MORPHOLOGY AND LABORATORY CULTIVATION OF ECHINOSTELIUM MINUTUM

Alexopoulos , Constantine J. (State U. Iowa, Iowa City.) Morphology and laboratory cultivation of Echinostelium minutum. Amer. Jour. Bot. 47(1): 37—43. Illus. 1960.—The morphology of the sporangium, spores, swarm cells and Plasmodium of the white form of Echinostelium minutum is described. A peridium is present in the early stages of sporangial formation. It eventually disappears leaving only a small collar at the base of the columella. The structure of the spore wall is unique in this genus. The spore case may be described as consisting of a thin wall with several thickened portions distributed over its surface. These are particularly evident in germinated spores. Spore germination and swarm cells are described for the first time. Swarm cells are biflagellate with two long anterior flagella of nearly equal length. The Plasmodium remains microscopic until fruiting time, when it gives rise to but a single sporangium. The plasmodial protoplast never becomes differentiated into veins but remains more or less homogeneous. It exhibits almost imperceptibly slow, irregular streaming instead of the reversible, rapid, rhythmic motion characteristic of plasmodia of most other Myxomycetes which have been studied. It typifies, therefore, a third type of Plasmodium which may be placed alongside that of the Physarales, and that of Stemonitis flavogenita. The laboratory cultivation of E. minutum from spore to spore on agar media is reported here for the first time.     

SPORES OF BOTRYOPTERIS GLOBOSA AND BOTRYOPTERIS AMERICANA FROM THE PENNSYLVANIAN

A study of spores from fertile pinnae of Botryopteris from middle and upper Pennsylvanian coal balls from Iowa, Illinois, and Kansas indicates that there are two distinct species, Botryopteris globosa and B. americana. The organization and attachments of fertile pinnae and the sporangial morphology and dimorphism are identical in the two species. Data are given on fertile pinnae dimensions, attachments of six fertile pinnae, spore counts from individual sporangia, and spore morphology. The ornamentation of B. americana spores is verrucate to rugulate with verrucae fusing to a variable extent to form bars and convolute ridges; B. globosa spores are vermiculate or fossulate to densely rugulate with scattered verrucae. Comparisons are made with B. forensis and a re-interpretation of the spore forms of B. forensis is suggested.     

MORPHOGENESIS OF THE SPORANGIUM OF COMATRICHA

Goodwin , Donna C. (State U. Iowa, Iowa City.) Morphogenesis of the sporangium of Comatricha. Amer. Jour. Bot. 48(2): 148–154. IIlus. 1961.—Three species of the myxomycete genus, Comatricha, were studied: Comatricha nigra, C. fimbriata, and C. elegans. The sporangia developed on living bark of Ulmus americana in moist chamber. The hypothallus is formed under the homogeneous protoplasmic mass of the sporangial initial. The fibrous threads of the hypothallus bend upward, lengthening at the apices to become the fibers of the stalk and columella. The undifferentiated protoplasm is carried upward as the stalk elongates. When the columella has attained its mature height, threads bend out from the columella and grow toward the periphery of the sporangium. These threads form the capillitium. Simultaneous with the appearance of the capillitial initials, the peridium, a delicate membrane, forms. After the capillitium is mature, the protoplast cleaves into many cells, the future spores. The peridium evanesces early in the stage of spore maturation. Cellulose is present in the stalk, capillitium, and spore walls but is not found in the peridium or hypothallus. The capillitium of these species follows a developmental pattern designated as the “Comatricha-type” by Ross (1957) from a study of Comatricha typhoides. The taxonomic implications of the sporangial developmental pattern are discussed.     

Relationship between agitation speed and the morphological characteristics of Verticillium lecanii CS-625 during spore production

Verticillium lecanii is recognized as an entomopathogenic fungus, and has high potential in the biological control of pests. In this study, it was investigated that the relationship between agitation speed in a 2.5 L stirred tank reactor (STR) at 25°C and initial pH 5.5, and the morphological characteristics of V. lecanii CS-625, such as hyphal length/width, spore length/width, and the number of tips during spore production. The agitation speed affected the hyphae patterns and the number of tips. The number of spores rapidly increased at 48 to 60 h of cultivation, and the highest spore productivity (2.5 × 10¹⁰ spore/L·h at 60 h) occurred with an agitation speed of 350 rpm and an aeration rate of 1.0 vvm. The number of tips increased in proportion to the increase in spore production during the same culture time. The highest number of tips (4.8 × 10⁸ tipJ.mL) was obtained at 72 h of cultivation. The shortest mean spore length (2.8 μm) was obtained at 60 h of cultivation. Therefore, it was determined that the increased number of tips and decreased mean spore length were closely related to the production of V. lecanii spores.     

