A gene encoding alanine racemase is involved in spore germination in <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis>

Alanine racemase is a major component of the exosporium of Bacillus cereus spores. A gene homologous to that of alanine racemase (alrA) was cloned from Bacillus thuringiensis subsp. kurstaki, and RT-PCR showed that alrA was transcribed only in the sporulating cells. Disruption of alrA did not affect the growth and sporulation of B. thuringiensis, but promoted l-alanine-induced spore germination. When the spore germination rate was measured by monitoring DPA release, complementation of the alrA disruptant reduced the rate of l-alanine-induced spore germination below that of even wild-type spores. As previously reported for spores of other Bacillus species, d-alanine was an effective and competitive inhibitor of l-alanine-induced germination of B. thuringiensis spores. d-cycloserine alone stimulated inosine-induced germination of B. thuringiensis spores in addition to increasing l-alanine-induced germination by inhibiting alanine racemase. d-Alanine also increased the rate of inosine-induced germination of wild-type spores. However, d-alanine inhibited inosine-induced germination of the alrA disruptant spores. It is possible that AlrA converted d-alanine to l-alanine, and this in turn, stimulated spore germination in B. thuringiensis. These results suggest that alrA plays a crucial role in moderating the germination rate of B. thuringiensis spores.     

Investigation of factors influencing spore germination of Paenibacillus polymyxa ACCC10252 and SQR-21

Bioorganic fertilizer containing Paenibacillus polymyxa SQR-21 showed very good antagonistic activity against Fusarium oxysporum. To optimize the role of P. polymyxa SQR-21 in bioorganic fertilizer, we conducted a study of spore germination under various conditions. In this study, l-asparagine, glucose, fructose and K⁺ (AGFK), and sugars (glucose, fructose, sucrose, and lactose) plus l-alanine were evaluated to determine their ability to induce spore germination of two strains; P. polymyxa ACCC10252 and SQR-21. Spore germination was measured as a decrease in optical density at 600 nm. The effect of heat activation and germination temperature were important for germination of spores of both strains on AGFK in Tris–HCl. l-Alanine alone showed a slight increase in spore germination; however, fructose plus l-alanine significantly induced spore germination, and the maximum spore germination rate was observed with 10 mmol l⁻¹ l-alanine in the presence of 1 mmol l⁻¹ fructose in phosphate-buffered saline (PBS). In contrast, fructose plus l-alanine hardly induced spore germination in Tris–HCl; however, in addition of 10 mmol l⁻¹ NaCl into Tris–HCl, the percentages of OD₆₀₀ fall were increased by 19.6% and 24.3% for ACCC10252 and SQR-21, respectively. AGFK-induced spore germination was much more strict to germination temperature than that induced by fructose plus l-alanine. For both strains, fructose plus l-alanine-induced spore germination was not sensitive to pH. The results in this study can help to predict the effect of environmental factors and nutrients on spore germination diversity, which will be beneficial for bioorganic fertilizer storage and transportation to improve the P. polymyxa efficacy as biological control agent.     

Die Beeinflussung der Farnsporen-Keimung [Osmunda cinnamomea (L.) undO. claytoniana (L.)] über das Phytochromsystem und die Photosynthese

Summary The control by light of the spore germination ofOsmunda cinnamomea L. andO. claytoniana L. has been investigated.This light control is complex, and in order to investigate this control quantitatively the process of germination had to be divided into at least two stages. The first stage of germination leads to the rupture of the exospore, the second step is characterized by the outgrowth of a rhizoid. In a very few percent of our spore population the first stage can occur in total darkness, the second step however does not ever occur in darkness.In the present paper it has been demonstrated, usingO. cinnamomea spores, that the first step of germination is phytochrome controlled, whereas the second step is under photosynthetic control. This was observed withO. claytoniana by a rough action spectrum and in experiments with a CO₂-less atmosphere.The germinating spores of these two species ofOsmunda show a very similar behaviour as the spores of the mossFunaria hygrometrica (L.) Sibth. (Bauer andMohr 1959). They behave very differently compared with spores of other ferns, e.g.Dryopteris filix-mas (L.) Schott, where the whole process of germination is exclusively phytochrome controlled (Mohr 1956).

