Lobosporangium, a new name for Echinosporangium Malloch, and Gamsiella, a new genus for Mortierella multidivaricata

Lobosporangium is proposed as a new name for Echinosporangium Malloch, a later homonym of Echinosporangium Kylin. Lobosporangium transversale was isolated from arid soils on three occasions between 1964 and 1968 but has not been reported again. Observations on sporangium development in culture revealed rapid sporangiospore germination, rapidly growing hyphae forming anastomoses, threefold dichotomously branching aerial sporangiophores and formation of clusters of eight sporangia. The sporangia of L. transversale are illustrated, and the placement of Lobosporangium in the Mortierellaceae is discussed. A new genus, Gamsiella, is proposed that is based on Mortierella multidivaricata. Sporangial ontogeny of Gamsiella is compared with that presented here for Lobosporangium.     

The host guides morphogenesis and stomatal targeting in the grapevine pathogen <Emphasis Type="Italic">Plasmopara viticola</Emphasis>

The oomycete grape downy mildew (Plasmopara viticola Berk. & Curt. Ex de Bary) is a serious pathogen of grapevine and spreads by extremely efficient cycles of asexual propagation. The high efficiency must involve efficient sensing of the host. We therefore analyzed the time course and morphology of the early development of this pathogen in a host system, by infection of leaf discs of grapevine (Vitis vinifera L. cv. Müller-Thurgau), and in a host-free system. Host factors were demonstrated to influence pathogen development in the following ways: (i) the release of zoospores from mature sporangia was accelerated, (ii) the morphogenesis of the germ tube was coordinated, and (iii) the zoospores were targeted to the stomata by factors that depended on stomata closure. The findings show that the early development of P. viticola is regulated, specifically and coordinately, by factors originating from the host plant.     

Developmental stage-dependent response ofPilobolus crystallinus sporangiophores to gravitative and centrifugal stimulation

Gravitropic response of sporangiophores ofPilobolus crystallinus was studied by successive microscopic observation of the sporangiophores horizontally placed in the dark (red light) and by analysis of sporangiophore response to centrifugal stimulation. Negative tropism against the gravitative and also centrifugal stimulation was found only in mature sporangiophores after development of sporangium and after the resumption of elongation beneath the fully-developed subsporangial vescle, but there was no response in younger sporangiophores, implying that the gravitative perception system of the sporangiophores is dependent on their developmental stages.     

In vitro evidence of mycoparasitism of the ectomycorrhizal fungus <Emphasis Type="Italic">Laccaria laccata</Emphasis> against <Emphasis Type="Italic">Mucor hiemalis</Emphasis> in the rhizosphere of <Emphasis Type="Italic">Pinus sylvestris</Emphasis>

Interactions between the ectomycorrhizal fungus Laccaria laccata and the soil fungus Mucor hiemalis f. hiemalis in co-culture, and in the rhizosphere of in vitro-grown Pinus sylvestris seedlings were investigated by light- and scanning electron-microscopy. In co-culture, mycelial growth away from the L. laccata colony reduced the number of aerial hyphae at the contact zone and increased the density and compactness of the mycelium-characterized gross morphology of the saprobic fungus. Although the growth of M. hiemalis was suppressed, no penetration of M. hiemalis hyphae after the colony was entered by L. laccata was observed. Instead, dense coiling of L. laccata hyphae around sporangiophores, overpowering them and causing them to disappear, was quite common. On nonmycorrhizal roots, sporangiospores germinated heavily and formed long hyphae for 2 days post inoculation, whereas their germination was totally inhibited on mycorrhizal roots. At 3 days after inoculation, only sporangia were seen with mycelial mats firmly attached to the roots by the mantle hyphae, whereas some remnants of sporangiophores, ruptured sporangial walls and degraded hyphae of M. hiemalis were overgrown by the mantle hyphae. During the next 3 days, the mantle-hyphae-invading sporangia formed short, thin branches that grew directly towards individual spores, tapering off upon contact.     

