天麻抗真菌蛋白对木霉菌丝的作用位点

天麻抗真菌蛋白(Gastrodia Antifungal Protein,GAFP)能强烈抑制腐生真菌菌丝的生长,在天麻限制和防止蜜环菌[Armillariella melles (Vahl.ex Fr.) Karst.]侵染球茎的防卫机制中起重要作用。本文报告GAFP抗菌机理研究的部分内容——GAFP对木霉菌丝的作用位点。用荧光试剂异硫氰酸荧光素(Fluorescein isothiocyanate,FITC)标记GAFP,试验表明,标记后的GAFP与未标记的GAFP对木霉菌丝生长均有抑制作用。在荧光显微镜下观察GAFP在木霉菌丝上的作用位点,发现被“标记GAFP”作用后的菌丝边缘有荧光,并主要集中在木霉菌丝的顶端和菌丝横隔处,表明GAFP对木霉的作用位点在菌丝的细胞壁上。     

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.     

Transmembrane H + and Ca 2+ fluxes through extraradical hyphae of arbuscular mycorrhizal fungi in response to drought stress

丛枝菌根真菌(AMF)能够和大多数陆地植物形成共生体系, 对于植物生长发育和适应各种逆境胁迫具有重要作用。很多研究表明干旱胁迫下AMF能够促进宿主植物对水分的吸收从而增强植物抗旱能力, 但目前针对AMF根外菌丝响应水分胁迫的生理变化以及AMF与宿主植物逆境信号交流的研究并不多。该研究利用AMF Rhizophagus irregularis和胡萝卜(Daucus carota var. sativa)毛状根双重无菌培养体系获得纯净根外菌丝, 向培养基添加聚乙二醇(PEG)模拟干旱胁迫, 运用场发射扫描电子显微镜(FE-SEM-EDS)观察干旱胁迫对AMF根外菌丝形态的影响, 同时采用非损伤微测技术(NMT)观测根外菌丝跨膜H ⁺和Ca ²⁺离子流变化。结果发现, PEG处理1 h后菌丝尖端和侧面发生H ⁺外流和强烈的Ca ²⁺内流, 荧光探针分析也显示菌丝胞内pH值显著上升、Ca ²⁺浓度增加; PEG处理24 h后菌丝形态发生明显变化, 培养基pH值降低, P、Ca、Fe等元素在菌丝际积累。这些试验结果表明, 干旱胁迫下AMF根外菌丝跨膜H ⁺和Ca ²⁺流发生变化, 促进了菌丝与环境之间的物质交换。菌丝酸化生长环境有利于养分吸收, 并促进AMF与宿主植物之间的信号交流以增强植物的耐旱性。     

Growth stimulation of a Shiro-like,mycorrhiza forming,mycelium of <Emphasis Type="Italic">Tricholoma matsutake</Emphasis> on solid substrates by non-ionic surfactants or vegetable oil

The incorporation of Tweens (1 %, 2 %, 5 %) or olive oil (1 %, 2 %) in soil or in soil-containing substrate strongly stimulated mycelial growth of the edible ectomycorrhizal mushroom Tricholoma matsutake (Matsutake) after 1 or 3 months, respectively. The growth responses to Tween 40 and Tween 80 were dose-dependent. Fungal biomass increased up to 15-fold as a result of olive oil incorporation. After 4 months of Matsutake/pine co-culture in the presence of olive oil (2 %), compact aggregates of substrate, hyphae, and surface-colonized roots were observed, recalling in some ways the mycelial mat structure of Matsutake in the field, i.e. Shiro. Olive oil did not prevent formation of well-developed Hartig net palmettis although those seemed rather less abundant than without oil addition. The incorporation of Tween 80 or olive oil (2 %) into nutrient agar induced the proliferation of peripheral hydrophilic-like hyphae penetrating the medium. Tricholoma matsutake growth stimulation, possibly related to the presence of fatty acids in surfactants and oil, could be a consequence of the higher hydrophilicity of treated hyphae, or of enhanced lytic enzyme excretion and activity. Parameters such as adjuvant type, concentration, and growth conditions will be further optimised to formulate culture substrates adapted to the co-culture of T. matsutake and its host plants.     

cAMP promotes hyphal branching in <Emphasis Type="Italic">Mucor globosus</Emphasis>

The growth pattern of Mucor globosus cultured on a medium with or without cyclic adenosine monophosphate (cAMP) was examined. Branching remarkably increased in a mycelium grown on an agar medium containing cAMP. In submerged culture containing cAMP, some sporangiospores grew spherically and formed yeast-like cells, and others showed hyphal growth. These hyphae showed septation and swelling and formed spore-like structures. When these hyphae were transferred to cAMP-free medium, a germ tube emerged from each compartment. These results show that cAMP has two different effects on the development of hyphae: one is the promotion of branching, and the other is the suppression of polarized growth.     

