Ethylene Glycol-Induced Alteration of Conidial Germination in Neurospora crassa

In nutrient medium containing 3.22 M ethylene glycol or glycerol, conidia of Neurospora crassa grow as single cells, without forming the germ tubes characteristic of normal morphological germination. Ethylene glycol is more effective than glycerol in producing this response. After growth in ethylene glycol medium for a suitable time, the cells are easily disrupted by an abrupt decrease in osmotic pressure. Osmotic disruption yields intact nuclei and mitochondria, although mitochondrial fractions obtained in this way show significantly reduced concentrations of cytochromes c + c(1), as compared to those observed for comparable fractions obtained from vegetative hyphae. Cell cultures gradually adapted to lower concentrations of the glycol show a much higher degree of synchrony in the formation of germ tubes than do untreated conidia.     

Modelling the effects of temperature and wetness on the polycyclic phase of Stemphylium vesicarium,the pathogen causing purple spot on asparagus (Asparagus officinalis L.)

The polycyclic phase of Stemphylium vesicarium is the key factor for the forecast and integrated control of purple spot on asparagus. The annual dynamics of airborne conidia were determined under field conditions by conidia traps. From 2013 to 2015, conidia became airborne at the earliest at mid‐July, but the number trapped was considerably enhanced only after mid‐August, early September. The cumulative percentage of trapped conidia was best described using a logistic function depending on the daily temperature sum (base 0°C) accumulated only on days with >0.2 mm of rainfall (R² = .81). The germination of conidia was modelled by a generalized beta‐modified Chapman Richards function, and the germ tube length was modelled by a generalized beta‐power function. Conidia germinated in a wide temperature range, with an optimum at 23.3°C, whereas germ tube length had a narrow nearly optimum temperature range around 28.7°C, which indicates that infection by conidia is more restricted by germ tube growth than by germination. The effect of temperature on the number of lesions produced by two strains on green asparagus spears had the narrowest optimum range (optimum at 21.9°C) of all parts of the polycyclic phase. In plant tissue, the spread of the fungus depends on the mycelium growth. The mycelium growth of the four strains, which was modelled with data from a petri dish experiment, had an optimum temperature at 24.7°C.     

Synchronization of DNA synthesis in Neurospora crassa by 2′-deoxyadenosine and spore selection

2-Deoxyadenosine (2 mM), a DNA inhibitor, was used to synchronize DNA synthesis in cultures of Neurospora crassa lys 3. The cultures recovered spontaneously from the inhibitor which had little or no effect on the synthesis of RNA, protein or carbohydrate or on the specific growth rate of the mould. The degree of synchrony of DNA synthesis obtained with 2-deoxyadenosine varied directly with the organism's specific growth rate when the latter was altered by temperature changes. A direct relationship was observed between the rate of synthesis of DNA during the S period and the organism's specific growth rate.Conidia of Neurospora crassa lys 3 were separated into different density classes using urografin gradients; the separation treatment did not have an appreciable effect on the subsequent germination or growth of conidia. Populations of large, less dense conidia produced germ tubes more rapidly and more synchronously than populations of small, dense conidia. Cultures inoculated with the large conidia displayed continuous synthesis of RNA and protein but discontinuous synthesis of DNA.     

Molecular and cellular events during the germination of conidia of Sporothrix schenckii

Hyaline, non pigmented microconidia of Sporothrix schenckii were harvested and allowed to form germ tubes in a basal medium with glucose at pH 4.0 and 25 °C. These conditions supported only the development of the mycelial form of Sporothrix schenckii in a reproducible, synchronized manner which allowed further analysis of the early cellular events ocurring during the germination of the conidia. The relationship between macromolecular synthesis (DNA, RNA and protein synthesis) and nuclear division, hyphal growth and septum formation were established. Following inoculation, protein synthesis was observed after 10 minutes followed by RNA synthesis, after 1 h and DNA synthesis after 2 h. The first nuclear division was observed during the 9 to 12 h interval after inoculation. Germ tube formation slightly preceeded nuclear division and was first evidenced 9 h after the induction of germination but was not completed until 12 h after inoculation. Septation was first observed in the germ tubes 0.25 m from the mother cell-germ tube function 9 h after induction of germination.     

