.     

Differential effect of temperature on mitrochondrial activity in shoots from freshly harvested and moderately aged kernels of maize (Zea mays L.)

This paper reports on a study of mitochondrial activity in etiolated shoots of freshly harvested and moderately aged kernels of maize. Activity was investigated after incubation at a favourable temperature (25°C), sub-optimal temperature (13°C) and after a heat shock (46°C for 2h). Although impaired mitochondrial activity in shoots from moderately aged maize kernels was not detected at 25°C, deficiencies became evident under low temperature stress (13°C). State 3 oxygen uptake, cyanide-insensitive oxygen uptake and cytochrome oxidase activity were lower in mitochondria from these shoots at 13°C than in mitochondria from shoots of freshly harvested kernels at this temperature. After a heat shock, cyanide-insensitive oxygen uptake was higher, and cytochrome oxidase activity lower, in shoots of aged kernels than in shoots of fresh kernels. No significant differences in ADP: O ratio or succinate dehydrogenase activity occurred between mitochondria from shoots of the two seed lots in any of the temperature treatments.     

Microcycle conidiation in Penicillium urticae: an ultrastructural investigation of conidiogenesis.

A cultivation system has been developed for Penicillium urticae which yields 'microcycle' conidiation in submerged culture. Spherical growth of spores was initiated by incubation at 37 degrees C in a growth-favoring medium. Transfer of these enlarged spores to a nitrogen-poor medium at 35 degrees C results in synchronous germination and limited outgrowth followed by roughly synchronous conidiation. A study of the conidiation stage showed that a phialide and an immature conidium began to form at the tip of all germ tubes 18 h after the temperature shift. By 24 h additional phialides commonly appeared as a branch near the tip of the germ tube and the more mature conidia exhibited increasing refractility. The earliest ultrastructural signs of conidiation were various round invaginations in the plasma membrane and a thickening and rounding of the new spore wall which appeared as an inner extension of the phialide cell wall. Upon segregation of the conidium from the phialide cell by conidial wall formation, 'trench-like' invaginations gradually appeared in the plasma membrane and a disorganized rodlet pattern was formed on the outer surface of the maturing conidial wall. Continued maturation involved the formation of chains of conidia and phialide senescence which was characterized by a general degradation of intracellular structure. A comparison with standard surface and submerged culture conidiation indicated that 'microcycle' conidiation, while less prolific, was essentially identical.     

Mechanisms involved in the increased sensitivity ofEscherichia coli to microcin 15m at 42°C

Escherichia coli cells show a markedly increased sensitivity to the antibiotic microcin 15m when briefly treated at 42°C as compared to the effect at 37°C. Furthermore, mutants resistant to the microcin at 37°C become sensitive at 42°C at microcin concentrations that are inactive at 37°C. This effect can be overcome byl-methionine. The mechanism involved seems to be based on an apparent inactivation of the homoserine-O-transsuccinylase activity. As previously established, this enzyme suffers a reversible partial inactivation when the cells are shifted to 42°C and the action of the microcin at this temperature seems to bring this process to a virtually irreversible stage. In mixed cultures of the microcin-producing strain and oneE. coli strain sensitive to the antibiotic, a much stronger growth inhibition of the latter strain has been observed at 42°C than at 37°C.