Growth inhibition and excessive branching in <Emphasis Type="Italic">Aphanomyces cochlioides</Emphasis> induced by 2,4-diacetylphloroglucinol is linked to disruption of filamentous actin cytoskeleton in the hyphae

We observed that 2,4-diacetylphloroglucinol (DAPG), a major antimicrobial metabolite produced by a rhizoplane bacterium Pseudomonas fluorescens ECO-001 inhibited mycelial growth of a damping-off phytopathogen Aphanomyces cochlioides AC-5 through inducing excessive branching and curling in the hyphae. This study aimed to unravel the mode of action of DAPG caused excessive branching, curling and growth inhibition of AC-5 hyphae by detecting localized changes in the cortical filamentous actin (F-actin) organization by rhodamine-conjugated phalloidin. Confocal laser scanning microscopic observations revealed that both living bacteria and DAPG severely disrupted the organization of F-actin in the A. cochlioides hyphae in a similar manner. Furthermore, an inhibitor of F-actin polymerization, latrunculin B also induced similar growth inhibition, excessive branching and caused disruption of F-actin in the AC-5 hyphae. Our results suggested that growth inhibition and excessive branching induced in A. cochlioides by DAPG is likely to be linked to the disruption of F-actin cytoskeleton in the affected hyphae. This is the first report on disruption of cytoskeleton of a eukaryotic A. cochlioides by a well-known biocontrol metabolite DAPG secreted from a prokaryotic bacterium ECO-001.     

In vitro efficacy of <Emphasis Type="Italic">Hyptis suaveolens</Emphasis> L. (Poit.) essential oil on growth and morphogenesis of <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> f.sp. <Emphasis Type="Italic">gladioli</Emphasis> (Massey) Snyder &; Hansen

Hyptis suaveolens L. (Poit.) essential oil was tested in vitro on the growth and morphogenesis of Fusarium oxysporum f.sp. gladioli (Massey) Snyder & Hansen, which causes Fusarium corm rot and yellows in various susceptible cultivars of gladiolus. The fungitoxicity of the oil was measured by percentage radial growth inhibition using the poisoned food technique (PF) and volatile activity assay (VA). The mycelial growth of the test fungus was completely inhibited at 0.998 and 0.748 μg ml⁻¹ concentration of oil in PF and VA, respectively. Essential oil was found to be fungicidal in nature at 1.247 and 0.998 μg ml⁻¹ concentration of oil in PF and VA, respectively. Determination of conidial germination in the presence of oil was also carried out and it was found that the oil exhibited 100% inhibition of conidial germination at 0.450 μg ml⁻¹ concentration. The effect of essential oil on the yield of mycelial weight was observed and it was found that at 0.873 μg ml⁻¹ concentration no mycelium was recorded and 100% inhibition was observed. The fungitoxicity of oil did not change even on exposure to 100°C temperature or to autoclaving, and the oil also retained its fungicidal nature even after storage of 24 months. The main changes observed under light microscopy after oil treatment were a decrease and loss of conidiation and anomalies in the hyphae such as a decrease in the diameter of hyphae and granulation of cytoplasm. The treatment of the oil also showed highly reduced cytoplasm in the hyphae, showing clear retraction of the cytoplasm from the hyphae and ultimately in some areas hyphae without cytoplasm were also found. GC-MS studies of the essential oil revealed that the oil consisted of 24 compounds with 1,8-cineole as major component accounting for 44.4% of the total constituents.     

Hyphal healing mechanism in the arbuscular mycorrhizal fungi Scutellospora reticulata and Glomus clarum differs in response to severe physical stress

The hyphal healing mechanism (HHM) has been shown to differ between Gigasporaceae and Glomeraceae. However, this process has not been considered under (severe) physical stress conditions. Scutellospora reticulata and Glomus clarum strains were cultured monoxenically. The impact of long distance separating cut extremities of hyphae and of multiple injuries within hyphae on the HHM was monitored. For long distances (>5000 microm) separating cut extremities, hyphae healing was observed in half the cases in S. reticulata and was absent in G. clarum. For multiple-injured hyphae, the HHM was always oriented towards the complete recovery of hyphae integrity in S. reticulata, while in G. clarum, the growing hyphal tips (GHTs) could indifferently reconnect cut sections, by-pass cut sections or develop into the environment. Hyphae behaviour under severe physical stress clearly differentiated S. reticulata from G. clarum, suggesting that both fungi have developed different strategies for colony growth to survive under adverse conditions.     