Effect of cultivation conditions on spore production from Bacillus amyloliquefaciens B128 and its antagonism to Botrytis elliptica

Aims:  To maximize spore production by Bacillus amyloliquefaciens B128, and its antagonism to the fungal pathogen Botrytis elliptica B061. Methods and Results:  In the 5-l stirred-tank bioreactor (STR), with the 0·5 vvm aeration rate, an agitation rate of 200 rev min⁻¹ significantly enhanced the spore yield compared to the same in 300 rev min⁻¹ cultivations. In a 20-l airlift bioreactor (ALR) the maximal spore production was further increased with a controlled aeration rate of 2·5 vvm operated in a 24-mesh net-draft tube mode, and no pH control cultivation. This spore yield in the 20-l ALR was five- and eightfold higher; in addition the cultivation period was 19 h shorter, compared to that obtained from shaker flask and in the 5-l STR cultivations respectively. Conclusions:  Although culture conditions are still to be optimized, by using an ALR with net-draft tube, a scaling up from shaker flasks and STR to ALR of spore production by the strain B128 is technically feasible. Significance and Impact of the Study:  The spore yields obtained using bioreactors were much higher than those previously reported. The freshly produced spore preparations from the B128 strain significantly antagonized the grey mould pathogen B. elliptica.     

Inhibitory effect of botanical extracts against Alternaria alternata of aloe vera dry rot

Aloe vera dry rot caused by Alternaria alternata, is one of the most serious fungal diseases affecting the commercial cultivation of aloe vera in North India. Control of this disease through chemical is quite expensive and not ecofriendly. The plant extracts as biopesticdes act as a vital components for the management of this disease. Evaluation of some plant extracts was carried out against A. alternata in laboratory conditions. The extracts were prepared at 5% and 10% concentration and were evaluated through inhibition in radial growth (food poison technique) and spores (conidia) germination (hanging drop technique) against A. alternata. Neem leaf extract gave 58.6% inhibition in radial growth and 56.5% in spore germination at 10% concentration followed by Ocimum sanctum which was found effective and gave 54.7% inhibition in radial growth and 50.4% in spore germination over control.     

Characteristics of spore germination and protonemal development in Hypnum pacleseens

The spore germination, protonemal development, and gametophyte differentiation of Hypnum pacleseens were observed in cultivation. Photomicrographs showed that spore germination of Hypnum pacleseens occured within the exospore. Its protonema is massive with filamentous chloronema formed inside. The terminal part of the chloronema differentiated into filamentous caulonema and its rhizoid was derived from the apical cell of the filamentous chloronema. The initial cell of gametophyte differentiated from chloronema and caulonema. Sporeling-type of Hypnum pacleseens is developmentally similar to Glyphmitrium-type.     

Germination and survival of tetraspores of Kappaphycus alvarezii var. alvarezii (Solieriaceae, Rhodophyta) introduced in subtropical waters of Brazil

The carrageenophyte Kappaphycus alvarezii was introduced in 1995 and vegetatively propagated in Ubatuba, São Paulo State, Brazil, for the purpose of commercial cultivation. This species produces tetraspores mainly in the austral summer and fall. Tetraspore germination and survival were studied under different conditions of temperature, photon flux density, and photoperiod in the laboratory. Field experiments were also carried out. Although tetraspores of K. alvarezii germinated, they had low survival rates, most dying after 20 days. Recruitment of K. alvarezii tetraspores did not occur in experiments conducted in the field. The results indicated that the establishment of K. alvarezii via spore production in the natural environment of the south‐east coast of Brazil is rather remote.     