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Allomyces neo-moniliformis gametogenesis

Summary InAllomyces neo-moniliformis meiosis takes place during resting sporangium germination. The meiospores are characteristically binucleate and biflagellate as described byEmerson (1938) andTeter (1944). A variation in the number of nuclei and flagella per meiospore from two is correlated with germination of the resting sporangia under reduced oxygen tension. The meiospores are extremely poor swimmers and are typically amoeboid. At encystment the gamma bodies of the cell are mobilized and appear involved in cyst wall synthesis. A single mitotic division of each nucleus gives rise to four nuclei. Gamete cleavage is as described for spore cleavage inBlastocladiella (Lessie andLovett 1968). The assembly of the nuclear cap and side body complex of the spore are extremely late processes in gametogenesis. The gametes are released when the single papilla dissolves. The gametes fuse in pairs and after zygote formation the cell is uninucleate with two flagella. The biflagellate zygote is an active swimming cell. The presence of homothallism or hetero-thallism inA. neo-moniliformis is discussed.     

Megaspore wall growth inSelaginella (Lycopodiatae)

Different stages of megaspore and megasporangial development inSelaginella argentea (Wallich)Spring,S. bigelowii Unerw., andS. kraussiana (Kze.)A. Br. have been seen and studied. Megaspore wall units give positive reactions for polysaccharides and protein in young megaspores, and become the thick and resistant wall typical of the genus only later.—Units forming the exospore and the spaces between units enlarge from widths of 5–10nm early during development up to over 200 nm at pregermination stages. The spaces enlarge first. Initially they are circular and mostly about 70 nm in diameter. Later, spaces toward the inner part of the exospore enlarge more than those near the outer surface. During pregermination, wall spaces range in size from 4 to 50 times the width of units with the larger spaces located near the inner surface. As a result the exospore would be under tension to spring outward during germination when the laesurae are lysed.—A gap in the exospore, shaped like a half-moon in polar sections, forms in equatorial and distal portions of the spore. This gap becomes enormous, three times the volume of the central space plus the mesospore, and is filled with lipids and other nutrients. Late in development, during the period of tapetal cell degeneration, the gap contents are moved into the central space and the gap is closed.—Late in development the mesospore is degraded. Its products, along with gap contents, seem to be added to the contents of the central cavity and appear as reserve storage globules. A primary wall-like endospore is formed during this period, at the inner surface of the exospore. During germination this endospore develops further at its inner surface.—Changes in the size and shape of megasporangia occur independently of the size of megaspores.Megaspore development inSelaginella. II. For first part seeMorbelli & Rowley (1993).     

Carbon Requirements of Some Nematophagous, Entomopathogenic and Mycoparasitic Hyphomycetes as Fungal Biocontrol Agents

Thirty-three carbon sources were evaluated for their effects on spore germination, hyphal growth and sporulation of 11 fungal biocontrol agents, i.e. the nematophagous fungi Paecilomyces lilacinus, Pochonia chlamydosporia, Hirsutella rhossiliensis, H. minnesotensis and Arkansas Fungus 18, the entomopathogenic fungi Lecanicillium lecanii, Beauveria bassiana and Metarhizium anisopliae, and the mycoparasitic fungus Trichoderma viride. Variations in carbon requirements were found among the fungal species or strains tested. All strains studied except for T. viride grew on most carbon sources, although B. bassiana had more fastidious requirements for spore germination. Monosaccharides and disaccharides were suitable for fungal growth. For most isolates, d-glucose, d-mannose, sucrose and trehalose were superior to pectin and soluble starch among the polysaccharides and lactic acid among the organic acids. Both ethanol and methanol could accelerate growth of most isolates but not biomass. d-mannose, d-fructose and d-xylose were excellent carbon sources for sporulation, while d-glucose, sucrose, cellobiose, trehalose, chitin, dextrin, gelatin and lactic acid were better for some isolates. Neither sorbic acid nor linoleic acid could be utilized as a single carbon source. These findings provided a better understanding of the nutritional requirements of different fungal biocontrol agents that can benefit the mass production process.     