Transcellular ion currents during sporangium development in the water mould Achlya bisexualis

Changes in the pattern of electric currents that accompany the transformation of growing hyphae of Achlya bisexualis into sporangia have been examined. When hyphae were transferred to a non-nutrient buffer, they continued to extend for several hours and then gave rise to sporangia. Throughout this process, current (positive charge) flowed into the apical region that corresponds approximately to the future sporangium. The current ceased after the crosswall appeared. The sporangium then remained electrically quiescent, except for a brief intense burst of outward current at the 'homogeneous' stage of spore cleavage. The inward current during sporangium formation largely represents an influx of protons. Addition of nitrate abolished the flow of electric current with little effect on sporulation. The late burst of outward current is most probably an artefact, generated by the discharge of salts from the sporangial vacuole. The transcellular electric current apparently plays no role in sporangium formation or in spore cleavage. Calcium ions, however, are required and may traverse the plasma membrane.     

The Distribution of Cytoplasmic Vesicles, Multivesicular Bodies and Paramural Bodies in Elongating Sporangiophores and Swelling Sporangia of Thamnidium elegans Link

In Thamnidium elegans Link, cytoplasmic vesicles of variablesize were present in large numbers in sporangiophore apicesin agar and in smaller numbers in sporangiophore apices in air.Golgi-like cisternae were associated with small, sub-apicalclusters of vesicles. Apices in agar contained a nucleus andmitochondrion-free tip zone in which vesicles were concentrated.This zone was almost completely absent from apices in air. Inswelling sporangia, vesicles were sparce and were not concentratedagainst the wall. Rates of surface area increase were similarfor sporangiophores elongating, respectively, in agar and inair. Rates of surface area increase during sporangium swellingwere equal to or greater than rates of surface area increaseduring sporangiophore elongation. Vesicles were associated withformation of a secondary wall layer in swollen sporangia. Paramuralbodies and multivesicular bodies were present at all stagesof sporangiophore elongation and sporangium swelling. Isolatedhalo bodies (apical corpuscles) were present in walls at sporangiophoretips, and clusters of similar bodies were present in side wallsof sporangiophores.     

Diameter and amount of β-carotene determine the maximal phototropic bending angle ofPhycomyces sporangiophores

Wild-type sporangiophores at stage IVb (final developmental stage after sporangium formation) ofPhycomyces show a pronounced positive phototropism to unilateral white light. We found that the maximal bending angle was larger in thin sporangiophores than in thick ones, and larger in the sporangiophores containing a small amount of β-carotene than in those containing a large amount of it. These phenomena probably occur because of the increase in length of intracellular light path or in the intracellular light-attenuation coefficient, as supported theoretically.     

Antibacterial Activity and Mechanisms of Ethanol Extracts from <i>Scutellaria baicalensis</i> Georgi and <i>Magnolia officinalis</i> against <i>Phytophthora nicotianae</i>

Phytophthora nicotianae causes substantial economic losses in most countries where tobacco is produced. At present, the control of P. nicotianae mainly depends on chemical methods, with considerable environmental and health issues. We investigated the effects of ethanol extracts from Scutellaria baicalensis Georgi (SBG) and Magnolia officinalis (MO). On mycelial growth, sporangium formation, and zoospore release of P. nicotianae. Both extracts inhibited the growth of P. nicotianae, with mycelial growth inhibition rates of 88.92% and 93.92%, respectively, at 40 mg/mL, and EC50 values of 5.39 and 5.74 mg/mL, respectively. The underlying mechanisms were the inhibition of sporangium formation, the reduction of zoospore number, and the destruction of the mycelium structure. At an SBG extract concentration of 16.17 mg/mL, the inhibition rates for sporangia and zoospores were 98.66% and 99.39%, respectively. At an MO extract concentration of 2.87 mg/mL, the production of sporangia and zoospores was completely inhibited. The hyphae treated with the two plant extracts showed different degrees of deformation and damage. Hyphae treated with SBG extract showed adhesion and local swelling, whereas treatment with MO extract resulted in broken hyphae. Mixture of the extracts resulted in a good synergistic effect.     

Cytology of Thamnidium elegans link

The resting nuclei in hyphae, sporangiophores and sporangiospores of sporangia and sporangiola of Thamnidium elegans consist of a large centrals nucleolus and a shell of chromatin surrounding the nucleolus. Division of the nucleus in hyphae and sporangiospores is achieved by elongation and constriction.     

