Spore age and coverslip placement affects germination and post-germination development of Colletotrichum dematium var circinans

Spore suspensions from young (10–14 da; young spores) and old (4 mo; old spores) colonies of PColletotrichum dematium var circinans were placed on slides. Coverslips were left off, placed on in the normal manner, or supported on shims. Slides were placed in moist chambers and incubated in light or dark for up to 48 hrs. Germination and post-germination development were studied. Shimming had some beneficial effect on germination, especially for old spores in dark. In general, more germ-tubes and appressoria were produced on spores under shims than spores with other coverslip treatments. By 48 hrs more old spores under shims germinated, and greater numbers of germ-tubes and appressoria were produced than on other old spores under different coverslip treatments. However, numbers produced were lower than those predicted for comparably treated young spores. Spore age, incubation regime, and placement of coverslips did not affect germ-tube initiation. For all treatments more germ-tubes were initiated from spore tops than bottoms or tips. Fewer germ-tubes were initiated from spore centers than other locations on tops and bottoms, and from both tips than one tip. Approximately 26 % of all appressoria were produced sessile. A higher percentage of sessile appressoria were produced on old spores (80 %) than on young spores (20%).     

Ultrastructure and elemental composition of dormant and germinating Diplodia maydis spores.

The ultrastructure of Diplodia maydis spores was studied in thin sections with a transmission electron microscope. Storage vacuoles were evenly distributed in the two cells. Some of the vacuoles that contained a dense osmiophilic sphere(s) were surrounded by a membrane, and had membranous aggregates around their periphery. The sport wall was composed of an electron-dense layer and an electron-translucent layer. An inner cytoplasmic membrane was present. Dormant and germinating spores were studied with scanning electron microscopy and also with a Si (Li) energy-dispersive X-ray analyzer. The dormant spore was ovate and usually two-celled with a central septum. Germination proceeded via a germ tube from the side of one end of the cell. Of several methods for preparation of specimens for X-ray analysis studied, freeze-dried spores mounted on carbon stubs and then further carbon coated gave the best results. X-ray analyses revealed that spore populations contained large amounts of Si, P, Cl, and K, smaller amounts of S and Ca, and trace amounts of Mg and Al. Analyses of single spores revealed high K and Cl and low P and Mg at one end of the cell with concomitant low K and Cl and high P and Mg in the central portion and other end of the cell. In two-celled germinating spores, high K and Cl occurred in the end of the nongerminating spore cell, whereas the germinating cell contained high P and Mg and low K and Cl. X-ray image maps revealed that K and Cl were located together at one end of the spore.     

Domain protolife

We propose the Thermal Protein First Paradigm (protocell theory) that affirms that first life was cellular. The first cells emerged from molecular (chemical) evolution as protocells (heated amino acids self-order in copolymerization reactions to form thermal proteins which self-organize when in contact with water to form protocells). Metaprotocells are specialized protocells capable of synthesizing ATP (light energy conversion to chemical energy), polypeptides, and polynucleotides. Aggregations of protocells in thermal protein matrices form distinctive morphologies (protocellular networks). Prokaryotic cells emerged from metaprotocells. We classify protocells and metaprotocells as members of the Domain Protolife. We revised the cell theory to include protolife.     

Dry mass changes in germinating spores of Diplodia maydis

Summary The dry mass of two-celled Diplodia maydis spores was measured both before and after germination by quantitative interference microscopy. The dry mass of spores declined approximately 50% during germination. However, the dry mass of germinating spores plus the dry mass of their germ tubes was greater than the dry mass of spores before germination. We conclude that the germinating spores absorbed nutrients released from non-germinating spores.The dry mass of fungal spores can be estimated by weighing large numbers of spores and determining the mean from sample spore counts. Mumford and Pappelis(4) determined the total dry mass of individual spores of Fusarium roseum and the contained lipid bodies before and after spores germinated using quantitative interference microscopy. The mean spore dry mass before germination was 57 pg. Lipid bodies accounted for about 61% of that mass and decreased as spores germinated. The total dry mass of the spore and germ tube 24 hr later greatly exceeded that of the spore before germination. Quantitative interference microscopy has been used to measure the dry mass of various types of cells. Kulfinski and Pappelis (3) recently reviewed how this technique has been applied to plant cells. Technical aspects of interference microscopy have been described by Ross (6).The purpose of this study was to examine the dry mass changes in Diplodia maydis (Berk.) Sacc. with and without germ tubes through the use of interference microscopy.     

Mycelial elongation and sporulation of two fungi on amended media in light or dark

Botrytis allii andCollectotrichum dematium are onion pathogens which can infect in the field and cause decay in storage. Some phenolics can hinder development of these fungi, but the effect of cytokinins is not clear. Cytokinins (kinetin or 6-benzyladenine) or phenolics (caffeic or chlorogenic acids) were added to agar at concentrations of 0 to 10^–3 M. Cultures were continuously irradiated with fluorescent light or maintained in the dark for 6 days. On unamended media, final mycelial elongation was 45 or 17.8 mm and sporulation was 28 or 10.6 × 10⁴ spores/ml forBotrytis andColletotrichum, respectively. ForBotrytis, mycelial elongation was slightly (5%) but significantly increased and sporulation increased by 21% by incubation on phenolics as compared to cytokinins. Mycelial extension ofColletotrichum was not affected by amendment. Sporulation ofColletotrichum on kinetin was 16 to 28% greater than on the other amendments. As amendments concentration increased elongation of mycelia of both fungi decreased. Sporulation ofBotrytis increased by 60% as amendment concentration increased from 0 to 10^–5 M and then decreased 25% at 10^–3 M. As amendment concentration increased from 0 to 10^–3 M, sporulation ofColletotrichum increased by 45%. Incubation in light increased mycelial extension 3 to 17% forBotrytis andColletotrichum respectively, and sporulation was increased approximately 78% for both fungi. These compounds do not appear to inhibit development of theseBotrytis orColletotrichum species in culture.     