An account of some physiological studies in two species of phytophthora

Summary The paper deals with some physiological studies of two typical strains ofPhytophthora palmivora Butl. andP. parasitica Dast. var.macrospora Ashby causing severe fruit rots ofAchras sapota L. andAnona squamosa L. respectively. The effect of temperature on the germination of sporangia had a marked effect. Maximum percentage of indirect germination (by formation of zoospores) occurred at 20° C, whereas direct germination of sporangia (by formation of germ tube) was at 30° C, which further continued upto 37° C. Maximum indirect germination at 20° C shows the favourable cool temperature for the epidemic of the diseases (Fruit rots) in nature. The best substrate (medium) for germination of sporangia was found to be tap water. Next to this was the host decoction or extract. Glucose solution also accelerated sporangial germination. The effect of two dyes viz., Malachite green and crystal violet was also studied in relation to growth and sporulation ofPhytophthora isolates. Their addition to the medium in various fractional dilutions had a profound influence in the rate of growth and sporulation. An interesting observation noted was that growth of the isolates was inversely porportional to the various concentrations of the dyes, under study. An attempt was also made to study the influence of various vitamins. In all, six vitamins were included in the study. Out of these, thiamine and riboflavin were found to be the best sources promoting good growth and sporulation of both the species ofPhytophthora under study.Forms a part of Senior author's M. Sc. (Agric.) Thesis, University of Poona, Poona (India).Respectively, Ex-Junior Research Fellow, I.C.A.R., New Delhi; Professor of Plant Pathology and Principa, College of Agriculture, Junagad (Gujarat); Plant Pathologist, Wheat Rust Research Station, Mahabaleshwar, Poona, India.     

Pollen germination and pollen tube growth in Fraxinus pennsylvanica

With regard to adaptation of green ash (Fraxinus pennsylvanica Marshall) to ecological conditions in Croatia, pollen germination and pollen tube length after 2, 4 and 6 hours were examined in vitro at 10, 15, 20 and 25°C during two years 2001 and 2002. Narrow leaved ash (F. angustifolia Vahl) pollen served as a control in 2002. The year, time and temperature, and the interaction between time and temperature were significant for both germination percentage and pollen tube length. Interactions year × temperature and year × time were significant for pollen tube length only. The highest germination percentage (17.86% in 2001 and 19.40% in 2002) of green ash pollen was at 15°C after 6 hours. The pollen tube length was greatest at 20°C (393.46 μm) in 2001 and 25°C (899.50 μm) in 2002 after 6 hours. Narrow leaved ash pollen had the highest germination percentage (19.22%) at 20°C after 6 hours and was significantly reduced at 25°C. The pollen tube length was greatest at 25°C (518.90 μm) after 6 hours. It can be concluded that green ash pollen has satisfactory germination in ecological conditions in Croatia and that the optimum temperature for pollen germination is higher than 20°C.     

From growth to autolysis: the murein hydrolases inEscherichia coli

Murein hydrolases cleave bonds in the bacterial exoskeleton, the murein (peptidoglycan) sacculus, a covalently closed bag-shaped polymer made of glycan strands that are crosslinked by peptides. During growth and division of a bacterial cell, these enzymes are involved in the controlled metabolism of the murein sacculus. Murein hydrolases are believed to function as pacemaker enzymes for the enlargement of the murein sacculus since opening of bonds in the murein net is needed to allow the insertion of new subunits into the sacculus. Furthermore, they are responsible for splitting the septum during cell division. The murein turnover products that are released during growth are further degraded by these hydrolases to products that can be recycled by the biosynthetic enzymes. As potentially suicidal (autolytic) enzymes, murein hydrolases must be strictly controlled by the cell, Inhibition of murein synthesis, for example by penicillin, triggers an unbalanced action of murein hydrolases causing bacteriolysis. InEscherichia coli, 14 different murein hydrolases have so far been identified, includingN-acetylmuramyl-l-alanine amidases,dd-endopeptidases,dd-carboxypeptidases,ld-carboxypeptidases, andN-acetylglucosaminidases. In addition lysozyme-like enzymes, called “lytic transglycosylases,” produce (1→6)-anhydromuramic acid derivatives by an intramolecular transglycosylation reaction.     