Fine Structure of Germinating Botrytis fabae Sardina Conidia

The fine structure of germinating Botrytis fabae conidia wasstudied using both chemically stained sections and freeze-etchedreplicas. Germinating conidia have fewer organelles than restingconidia, glycogen is absent, and prevacuoles have disappeared.Endoplasmic reticulum which occurs as small strands close tothe cell wall of resting conidia becomes, on germination, multiplesheets surrounding the nuclei. A cross wall is formed at thebase of the germ tube soon after germination commences. Thenew wall material which appears to be continuous with this septalwall is produced, at least partly, from a new wall layer laiddown in the centre of the old conidial wall. An apical corpuscleis present at the apex of young germ tubes. Freeze-etched preparationsshow the formation of lomasomes by the passage of vesicles throughthe plasmalemma of conidia and germ tubes. In young hyphae lomasomescontain a complex arrangement of branching tubules. Some ofthe particles on the outer plasmalemma of young hyphae are arrangedin a geometrical pattern.     

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.     

CYTOLOGICAL INVESTIGATIONS OF KABATIELLA CAULIVORA

Cole , Herbert , Jr ., and Houston B. Couch . (Penn. State U., University Park.) Cytological investigations of Kabatiella caulivora. Amer. Jour. Bot. 46(1) : 12-16. Illus. 1959.—Initial growth of K. caulivora on artificial media is characterized by budding, yeast-like, conidia, exclusive of mycelia. After 14 days growth, at 20°C., mycelial growth becomes macroscopically evident. A study of the germination behavior of a total of 5500 conidia from the initial yeast-like growth stage showed 59 to germinate by the production of mycelia, while the balance germinated by budding. Five thousand conidia of the mycelial growth type were studied in a successive, single-spore transfer series, and, in all cases, conidial germination continued to be by means of germ tubes. Conidia of both growth forms were studied by means of bright field and phase contrast microscopy, and found to be multinucleate—possessing variable nuclear numbers, ranging from 1-8 per cell. Mean nuclear number for both the mycelial and conidial types was 2.8. All nuclei of both growth types appeared to contain the same chromosome complement. The cultural variability exhibited by K. caulivora cannot be reconciled with the concept of dual phenomenon. It is suggested, rather, that the mycelial homotype probably arises as the result of a unidirectional mutation within the conidial growth form.     

A self-inhibitor of protein synthesis in the conidia of Glomerella cingulata

Summary A diffusible self-inhibitor of germination of conidia of Glomerella cingulata appears to act as a regulator of protein synthesis. Both uptake of labeled amino acids and their incorporation into protein are reduced by the inhibitor or by crowding. Compared to conidia incubated without self-inhibitor, conidia incubated with self-inhibitor incorporated no labeled amino acids into protein in the first hour and 80% less in 6h. Thoroughly washed conidia were more permeable to amino acids and incorporated 6 times more precursor into proteins than unwashed conidia. At high density in nutrient medium, conidia of G. cingulata preferentially form secondary conidia instead of germ tubes and a mycelium. This inhibition of germination of conidia and regulation of development is mimicked by exposing them to an auto-inhibitor extracted from used culture medium and conidial washings. Germination of conidia of G. cingulata involves two steps, an initial step of 5 h duration which continues unaffected by crowing (1.7×10⁸/ml) and a subsequent 2 h step which conidia do not take unless they are sufficiently diluted. It is this step for which protein synthesis may be required.Non-Standard Abbreviations CHM cyloheximide - NM Neurospora minimal medium - psi pound per square inch - RPH reconstituted algal protein hydrolysate - TCA trichloroacetic acid     

Regulation and glutamic acid decarboxylase during Neurospora crassa conidial germination.