Lag period for phototropism inPilobolus crystallinus sporangiophores

The lag period for the second positive curvature was examined inPilobolus crystallinus sporangiophores. The lag period for curvature development was 20–30 min at lower fluence rates than 6.32 nmol/m²s but greatly extended at higher fluence rates. When a 20-min symmetrical irradiation with blue light was applied before a 20-min unilateral blue light irradiation, sporangiophores bent as much as those unilaterally and continuously irradiated for 40 min. However, when a 20-min unilateral irradiation was followed by a 20-min symmetrical irradiation, sporangiophores did not show any curvature. That is, the reaction during the first 20 min of the lag period is independent of light direction. This light-direction-independent lag period is considered to be the duration required for adaptation. The lag period for phototropism was also extended when fluence rate was reduced after the start of irradiation. These results suggested that an adaptation process is involved in phototropism ofPilobolus.     

In vitro studies on the mechanisms of endospore release by Rhinosporidium seeberi

Studies of Rhinosporidium seeberi have demonstrated that this organism has a complex life cycle in infected tissues. Its in vivo life cycle is initiated with the release of endospores into a host's tissues from its spherical sporangia. However, little is known about the mechanisms of sporangium formation and endospore release since this pathogen is intractable to culture. We have studied the in vitro mechanisms of endospore release from viable R. seeberi's sporangia. It was found that watery substances visibly stimulates the mature sporangia of R. seeberi to the point of endospore discharge. The internal rearrangement of the endospores within the mature sporangia, the opening of an apical pore in R. seeberi's cell wall, and the active release of the endospores were the main features of this process. Only one pore per sporangium was observed. The finding of early stages of pore development in juvenile and intermediate sporangia suggested that its formation is genetically programed and that it is not a random process. The stimulation of R. seeberi's sporangia by water supports the epidemiological studies that had linked this pathogen with wet environments. It also explains, in part, its affinities for mucous membranes in infected hosts. The microscopic features of endospore discharge suggest a connection with organisms classified in the Kingdom Protoctista. This study strongly supports a recent finding that placed R. seeberi with organisms in the protoctistan Mesomycetozoa clade. This revised version was published online in June 2006 with corrections to the Cover Date.     

Electron microscopy of primary zoosporogenesis inPlasmodiophora brassicae

Primary zoosporogenesis in resting sporangia ofPlasmodiophora brassicae that had been incubated for 14 d in culture solution containing turnip seedlings was examined by transmission electron microscopy. A single zoospore differentiated within each sporangium, the differentiation being initiated by the emergence, of two flagella in the tight space formed by invagination of the plasma membrane within the sporangium. The differentiazing zoospore was similar in intracellular aspects to sporangia within clubroot galls. Then a deep groove formed on the zoospore cell body by further invagination of the plasma membrane. Two flagella appeared to coil around the zoospore cell body in parallel along this groove. Thereafter, the cell body lost the groove and became rounded following the protoplasmic condensation (contraction of cell body) during late development, and assumed an irregular shape at the stage of maturation. Intracellular features in, developing and mature zoospores were complicated, being characterized by electron-dense nuclei and mitochondria, microbodies, cored vesicles and various unidentified cytoplasmic vesicles and granules. A nucleolus-like region was observed only in the nucleus of the mature zoospore. A partially opened germ, pore was also seem in the sporangium containing the mature zoospore.     

A STUDY OF LIGHT INTENSITY,PERIODICITY, AND WAVELENGTH ON ZOOSPORE PRODUCTION BY PROTOSIPHON BOTRYOIDES KLEBS12

When mature Protosiphon cells were placed in darkness, zoospore production was more extensive and was completed in a shorter time at a temperature of 27 C than at 22 or 15 C. Cool-white fluorescent (Sylvania) light inhibited the process measurably at a radiation intensity of 0.6±10³ ergsjcm²-sec; inhibition was 96% complete at 14±10³ ergs/cm²-sec. For mature cells previously grown under repeated 12-12 hr light-dark cycles, a dark period of approximately 2 hr at 22 C allowed cell division to proceed to a stage such that reillumination did not inhibit continued development of zoospores. Monochromatic light from 402 to approximately -494 nm, as compared to darkness, inhibited zoospore formation; maximal inhibition was at 432-461 nm. In contrast, monochromatic light from 522 to 726 nm stimulated zoospore formation relative to darkness. Synchronous zoospore production was obtained using the following regimes: (A) 12 hr cool-white alternated with 12 hr yellow, (B) 12 hr cool-white alternated with 12 hr blue. Under regime A synchronous zoospore release (following synchronous production) occurred near the end of the yellow irradiation period, while under regime B it occurred near the end of the cool-white irradiation period. The significance of this in terms of photoprocesses and possible photoreceptors is discussed.     