碎米藓属两种藓类植物孢子萌发与原丝体发育特征研究

为获取其孢子萌发类型与该属植物系统发育、生态选择以及生殖策略选择的相关性,该研究通过室内人工培养的方式,在微米量级下观察并描述了碎米藓属(Fabronia)碎米藓(F.pusilla)和东亚碎米藓(F.matsumurae)两种藓类植物孢子萌发、原丝体发育和配子体发生的过程。结果表明:(1)两种藓类植物孢子均为壁外萌发,均产生由1~15个半圆球形细胞组成的绿丝体(chloronema)短枝;(2)碎米藓在绿丝体顶端分化产生轴丝体细胞,东亚碎米藓未分化产生轴丝体(caulonema);(3)两种藓类植物配子体原始细胞均在绿丝体上分化产生。参照Nishida对藓类植物孢子萌发型划分标准,分析并确定了碎米藓属两种藓类植物孢子萌发型均为蓑藓型(Maromitrium-type),为碎米藓属的系统分类提供了发育学证据。     

Evaluation of inoculum performance for enhancing gamma-linolenic acid production from Mucor rouxii

Aims: To facilitate a cost‐effective preparation of spore inoculum with good capacity for gamma‐linolenic acid (GLA) production from Mucor rouxii. Methods and Results: Sporangiospore production, mycelial growth ability and fatty acid composition of M. rouxii were determined. Compared with fungal cultivation on solid semi‐synthetic media, high spore production was achieved from M. rouxii grown on rice grains, particularly polished rice (30·7 g kg^?1 initial substrate). Variations in the fatty acid profiles were found in the spores grown on different types of solid media, whereas the spores obtained at different ages from cultivated polished rice showed a similar fatty acid profile. Using the inocula from different spore‐forming media and culture ages, and low temperature storage, not much change in the vegetative growth of submerged cultures or fatty acid composition of mycelia was observed. Conclusion: The spores generated on polished rice exhibited a high performance for GLA production. Age of spore and timing of spore storage at low temperature did not affect on fatty acid profile of the mycelial cultures. Significance and Impact of the Study: The simple, low cost method of inoculum preparation can be applied for large‐scale production of GLA‐rich oils, which reduce a time constraint and variation in fatty acid composition.     

SPORE RELEASE IN ACROCHAETIUM SP. (RHODOPHYTA) IS BACTERIALLY CONTROLLED1

The facultative red algal epiphyte Acrochaetium sp. liberated spores preferentially and recruited more successfully in laboratory cultures when its host Gracilaria chilensis C. J. Bird, McLachlan et E. C. Oliveira was present. The same effect was also induced by cell‐free medium from G. chilensis, suggesting it contained a molecular signal. Antibiotics prevented spore release in Acrochaetium sp., even when G. chilensis was present, suggesting a prokaryotic origin of the signal. Simultaneous application of N‐butyl‐homoserine‐lactone (BHL) restored the spore‐release capacity, which demonstrated that spore release was not directly inhibited by the antibiotics and indicated that bacterially generated N‐acyl‐homoserine‐lactones (AHLs) regulate spore release. An involvement of AHL was further indicated by the fact that two different halofuranone inhibitors of AHL receptors also inhibited spore release when they were applied at relatively low concentrations. Of seven different AHLs tested, only BHL induced the effect. However, BHL was only active at relatively high concentrations (100 μM), and it was not detected in spore‐release‐inducing medium of G. chilensis. Another water‐soluble AHL or an AHL structure analog is therefore probably the active compound in G. chilensis cultures. The data presented demonstrate that life cycle completion in Acrochaetium sp. strongly depends on bacteria, which are not always present in sufficient numbers on the alga itself. Exogenous bacteria that are associated with G. chilensis or with other potential substrates may therefore trigger timely spore liberation in Acrochaetium sp., provided that the necessary concentration of AHL is reached. This first finding of AHL perception in a red alga confirms that AHL signalling is more widespread among eukaryotes than was thought until recently. However, spore release of a second red alga, Sahlingia subintegra (Rosenv.) Kornmann, was unaffected by AHL, and the reaction observed is therefore not universal.     

Influence of ultraviolet radiation on spore liberation in marine macroalgae Ulva fasciata (Ulvales,Chlorophyceae) and Gracilaria corticata (Gracilariales,Rhodophyceae)