Influence of temperature upon growth and sporulation in two species of Phytophthora

The paper deals with the influence of temperature on the growth and sporulation of two species ofPhytophthora, viz.,P. palmivora Butl. andP. parasitica Dast. var.macrospora Ashby, the causal agents of fruit rots ofAchras sapota L. andAnona squamosa L. respectively. Germination of sporangia at different temperatures were also undertaken. There was marked variation in growth and sporulation of these two organisms. Isolate C (Phytophthora palmivora) showed no growth at 5° and 35°C, scanty growth at 10° and 32.5° with an optimum temperature between 26–28°C. On the other hand, Isolate S (Phytophthora parasitica var.macroscora) showed no growth at 10°C, but slight growth even at 37°C. Eight days exposure at 37°C completely stopped the growth of this Isolate. It showed best growth at 30°C and hence this was its optimum temperature. In general, Isolate C sporulated abundantly at all temperatures tested but reached its maximum at 25°C. On the other hand Isolate S showed best growth but failed to sporulate at any of the temperatures in 98 hours growth, although it sporulated freely when the incubation period extended up to two weeks. On the basis of temperature toleration the twoPhytophthora isolates are distinguished from each other as two different species. This confirms the earlier observations and nomenclature criterion as emphasized and formulated byTucker (1931). In the germination studies, it was observed that the indirect germination with the formation of abundant zoospores started from 5° and continued even up to 35°C, reaching maximum at 20°C. High temperature was not favourable for indirect germination. As the temperature proceeded increasing, the percentage of direct germination by formation of germ tubes also increased. Direct germination was observed from 10° which continued up to 37°C, with a maximum reach at 30°C. This confirms the epidemic of fruit rots in nature during monsoon season which is prevalent with the persistence of high humidity and rainfall.Taken from a thesis submitted by the author for the degree of Master of Science in the Faculty of Agriculture, Poona University, India.     

Ionic and non-ionic compounds in the germination of spores of bacillus megaterium texas

Summary Germination requirements of suspensions of spores of Bacillus megaterium, Texas strain, an l-alanine-inosine type, have been examined employing a decrease in optical density as the criterion of germination. In deionized water, l-alanine and inosine were devoid of germinative powers. They were effective only in conjunction with any one of a large variety of salts. Data are given for germination by the monovalent and divalent alkali metal chlorides. The potassium halides were germinative; potassium fluoride was the best. Salts of organic acids, including fatty acids and polycarboxylic acids, were germinative. The need for inosine could be bypassed by various salts, e.g., ammonium propionate or salts of dipicolinic acid. Also, l-alanine was replaceable by a variety of amino acids, provided suitable ions were present. In the presence of magnesium chloride, sodium dipicolinate could substitute for either inosine or l-alanine, but not both. Salts of n-hexylamine and n-heptylamine bypassed the need for both l-alanine and inosine. A primary role for ions in germination is proposed and a secondary, augmentative action is attributed to l-alanine and inosine.     

Kinetics,nutrition and inhibitor properties of basidiospore germination in schizophyllum commune

Summary The kinetics, nutritional requirements and inhibitor properties of basidiospore germination in the wood-rotting mushroom Schizophyllum commune were investigated. Measurements of changes in absorbancy and dry weight showed a lag period of approximately 15–20 hrs, followed by an abrupt increase in the rate of both processes. Individual basidiospore elongation also showed a lag phase and population changes were heterogenous in this regard.Carbohydrates active for basidiospore germination were grouped into four categories. Those sugars active between 15 and 20 hrs included glycogen, turanose, cellobiose, maltose, sucrose, glucose, fructose, mannose, galactose and xylose. Several sugar alcohols were only active between 30 and 60 hrs incubation and these included mannitol, sorbitol, ribitol, xylitol, arabitol, erythritol and glycerol. A third category of carbohydrates active for germination required prolonged incubation between 30 hrs and 7 days and included lactose, sorbose, raffinose, melezitose, trehalose, ribose and melibiose. Compounds without activity after 7 days included galactitol, inositol, acetate, succinate, gluconate, citrate, fumarate, rhamnose, fucose and inulin.Nitrogen sources active in basidiospore germination included complex organic nitrogenous substrates, asparagine, glutamine, arginine, urea and various ammonium salts.Germination was inhibited by cycloheximide, l-ethionine, p-fluoro-dl-phenylalanine, sodium azide, 2,4-dinitrophenol, phenylmercuric acetate and 2-deoxy-d-glucose. Alkali as a trapping agent arrested germination in glucose-(NH₄)₂SO₄ medium but was without ill-effect in glucose peptone broth.     