A spectrophotometric approach for determining sporangium and zoospore viability of Plasmopara viticola

A spectrophotometric method for determining the viability of sporangia and zoospores of the oomycete Plasmopara viticola (causal agent of grapevine downy mildew) is described and evaluated to overcome the limitations of currently available methods for assessing propagule viability. Sporangia produced on leaf discs in the laboratory were harvested at different days after the initiation of sporulation (DAS) to yield differences in sporangium viability. Sporangia were suspended in sterile water, the suspensions were placed in a cuvette, and sporangium germination was monitored in a spectrophotometer (λ = 600 nm) at 2- to 3-min intervals for 5 hr. Absorbance started to increase after sporangia were suspended in water for ~30–60 min followed by major peak(s) for younger sporangia (1–3 DAS), whereas low to no increase in absorbance was observed for senescent sporangia (>7 DAS). Microscopic observation confirmed that the increase in absorbance corresponded to the release and active swimming of zoospores, whereas absorbance decreased when zoospores encysted and settled. A positive correlation (r = .839, p = .0365) was observed when the time to the initial increase in absorbance was plotted against the age of sporangia. The time to the absorbance peak (marking the time of maximum zoospore movement) was shortest for immature sporangia (0 DAS), longest for young sporangia (2 DAS) and decreased for mature and senescent sporangia. A similar pattern was observed for the standardized area under the absorbance curve (indicating the overall quantity of zoospores released), for which values were lowest for immature and senescent sporangia, highest for young sporangia and intermediate for mature sporangia. Consistent patterns obtained across two independent experiments suggest that the method is reproducible and may be further developed for other zoospore-releasing pathogens.     

Secondary Sporangium Formation in Phytophthora Palmivora (Butl.) Butl

Very few sporangia of Phytophthora palmivora germinated directlyand produced secondary sporangia in distilled water and in solutionsof amino acids and carbohydrates at 30 °C. Although 1.0per cent (w / v) peptone and yeast-extract stimulated a highpercentage of germination by formation of germ tubes, less than1.0 per cent of the germinated sporangia produced secondarysporangia. Secondary sporangium formation was induced by transferringgerminated primary sporangia from a nutrient medium of sufficientlyhigh concentration to either distilled water or dilute solutionsof organic and inorganic compounds immediately after emergenceof the germ tubes. The percentage of germinated sporangia formingsecondary sporangia was influenced by both the nature and concentrationof the medium into which they were transferred. The secondarysporangia were significantly smaller than the primary sporangia. Phytophthora palmivora, germination, sporangium, Theobroma cacao L., cocoa     

In vitro studies of Coelomomyces punctatus from Anopheles quadrimaculatus and Cyclops vernalis

Attempts to grow mycelium of Coelomomyces punctatus from Anopheles quadrimaculatus larvae were made using more than 50 combinations of known vertebrate and invertebrate tissue culture media and microbiological media. Growth and/or differentiation of mycelium into sporangia were observed in several media. Significant growth of hyphal fragments and differentiation into young resting sporangia occurred in conditioned Mitsuhashi-Maramorosch insect tissue culture medium. This medium was conditioned by growth for 3 weeks in it of Varma's Anopheles stephensi tissue culture cells and was supplemented with 20% heat-inactivated fetal bovine serum and a synthetic tripeptide, glycyl-histidyl-lysine. Limited growth and elongation of lateral hyphal branches and subsequent development into resting sporangia with typical outer wall markings and pigmentation of mature forms were observed in a modified brain-heart infusion medium. Some media stimulated hyphae to develop into smooth-walled, spherical bodies of size and appearance typical of young sporangium initials but with no further maturity. In most media, no growth or development of mycelium occurred, but the fungus remained alive for 2–4 weeks. Mycelium of C. punctatus dissected from Cyclops vernalis did not grow and develop in any of the media utilized. However, in one case the mycelium differentiated into gametes shortly after dissection into modified brain-heart infusion medium.     

Effects of specific cations on aggregation and fruiting body morphology in the myxobacterium Stigmatella aurantiaca.

The cation requirements for fruiting body formation in the myxobacterium Stigmatella aurantiaca on agarose were determined. Calcium alone caused the cells to aggregate into interconnecting ridges. Under these conditions, stalk formation was severely depressed but sporangia frequently formed. The combination of magnesium and manganese was necessary for optimal formation of discrete aggregates (rather than ridges) and stalks. Manganese inhibited sporangium development. The inclusion of calcium into the magnesium-manganese medium overcame the inhibition by manganese and stimulated the production of multiple sporangia.     