Glutamic acid decarboxylase (GAD) from Neurospora crassa was assayed in dormant and germinating conidia that had been permeabilized by toluene and methanol. N. crassa conidia contained 10 times the GAD activity found in vegetativemycelia. During conidial germination, GAD activity rapidly decreased to low levels before germ tubes appeared. GAD activity in germinating conidia closely followed the decreasing rate of glutamic acid metabolism. Inhibiting protein synthesis partially blocked the decrease in GAD activity, but eliminating exogenous carbon sources did not alter the initial rate of decrease in this enzyme. However, when conidia were incubated for more than 3 h in distilled water, GAD activity began to increase and eventually reached levels comparable to those in dormant conidia. Either GAD was reversibly inactivated or this enzyme could be synthesized from endogenous storage compounds when conidia were incubated in distilled water. These results are consistent with the hypothesis that GAD is a developmentally regulated enzyme that is responsible for catalyzing the first step in the metabolism of the large pool of free glutamic acid during conidial germination.     

Visualizing nuclear migration during conidiophore development in Aspergillus nidulans and Aspergillus oryzae: multinucleation of conidia occurs through direct migration of plural nuclei from phialides and confers greater viability and early germination in Aspergillus oryzae

Nuclear migration is indispensable for normal growth, differentiation, and development, and has been studied in several fungi including Aspergillus nidulans and Neurospora crassa. To better characterize nuclear movement and its consequences during conidiophore development, conidiation, and conidial germination, we performed confocal microscopy and time-lapse imaging on A. nidulans and Aspergillus oryzae strains expressing the histone H2B-EGFP fusion protein. Active trafficking of nuclei from a vesicle to a phialide and subsequently into a conidium provided the mechanistic basis for the formation of multinucleate conidia in A. oryzae. In particular, the first direct visual evidence on multinucleate conidium formation by the migration of nuclei from a phialide into the conidium, rather than by mitotic division in a newly formed conidium, was obtained. Interestingly, a statistical analysis on conidial germination revealed that conidia with more nuclei germinated earlier than those with fewer nuclei. Moreover, multinucleation of conidia conferred greater viability and resistance to UV-irradiation and freeze-thaw treatment.     

Morphology and lipid body and vacuole dynamics during secondary conidia formation in Colletotrichum acutatum: laser scanning confocal analysis

Colletotrichum acutatum may develop one or more secondary conidia after conidial germination and before mycelial growth. Secondary conidia formation and germination were influenced by conidia concentration. Concentrations greater than 1x105 conidia/mL were associated with germination decrease and with secondary conidia emergence. Secondary conidia can form either alone or simultaneously with germ tubes and appressoria. Confocal analysis showed numerous lipid bodies stored inside ungerminated conidia, which diminished during germ tube and appressoria formation, with or without secondary conidia formation. They were also reduced during secondary conidia formation alone. While there was a decrease inside germinated conidia, lipid bodies appeared inside secondary conidia since the initial stages. Intense vacuolization inside primary germinated conidia occurred at the same time as the decrease in lipid bodies, which were internalized and digested by vacuoles. During these events, small acidic vesicles inside secondary conidia were formed. Considering that the conidia were maintained in distilled water, with no exogenous nutrients, it is clear that ungerminated conidia contain enough stored lipids to form germ tubes, appressoria, and the additional secondary conidia replete with lipid reserves. These results suggested a very complex and well-balanced regulation that makes possible the catabolic and anabolic pathways of these lipid bodies.     