Experimental retracement of the origins of a protocell

Although Oparin used coacervate droplets from two or more types of polymer to model the first cell, he hypothesized homacervation from protein, consistent with Pasteur and Darwin. Herrera made two amino acids and numerous cell-like structures (sulfobes) in the laboratory, which probably arose from intermediate polymers. Our experiments have conformed with a homoacervation of thermal proteinoid, in which amino acid sequences are determined by the reacting amino acids themselves. All proteinoids that have been tested assemble themselves alone in water to protocells. The protocells have characteristics of life defined by Webster's Dictionary: metabolism, growth, reproduction and response to stimuli in the environment. The protocells are able also to evolve to more modern cells including the initiation of a nucleic acid coding system.Principal spinoffs from the results are revised evolutionary theory, models for protoneurons and networks thereof, and numerous industrial applications of thermal polyamino acids. Life itself has thus been reaffirmed to be rooted in protein, not in DNA nor RNA, which are however crucial to inheritance in modern life as instruction manual (Kornberg).Recognition of the advances have been considerably delayed by the deeply held assumption that life began by chance from random polymerization of amino acids, in contrast to the experimental findings. The concepts of DNA/RNA-first and protein-first are reconciled by a rise-and-fall progression as often seen in biochemical and biological evolution.The fact that amino acids order themselves explains in turn that thermal copolyamino acids are finding numerous applications. The entire sequence of processes in the proteinoid origins theory is now seen to be highly deterministic, in close accord with Einstein.     

Morphological Observations of Diplodia maydis on Synthetic and Natural Substrates as Revealed by Scanning Electron Microscopy

Mycelial and spore morphology of Diplodia maydis were investigated by using scanning electron microscopy after growth on various media and natural substrates (oat and corn kernels, and corn husks). Of several specimen preparation methods studied, Parducz fixation followed by critical-point or freeze-drying gave adequate preservation for pycnidia, mycelia, and spores. Morphological characteristics were similar in rotary and reciprocal shaker cultures and differed from that found in stationary cultures in the amount of slime-like material produced and precipitated matter on the mycelial surfaces. In general, mycelial surfaces were smooth. Large areas of coalesced material were present in all samples examined. Slime-like material produced in liquid media appeared as a finely laced net, randomly appearing throughout the mycelia with bead-like structures present along the net. A fine netting also was observed interspersed among the spores inside the pycnidia obtained from oats. Slime-like material was observed to cover the pycnidia produced on oat and corn kernels. In the latter case, the spores were less protected by the outer slime-like covering. Thickened node-like structures were observed in mycelial mats produced in modified Fries 2 medium, on potato dextrose agar plates, and on infected oats. Round and ovate thickened node-like structures were observed in mycelium produced on corn kernels. In general, node-like structures were less abundant in mycelia from naturally infected substrates. Conidia were commonly rounded to tapered and two celled, with a distinctive ridged septum at the middle. Dried spores were collapsed in a characteristic flask-like fashion.     

Ultrastructure of Diplodia maydis grown on selected synthetic media

Of all the fixatives studied, glutaraldehyde followed by osmium was considered best. Fixation temperatures did not result in ultrastructural differences. Holdover for short periods (up to 7 days) in 70% alcohol and long periods (3 months) in glutaraldehyde did not result in ultrastructural differences from those processed without a hold-over period. Uranyl acetate increased contrast, as expected. Epon was selected over Epon-Araldite (more viscous) and Spurr's resin (less contrast). Generally, growth conditions did not alter gross hyphal morphology, although cytological changes were noted. Diplodia maydis is morphologically similar to ascomycetous fungi. It contained typical organelles as found in other fungi, e.g. cell wall, septa, Woronin bodies, nuclei, mitochondria, endoplasmic reticulum, lomasomes, plasma membranes, cytosomes, vacuoles, storage materials (lipids, carbohydrates and intracellular and extracellular electrondense substances), thickened mycelial strands, and intrahyphal hyphae. Bead-like structures and thickened nodes as observed by SEM were accounted for by structures observed by TEM. Mitochondria were dramatically elongated, and in one case, branched. Hyphae were organized in three zones: apical, subapical, and vacuolated. Apical vesicles were thought to account for cell extension. Evidence was presented that Woronin bodies originated from microbodies. It was suggested that the Golgi apparatus consists of isolated single cisternae. Free ribo- or polysomes (without ER) were suggested as the site of the majority of protein synthesis. Continuities and configurations of plasma and outer nuclear and mitochondrial membranes, ER, cytosomes, vacuoles and storage materials suggested that a system similar to Morre et al's endomembrane system is in operation in D. maydis to account for component flow and differentiation.