Effect of CO2 on the germination of conidiospores of Aspergillus niger

Summary The effect of different concentrations of CO₂ on the germination of conidiospores of Aspergillus niger A 5 has been studied using Pardee's buffer mixtures which maintain constant CO₂ tensions. The beneficial effect of CO₂ on germination is maximum at 0.5% CO₂ concentration, when 70–90% of the spores germinate within 6 hours, whereas in controls with air containing 0.03% CO₂ there is only 15–20% germination at 6 hours. At higher CO₂ concentrations this beneficial effect of CO₂ on germination diminishes and at 3% there is a complete inhibition of spore germination.The spore density and the ph of the medium have a noticeable effect on germination rates in presence of 0.5% CO₂. The germination rates decrease at spore densities higher than 5 · 10⁵/ml and at a ph of 6.8.Communication No. 431.     

Ultrastructural localization ofl -fucose residues in nuclei of root primordia of the green peaPisum sativum

Summary Sugars have been demonstrated in animal cell nuclei, but only a few studies have mentioned their presence in plant cell nuclei. In this studyl-fucose residues were localized at the ultrastructural level, usingUlex europeaus agglutinin I lectin, during the early stages of germination ofPisum sativum and in mature root tip cells. This sugar was present after 1 h of germination, and its concentration was found to vary during 3 to 6 h imbition; after 72 h of imbition its concentration had more than doubled. Furthermore, labelling was particularly abundant in the nucleolus, nucleolus-associated bodies and dense nuclear bodies. The possibility that some of thel-fucose residues are associated with proteins is discussed.     

Changes in fungi with age

Summary The uptake and incorporation of l-phenylalanine and l-leucine into various cell fractions of Rhizoctonia solani and Sclerotium bataticola decreased with cell age. As would be expected, most of the radioactivity from the amino acids was distributed in the soluble and protein fractions. Ratios of the radioactivity in the soluble fraction to that in the protein fraction indicated that in R. solani the mechanism for protein synthesis decreased with age. However, in S. bataticola, the decrease in protein synthesis with age may be due to a decrease in permeability to the amino acids.     

Spores of microorganisms

Calcium-dipicolinate (Ca-DPA)-rich and Ca-DPA-deficientBacillus cereus spores were incubated in a synthetic medium with germination stimulants and in bactopeptone medium with a fairly high calcium ion concentration. In the complex medium the germination of Ca-DPA-rich spores was completely blocked at a concentration of 0.5m CaCl₂, whereas the complete blockage of germination in the synthetic medium required higher concentrations (0.6–0.8m) of calcium chloride. Ca-DPA-deficient spores germinated more slowly and less completely in the synthetic medium than in the bactopeptone medium. The germination of these spores took place, however, even at higher calcium ion concentrations (0.6–0.8m). On the contrary, lower calcium chloride concentrations (0.1–0.4m) accelerated the germination of these spores in the synthetic medium and the final percentage of phase-dark and stainable spores was higher. “H-forms” of the Ca-DPA-rich and Ca-DPA-deficient spores prepared by acid titration germinated in both media. The germination of the latter spores being slower and proceeding less completely. “H-forms” germinated completely or partially in media with a high concentration of calcium chloride. The percentage of germinated spores, however, was strongly influenced by the concentration of this cation, especially the “H-forms” of Ca-DPA-deficient spores. Moreover, the germination of Ca-DPA-deficient spores in this medium was affected by the length of previous storage and, in the case of “H-forms” by the pH at which they were titrated. It was assumed that the increased permeability of calcium into the calciumundersaturated spore periphery in Ca-DPA-deficient and in “H-forms” of spores of both types co-determines (in the presence of germinants) the germinability of bacterial spores.     