Live-cell imaging of endocytosis during conidial germination in the rice blast fungus,Magnaporthe grisea

Although there is growing evidence that endocytosis is important in hyphal tip growth, it has not previously been shown to occur during fungal spore germination. We have analysed and characterized endocytosis during the germination of living conidia of the rice blast fungus, Magnaporthe grisea. Conidia treated with the endocytic markers Lucifer Yellow carbohydrazide, FITC-dextran, and FM4-64 were imaged by confocal microscopy. Internalization of these fluorescent marker dyes by conidia was blocked by chemical and temperature treatments that inhibit endocytosis, and the sequential staining of organelles by the membrane-selective dye FM4-64 was consistent with dye internalization by endocytosis. FM4-64 uptake occurred within 2-3 min of conidial hydration, more than 40 min before the emergence of the germ tube. The times at which each of the three conidial cells initiated dye internalization were different as were the rates of dye uptake by each cell. Using these techniques we have demonstrated for the first time that ungerminated and germinated spores of filamentous fungi undergo endocytosis. Furthermore, internalization of FITC-dextran and Lucifer Yellow carbohydrazide by germinating conidia provides the first direct evidence for fluid-phase endocytosis in a filamentous fungus. FM4-64 was internalized by both ungerminated conidia and conidial germlings on the rice leaf suggesting that endocytosis might play a significant role in spore germination and germ tube growth during the pre-penetration phase of infection.     

Microtubule dynamics during infection-related morphogenesis of Colletotrichum lagenarium.

Using a green fluorescent protein (GFP)-tubulin fusion protein, we have investigated the dynamic rearrangement of microtubules during appressorium formation of Colletotrichum lagenarium. Two alpha-tubulin genes of C. lagenarium were isolated, and GFP-alpha-tubulin protein was expressed in this fungus. The strain expressing the fusion protein formed fluorescent filaments that were disrupted by a microtubule-depolymerizing drug, benomyl, demonstrating successful visualization of microtubules. In preincubated conidia, GFP-labeled interphase microtubules, showing random orientation, were observed. At conidial germination, microtubules oriented toward a germination site. At nuclear division, when germ tubes had formed appressoria, mitotic spindles appeared inside conidia followed by disassembly of interphase microtubules. Remarkably, time-lapse views showed that interphase microtubules contact a microtubule-associated center at the cell cortex of conidia that is different from a nuclear spindle pole body (SPB) before their disassembly. Duplicated nuclear SPBs separately moved toward conidium and appressorium accompanied by astral microtubule formation. Benomyl treatment caused movement of both daughter nuclei into 70% of appressoria and affected appressorium morphogenesis. In conidia elongating hyphae without appressoria, microtubules showed polar elongation which is distinct from their random orientation inside appressoria.     

Hyphal tip extension in Aspergillus nidulans requires the manA gene, which encodes phosphomannose isomerase.

A strain of Aspergillus nidulans carrying a temperature-sensitive mutation in the manA gene produces cell walls depleted of D-mannose and forms hyphal tip balloons at the restrictive temperature (B.P. Valentine and B.W. Bainbridge, J. Gen. Microbiol. 109:155-168, 1978). We have isolated and characterized the manA gene and physically located it between 3.5 and 5.5 kb centromere distal of the riboB locus on chromosome VIII. The manA gene contains four introns and encodes a 50.6-kDa protein which has significant sequence identity to type I phosphomannose isomerase proteins from other eukaryotes. We have constructed by integrative transformation a null mutation in the manA gene which can only be maintained in a heterokaryotic strain with wild-type manA+ nuclei. Thus, a manA null mutation is lethal in A. nidulans. The phenotype of the mutation was analyzed in germinating conidia. Such conidia are able to commence germination but swell abnormally, sometimes producing a misshapen germ tube, before growth ceases. The reason for the lethality is probably the lack of synthesis of mannose-containing cell wall polymers that must be required for normal cell wall development in growing hyphae.     

DNA, RNA, protein and heterochromatin changes during embryo development and germination of soybean (Glycine max L.)