The effect of ultraviolet‐B (UV‐B) and UV‐A radiation on spore liberation in the intertidal marine macroalgae Ulva fasciata Delile (Chlorophyceae) and Gracilaria corticata J.Agardh (Rhodophyceae) was investigated. The two algae were exposed to UV‐A and UV‐B radiation separately for 10, 20, 30, 45 and 60 min and percentage inhibition of spore liberation was determined in controlled laboratory conditions. The spore liberation period in UV treated algae was extended for 4 days in U. fasciata and 9 days in G. corticata. UV‐B radiation inhibited spore liberation as much as 76.6% in U. fasciata and 55.5% in G. corticata at 60 min exposure. A significant positive correlation was observed between percentage inhibition of spore liberation and length of UV‐B exposure in both U. fasciata and in G. corticata. Similarly, UV‐A radiation also inhibited spore liberation as much as 75% in the former and 50% in the latter. There was a significant correlation between inhibition of spore liberation and length of UV‐A exposure in U. fasciata and in G. corticata. Analysis of variance results showed inhibition of spore liberation at 60 min of UV exposure differed significantly with that of other exposure lengths. The present findings reveal that UV‐A radiation also had an impact on spore liberation but to a lesser extent than UV‐B radiation. Thallus thickness and plant location on the shore determines their exposure to UV radiation. High UV impact was seen for U. fasciata growing in the upper parts of the intertidal region with a thin sheet like thallus and high surface area resulting in higher inhibition of spore liberation than in G. corticata.     

Licht- und elektronenmikroskopische Untersuchungen von Entwicklungsstadien der FlechteEndocarpon pusillum unter Kulturbedingungen

The lichenEndocarpon pusillum Hedw. was cultivated under laboratory conditions on agar, silica gel and soil substrate. Selected developmental stages of the life cycle (germination, contact between the symbionts, cortex, squamule and perithecia development) were studied by light and scanning electron microscope.—It could be shown that the spores had no rigid spore walls with germination colpies and the spore cells which are in contact with the substrate were formed directly into germination tubes.— Further studies showed that the initial contact between the components was thigmotropic and both the form and the gelatinous matrix around the algal cells play an important role in this process. — The development of the cortex occurs under reduced moisture conditions resulting in a reduced algal reproduction. The thickness of the cortex was dependent on light intensity during cultivation. The cortex originated from hyphae, which developed beyond the algal layer and were combined to a tight network.—Fruiting bodies with spores and hymenial algae were only formed in cultures on soil substrate.

Frau Prof. Dr.Elisabeth Tschermak-Woess zu ihrem 70. Geburtstag gewidmet.     

Sexual reproduction and soil tillage effects on virulence of Pyrenophora teres in Finland

Knowledge of the virulence of a pathogen population and recognition of the risks of changes in the virulence spectrum are essential in breeding crops for disease resistance. Sexual recombination in a pathogen increases the level of genotypic diversity and can influence the virulence spectrum. This study aimed to determine how sexual recombination can change virulence of the barley pathogen Pyrenophora teres and whether the barley cultivation system, no‐tillage or normal tillage, influences P. teres virulence. The inheritance of avirulence/virulence in P. teres following sexual reproduction was studied in three artificially created pathogen populations. The first was a product of crossing two net forms of the pathogen, and the second and the third were products of crossing net and spot forms. None of the progeny generated resembled the parents exactly. The average similarity of the progeny isolates of the net by net cross with the parental type, based on avirulence/virulence tests, was 92%. That for net and spot form progenies was 58% in comparison with the net form parents and 73% with the spot form parents. The virulence reactions of the progeny isolates did not correlate with morphological traits of the isolates: growth rate on agar, spore production, spore width, spore length and numbers of septa per conidium. To study the effect of the barley cultivation method on P. teres virulence, 313 single‐spore cultures were obtained from barley fields. Two hundred and seventy‐six of the isolates represented the spot form and 37 represented the net form of P. teres. No association was established between the tillage method and virulence for either the net form or the spot form isolates.     

Group Recruitment and Early Survival of Mazzaella Laminarioides

Several phycocolloid-producing Rhodophyta of significant economic importance are coalescing species, able to fuse with conspecifics during recruitment, reach larger sizes and increase their survival. In these species spores are needed to start cultivation (e.g. Gigartina, Mazzaella) or to increase the seed stocks, to renew senescent clones or to enlarge the base of genetic variation of vegetatively propagated species (e.g. Chondrus, Gracilaria, Eucheuma). This study uses Mazzaella laminarioides to evaluate some key features that influence recruitment success. Field measurements indicate that in any recruitment event a variable amount of the spores reaching a given place may form groups of 2 to over 100 coalescing spores, while field experiments support the idea that early recruitment success is a function of the number of coalescing spores forming the individual, as multisporic, coalescing recruits have higher survival rates than sporelings formed by one or a few spores. Therefore, group recruitment (spores settling and recruiting in close spatial proximity) appears as a prerequisite for sporeling coalescence and early recruitment success. In turn, laboratory experiments suggest that the frequency of group recruitment and coalescence increases with increasing spore abundance and with slight Ca⁺⁺ additions to the culture medium. These last two factors could be handled by farmers to improve the success of spore inoculations of coalescing species.     