Internal Changes in Hyphae of Rhizopus sexualis (Smith) Callen and Mucor hiemalis Wehm. associated with Zygospore Formation

The distribution of nucleic acids, nuclei, mitochondria, andreserve foods in vegetative hyphae, zygophores, and developingzygospores of Rhizopus sexualis and Mucor hiemalis were examinedby differential staining. The extreme tips and growing zones of vegetative hyphae containeda high concentration of RNA and numerous mitochondria. Nucleiwere not present at the extreme tip but were numerous just behindit. In older parts of the hyphae the concentration of RNA waslow and both nuclei and mitochondria were fewer than in thezone of elongation. Glycogen and lipids were present in all parts of the livinghyphae except the extreme tips and were more highly concentratedin the older parts of the hyphae. Young zygophores showed a much lower RNA/DNA ratio than thatfound in the vegetative hyphal tips. Transfer of colonies from20? C to temperatures of less than 10? C, which is known toprevent zygospore initiation, caused some but not all recognizablezygophores of R. sexualis, but not those of M. hiemalis, torevert to the RNA/DNA ratio characteristic of vegetative hyphae.Some zygophores of Rhizopus and most of those of Mucor developedinto sporangiophores at low temperature, retaining the relativelylow RNA/DNA ratio throughout development. It is suggested thata reduction in the RNA/DNA ratio is an early step in the changefrom the vegetative state to the reproductive one. At firstthis step is reversible, but soon becomes irreversible by anadditional step, the nature of which is unknown. For some timeafter this the reproductive hyphae are capable of either producingasexual sporangia or of conjugating to produce zygospores. Onceconjugation has taken place development either ceases or continuesuntil the spore is fully mature, but it cannot under any circumstancesthen be reversed. The development and maturation of the zygospore involves a greatincrease in number of both nuclei and mitochondria and in theconcentration of glycogen and lipids.     

Ontogeny of strobili, sporangia development and sporogenesis in Equisetum giganteum (Equisetaceae) from the Colombian Andes

Studies on the ontogeny of the strobilus, sporangium and reproductive biology of this group of ferns are scarce. Here we describe the ontogeny of the strobilus and sporangia, and the process of sporogenesis using specimens of E. giganteum from Colombia collected along the Rio Frio, Distrito de Sevilla, Piedecuesta, Santander, at 2200m altitude. The strobili in different stages of development were fixed, dehydrated, embedded in paraffin, sectioned using a rotatory microtome and stained with the safranin O and fast green technique. Observations were made using differential interference contrast microscopy (DIC) or Nomarski microscopy, an optical microscopy illumination technique that enhances the contrast in unstained, transparent. Strobili arise and begin to develop in the apical meristems of the main axis and lateral branches, with no significant differences in the ontogeny of strobili of one or other axis. Successive processes of cell division and differentiation lead to the growth of the strobilus and the formation of sporangiophores. These are formed by the scutellum, the manubrium or pedicel-like, basal part of the sporangiophore, and initial cells of sporangium, which differentiate to form the sporangium wall, the sporocytes and the tapetum. There is not formation of a characteristic arquesporium, as sporocytes quickly undergo meiosis originating tetrads of spores. The tapetum retains its histological integrity, but subsequently the cell walls break down and form a plasmodium that invades the sporangial cavity, partially surrounding the tetrads, and then the spores. Towards the end of the sporogenesis the tapetum disintegrates leaving spores with elaters free within the sporangial cavity. Two layers finally form the sporangium wall: the sporangium wall itself, with thickened, lignified cell walls and an underlying pyknotic layer. The mature spores are chlorofilous, morphologically similar and have exospore, a thin perispore and two elaters. This study of the ontogeny of the spore-producing structures and spores is the first contribution of this type for a tropical species of the genus. Fluorescence microscopy indicates that elaters and the wall of the sporangium are autofluorescent, while other structures induced fluorescence emitted by the fluorescent dye safranin O. The results were also discussed in relation to what is known so far for other species of Equisetum, suggesting that ontogenetic processes and structure of characters sporoderm are relatively constant in Equisetum, which implies important diagnostic value in the taxonomy of the group.