Gravity and light control of the developmental polarity of regenerating protoplasts isolated from prothallial cells of the fern Ceratopteris richardii

A procedure has been developed for isolating protoplasts from prothalli of Ceratopteris richardii which can be cultured and are capable of regeneration. Protoplasts were isolated from 2-week-old gametophytes in a medium containing wall-digesting enzymes in 0.5 m sucrose, followed by purification of the released protoplasts by floating them up into a 0.5 m sorbitol layer. Regeneration occurred over a period of 10–24 days, and, under optimal osmotic conditions, followed the developmental pattern seen during spore germination, in that the first division gave rise to a primary rhizoid. Thus, prothallial protoplasts are comparable to germinating spores as suitable models for studies of developmental polarity in single cells. As in germinating spores, the polarity of development in regenerating protoplasts is influenced by the vectors of gravity and unilateral light. However, the relative influence of light in fixing this polarity is greater in regenerating protoplasts, while in germinating spores, the influence of gravity is greater. Received: 26 September 1997 / Revision received: 17 January 1998 / Accepted: 7 February 1998     

Certain aspects of the pH dependence of triggering inBacillus megaterium spores

Incubation of unactivatedBacillus megaterium 14581 spores in glucose, or in glucose plusl-alanine, at or below pH 3.6 resulted in germination arrested somewhere before onset of stainability. However, triggering continued at this reduced pH, and spores thus triggered were fully capable of completing the germination sequence in the absence of the germinants once the pH was neutralized. The same spores could be triggered either by a mixture of glucose andl-alanine or by a larger concentration of glucose alone. From this it was concluded that triggering results from an adequate stimulus which can be generated in different ways.l-alanine action in triggering has a pH profile distinct from that of glucose, suggesting that these two germinants have different receptor sites as well. At a level of acidity at which a weak glucose concentration triggered relatively few spores, a much larger fraction was found apparently distributed over a range of sub-triggering levels. Some of these could be made to trigger on transfer to a secondary reagent, or mixture of reagents, which by themselves are not very efficient germinants of the strain studied. The degree of additional triggering was found to depend on the nature of the complementary germinants, as well as on the pH at which glucose stimulated them. Evidence that spores may occupy stimulated states for finite lifetimes is presented and discussed.     

Medicago truncatula f.laxicycla (Leguminosae), a new taxon with loose fruit coiling promoting rapid germination of the fruit-retained seeds

Medicago truncatula is a member ofMedicago sect.Spirocarpos, in which all 34 species have coiled fruit, usually tightly coiled, that permanently retain the seeds. A variant ofM. truncatula from Libya is unique for the species in possessing very loosely coiled fruits, and is here recognized as a new forma,M. truncatula f.laxicycla E. Small. In germination tests of a number of species ofMedicago, the unusual Libyan variant proved to be the only one in which rapid and simultaneous germination of most of the seeds occurred while still within a fruit. The loose coiling of the fruit of the Libyan variety appears to facilitate water entry and seedling escape. Its rapid germination might adapt it to respond quickly to intermittently available moisture, but at the cost of abandoning the strategy of intermittent germination adopted by other populations ofMedicago sect.Spirocarpos.     

Biochemical aspects on the germination of conidiospores of Aspergillus niger

Summary Biochemical events occurring in synchronously germinating spores of Aspergillus niger strain 1617 were investigated. The spores were found to require l-proline (or l-alanine), glucose and phosphate for the complete germination. The germination process in the above synthetic medium could be divided into three phases: endogenous swelling, exogenous swelling and sprouting. The first swelling phase was not influenced by the severe environmental factors so far tested, while the second phase was found to be affected by them, especially the CO₂ concentration. Rates of increase in cellular substances and in consumption of environmental substances changed markedly after germ tubes sprouted. The first cellular synthesis thus far detected was nucleic acid synthesis in the exogenous swelling phase. At the end of this phase accumulation of free amino acids, mainly glutamic acid and alanine, was observed. Protein synthesis then followed. A conspicuous increase in O₂-uptake commenced in parallel with the active synthesis of protein, when germ tubes began to sprout.During the course of germination a shift of metabolic pattern from that of the spore to the mycelium was indicated by the ratios of total nitrogen/dry weight, RNA/DNA, oxygen consumed/glucose consumed, and oxygen consumed/total nitrogen taken at various time intervals.Rosalie B. Hite Post-doctoral Fellow of the University of Texas.