DNA, RNA, protein and heterochromatin were measured cytophotometrically in developing soybean (Glycine max) seeds. The average 2C DNA content for the soybean genome was 2.64 pg. The amounts of nuclear DNA in embryo axes showed no significant change during embryo development, whereas the DNA content in cotyledon nuclei increased significantly from 3.58 pg to 5.49 pg. The number of endopolyploid nuclei increased from 26% to 48% and the DNA content from 4.45 to 5.49 pg after cessation of cell division. The changes in RNA and protein content during embryo development were in general similar to those in DNA content. This can be interpreted that increased DNA levels in soybean cotyledons generated during embryogeny increase the protein synthesizing capacity. During the first 15 days of germination, the number of endopolyploid nuclei in cotyledons declined from 46% to 4%, and this decline is interpreted as DNA degradation providing a ready source of nucleosides and phosphates during early embryo growth. A later decline, however, between 15 and 20 days after germination, was age related similar to leaf senescence, because the percentage of endopolyploid nuclei remained unchanged while the number of non-viable cells increased. In senescing cotyledons, 73% and 80% of RNA and protein but only 20% of DNA were lost, as compared to dormant cotyledons. The heterochromatin (condensed chromatin) measurements indicated that nuclei of metabolically inactive dormant and senescent cotyledon nuclei contained an average of 33% more heterochromatin than nuclei from the green cotyledons of seedlings.     

Fine structure of resting and germinatingPenicillium chrysogenum conidiospores

Summary The fine structure of resting and germinating conidia ofPenicillium chrysogenum has been examined by electron microscopy. In addition to enlargement of the cells, a number of changes in ultrastructure become evident as morphogenesis proceeds. The newly synthesized germ tube is continuous with the corresponding layers of the conidial wall. Some conidial wall layers, however, do not extend into the hyphal wall. Several sections showing initial septum synthesis suggest that a septal pore is not a necessary structural entity. A characteristic orientation of the initial septum formed after germination is described. Aside from numerical considerations, no significant changes occur in nuclei, mitochondria, or ribosomes. The electron micrographs illustrate the presence of spherosomes, lomasomes, and nucleoli; the possible significance of these structures is discussed.     

Ethylene production by Botrytis cinerea in vitro and in tomatoes

A laser-based ethylene detector was used for on-line monitoring of ethylene released by the phytopathogenic fungus Botrytis cinerea in vitro and in tomato fruit. Ethylene data were combined with the results of a cytological analysis of germination of B. cinerea conidia and hyphal growth. We found that aminoethoxyvinylglycine and aminooxyacetic acid, which are competitive inhibitors of the 1-aminocyclopropane-1-carboxylic acid pathway, did not inhibit the ethylene emission by B. cinerea and that the fungus most likely produces ethylene via the 2-keto-4-methylthiobutyric acid pathway. B. cinerea is able to produce ethylene in vitro, and the emission of ethylene follows the pattern that is associated with hyphal growth rather than the germination of conidia. Ethylene production in vitro depended on the L-methionine concentration added to the plating medium. Higher values and higher emission rates were observed when the concentration of conidia was increased. Compared with the ethylene released by the fungus, the infection-related ethylene produced by two tomato cultivars (cultivars Money Maker and Daniela) followed a similar pattern, but the levels of emission were 100-fold higher. The time evolution of enhanced ethylene production by the infected tomatoes and the cytological observations indicate that ethylene emission by the tomato-fungus system is not triggered by the ethylene produced by B. cinerea, although it is strongly synchronized with the growth rate of the fungus inside the tomato.     

Interactions of Fusarium species during prepenetration development

Interspecies interactions between Fusarium avenaceum, Fusarium culmorum, Fusarium graminearum, Fusarium poae, and Fusarium tricinctum were studied during early growth stages of isolates on model surfaces. Additionally, germination and germ tube growth of the pathogens were studied on attached and detached wheat leaves at 10 °C and 22 °C. Two-species interactions between Fusarium isolates during germination and germ tube growth were assessed after 8 hours of incubation. All species except F. tricinctum germinated and grew faster at higher than lower temperature. All species were able to germinate with more than one germ tube per conidium cell; and germination and germ tube growth were faster on leaves than on glass surface. Interactions among Fusarium species during germination and germ tube growth were predominantly competitive with macroconidia-producing species being more competitive. It is concluded that the type of conidia as well as environmental factors influence the competitiveness of Fusarium species during early stages of growth.