Behaviour of the hyphae of<Emphasis Type="Italic"> Laccaria laccata</Emphasis> in the presence of<Emphasis Type="Italic"> Trichoderma harzianum</Emphasis> in vitro

The growth rate and the behaviour of Laccaria laccata and Trichoderma harzianum hyphae in co-culture and in the rhizosphere of 3-month-old Pinus sylvestris seedlings grown in vitro were investigated. In the interaction zone, hyphae of L. laccata became more pigmented and formed short branches growing towards the hyphae of the saprobic fungus, coiled around them and penetrated sporadically. Vacuolated hyphae of T. harzianum showed protoplasm granulation and breaks in walls followed by release of protoplasts. In the rhizosphere, the mantle hyphae of L. laccata showed a tendency to surround conidia of T. harzianum. No obvious penetration of the conidial walls by the hyphae of the mycorrhizal fungus was observed by scanning electron microscopy. Instead, in rare cases, the hyphae of L. laccata showed marked wrinkles, and a partial degradation of a mucilaginous material covering the mantle appeared to occur.     

Structure and Development of the Endophyte in the Parasitic Angiosperm <Emphasis Type="Italic">Cuscuta japonica</Emphasis>

The endophyte, that is, the haustorial part within the tissues of the host plant Impatiens balsamina, of the parasitic angiosperm Cuscuta japonica was studied with light and electron microscopy. The endophyte consisted mainly of vacuolated parenchymatous axial cells and elongate, superficial (epidermal) cells. Then the elongate, epidermal cells separated from each other and transformed into filamentous cells, called searching hyphae. The hyphae grew independently either intercellularly or intracellularly in the host parenchyma. The apical end of the hyphal cells was characterized by conspicuous, large nuclei with enlarged nucleoli and very dense cytoplasm with abundant organelles, suggesting that the hyphal cells penetrating host tissue were metabolically very active. Numerous osmiophilic particles and chloroplasts were noted in the hyphae. The osmiophilic particles were assumed to be associated with elongation of the growing hyphe. Plasmodemata connections between the searching hyphal cells of the parasite and the host parenchyma cells were not detected. Hyphal cells that reached the host xylem differentiated into water-conducting xylic hyphae by thickening of the secondary walls. A xylem bridge connecting the parasite and the host was confirmed from serial sections. Some hyphal cells that reached the host phloem differentiated into nutrient-conducting phloic hyphae. Phloic hyphae had a thin layer of peripheral cytoplasm with typical features of sieve-tube members in autotrophic angiosperms, i.e., parallel arrays of smooth endoplasmic reticulum, mitochondria, and plastids with starch granules. Interspecific open connections via the sieve pores of the host sieve elements and plasmodesmata of the parasite phloic hyphae were very rarely observed, indicating that the symplastic translocation of assimilate to the parasite from the host occurred.     

Ultrastructural Study on the Interactions Between Three Respectively Different Disease- resistant Cucumber Cultivars and Phytophthora melonis

Ultrastructural changes during the process of interactions between three different diseaseresistant, susceptible, moderately resistant and resistant cucumber (Cucumis sativus L. ) cuhivars and Phytophthora melonis Katsura were respectively compared and the associated factors in the observed differences in colonization were determined. During the process of interaction in the susceptible cultivar, both inter- and intracellular growth of hyphae associated with extensive colonization of healthy hyphae and marked alteration of mesophyll cells, such as aggregation of cytoplasm and disintegration of cellular organelle were demonstrated. The general disorganization of host cell was observed in areas directly adjacent to the fungal hyphae and in cells several layers from the nearest visible hyphae. In the moderately resistant cultivar during interaction the host showed limited hypha growth with responses of resistance including 1 ) breakage of plasmodesmata to prevent the host cells to contact the hyphae, 2) increasing the quantity of endoplasmic reticulum and Golgi apparatus so as to increase the quantity of secretory protein, 3) a plasmalemma invagination containing granular material at the site in contact with the intercellular hyphae. The interaction in the resistant cuhivar was characterized as a hypersensitive reaction (HR) which means a rapid necrosis of the infected cells and the cells surrounding the invaded hyphae, resulting in death of the invaded hyphae. Formation of cell wall appositions right at the penetration sites of hyphae and haustorium-like structure was observed. The results indicate that the mechanism of host response to P. melonis invasion was differ- ent between the moderately resistant and the resistant cucumber cultivars.     