Effect of water activity and relative air humidity on the growth of Penicillium chrysogenum thom, Aspergillus repens (Corda) Sacc., and Trichoderma viride Pers. isolated from living spaces

Original data on the growth parameters of the fungi Penicillium chrysogenum Thom, Aspergillus repens (Corda) Sacc., and Trichoderma viride Pers. isolated from living spaces in Moscow are presented. Spore germination, fungal growth, and the radial growth rate of the colonies were investigated upon cultivation on agarized nutrient media with different water activity (a_w) values. Spore germination and fungal growth were studied in house dust under laboratory conditions at different relative air humidity (RH). It was shown that, at decreased a_w and RH, the spore germination time increased, as did the period from germination to mycelium and conidia formation, while the radial growth rate of colonies decreased. House dust was found to be a suitable growth substrate for A. repens and P. chrysogenum, supporting their complete life cycle. It was suggested that house dust is unsuitable as a substrate for the growth of T. viride. The a_w and RH ranges for development of these micromycetes were determined. On this basis, the A. repens, P. chrysogenum, and T. viride strains isolated from living spaces were identified as xerophilic, xerotolerant, and hygrophilic ones, respectively.     

Changes in the Lipid Composition of Mucor hiemalis Sporangiospores Related to the Age of the Spore-Forming Culture

Analysis of sporangiospore lipids of the fungus Mucor hiemalis F-1156 showed that alterations occur in the content of fatty acids and individual classes of lipids during long-term cultivation (for about 20 days). The changes in the lipid composition related to the age of the spore-forming mycelium suggest an important role of sporangiospore lipids in spore germination and in further development of the spherical cells formed in this processes. The M. hiemalis F-1156 sporangiospores with a lipid pool exhausted during long-term cultivation can give rise to both mycelial and yeastlike growth.     

梨蒴珠藓孢子萌发与原丝体发育特征研究

为了解梨蒴珠藓（Bartramia pomiformis）孢子萌发和原丝体发育特征,在显微镜下观察室内人工培养的梨蒴珠藓单倍配子体发育过程。结果表明,梨蒴珠藓孢子吸水膨胀5 d后,开始破壁萌发,原丝体系统以丝状绿丝体为主,轴丝体在绿丝体上分化产生。培养22 d后,配子体在轴丝体细胞上分化产生。参照Nishida的标准,梨蒴珠藓孢子萌发类型为真藓型（Bryum-type）。这为梨蒴珠藓的人工扩繁提供了发育学基础资料。     

Pilot production of Clonostachys rosea conidia in a solid‐state fermentor optimized using response surface methodology

The nonpathogenic, saprophytic fungus Clonostachys rosea is one of the most powerful fungal biological control agents (BCAs). However, the production of fungal BCAs is still a major constraint for their large‐scale use and commercialization. Here, we developed a novel solid‐fermentation reactor that is light transparent and ventilated both at the top and the bottom, and optimized C. rosea cultivation conditions in solid‐state fermentation using response surface methodology. The growth area of spores provided by the novel fermentor was two times that of the traditional one. A quadratic polynomial model was developed, which indicated the effects of variables on the conidia yield. The greatest spore production of 3.50 × 10¹⁰ spores/g‐dry‐matter was obtained after 11 days at the initial moisture content of 69.2% w/w, the medium thickness of 3.84 cm, and the porosity of 0.37%. The optimized spore yield was increased by one order of magnitude. The fermentation time was shortened from 15 to 11 days. With the novel solid‐fermentation reactor, increase in C. rosea spores production and decrease in fermentation time were achieved. Current data imply that both the novel solid‐fermentation reactor designed and the optimized fermentation conditions are suitable for industrial‐scale C. rosea spore production.     

Quality control of multisource aeroallergen data

Pollen and spore prevalence data from 14 air sampling stations were retrospectively analyzed for accuracy of taxa identification and counting. Pollen identification was excellent and levels calculated by our laboratory closely matched those reported by most stations. Neither identifications nor counts were routinely reliable for fungus spores. While Alternaria was usually counted correctly, Cladosporium was frequently undercounted, and ascospores and basidiospores, which often constituted more than half the total spore load, were either not counted or severely undercounted. Errors resulted from undercounting, poor recognition, and reporting (including naming) errors. Multisite aerobiological studies that include documentation of fungus spore levels must include pretraining for each technician, and carefully documented standardization of counting and reporting procedures.