The use of laser scanning confocal microscopy to characterize mycorrhizas of Pinus strobus L. and to localize associated bacteria

 Laser scanning confocal microscopy (LSCM), light microscopy (LM) and epifluorescence microscopy (FM) were used to observe the extramatrical hyphae, mantle patterns and associated bacteria on mycorrhizal tips of Pinus strobus L. seedlings grown in pot cultures. Laccaria sp. and Tuber sp. formed ectomycorrhizas with Pinus strobus, while Phialophora finlandia Wang & Wilcox and E-strain (sensu Danielson 1982) formed ectendomycorrhizas. Distinct mantle patterns and cystidia were observed with greater resolution using LSCM, and intracellular hyphae were visualized in three dimensions. Trypan blue penetrated fresh whole mounts to 20 μm and was an excellent stain for visualizing fungal hyphae and bacteria with LSCM. Fluorescein isothiocyanate and acridine orange were used in conjunction with LSCM and FM to localize bacteria on ectomycorrhizal tips. With LSCM, bacteria were visible in the surface mucigel, and optical sectioning through the root tip showed that bacteria were also present within the mantle. LSCM is a non-intrusive and fast method for visualizing mycorrhizal structures and their associated bacteria on fresh, whole root tips. Accepted: 17 July 1996     

The relation between turgor and tip growth inSaprolegnia ferax: Turgor is necessary,but not sufficient to explain apical extension rates

Linear growth rate ofSaprolegnia was reduced in direct proportion to increased osmotic pressure (II) of the medium, when sorbitol or PEG-400 was used as osmotica. However, increasing medium II reduced hyphal turgor only to a minimum positive level, which was maintained while extension rates continued to decline. TPA, a K⁺-channel agonist effective onSaprolegnia protoplasts, also caused dose-dependent linear growth rate reductions but did not substantially affect turgor. When turgor was compared with linear growth rate in the osmoticum experiments, there was a positive correlation only for hyphae growing faster than 12 μm/min; below this, there was a twofold range in extension rate despite essentially constant turgor. As well, TPA-treatments produced a twofold reduction in hyphal extension rate without substantially affecting turgor. Turgor should be consistent within a coenocyte, and is steady under constant growth conditions. However, under such conditions, we found average variations of fivefold in extension rate between hyphae, and twofold for hyphae over time. These results suggest that turgor is not the prime determinant of tip extension rate, and they are consistent with cytoskeletal regulation of that rate. Linear growth rates ofSaprolegnia colonies were similar on basal medium containing 1% (w/v) glucose, sorbitol, or PEG and only slightly faster than without added carbohydrate. Increasing medium II with glucose also reduced hyphal extension rate.     

黄瓜不同抗病品种与疫霉菌相互作用的超微结构研究

电镜观察发现,黄瓜（ＣｕｃｕｍｉｓｓａｔｉｖｕｓＬ．）的不同抗病品种在与疫霉菌（ＰｈｙｔｏｐｈｔｈｏｒａｍｅｌｏｎｉｓＫａｔｓｕｒａ）的相互作用过程中有不同的表现。感病性品种易被疫霉菌侵染,被侵染的叶肉组织及周围细胞中胶层解离、胞质凝聚、细胞器解体,叶片组织内有大量胞间及胞内生长菌丝。中抗病性品种也被疫霉菌侵染,但表现出胞间连丝断裂、内质网和高尔基体增多等抗病性反应,与菌丝相接触的细胞出现质膜内陷。抗病性品种出现过敏性坏死反应,叶肉细胞与入侵菌丝一同解体死亡,胞间菌丝向细胞内穿透处形成壁附加物。中抗病性品种和抗病性品种在与疫霉菌的相互作用过程中表现出不同的抗性机制。     

Inhibition of tumor growth by poly(ethylene glycol) derivatives of anti-ErbB2 antibodies

Poly(ethylene glycol) (PEG) modification of substances with antitumor activity was shown to enhance penetration into growing solid tumors and extend antitumor effects. Accordingly, PEG was introduced as a modifier to two types of monoclonal antibodies (N12 and L26) specific to the ErbB2 (HER2) oncoprotein. These antibodies suppress the growth of tumors overexpressing ErbB2 (e.g. N87 human tumor) and the effect of PEG on their antitumor activity was evaluated. Methoxy-PEG-maleimide conjugated to sulfhydryl groups at the hinge region of the antibodies impaired their antibody binding to N87 tumor cells and did not enhance the antitumor inhibitory activity in tumor-bearing mice. A branched N-hydroxysuccinimide-activated PEG (PEG2), conjugated through amino groups of the protein, was used for binding to the whole antibody (Ab) or to its monomeric Fab′ fragment. When tested against N87 cells in vitro, the binding activity and antitumor cytotoxic effects of Ab-PEG2 were mostly preserved. PEG2 modification did not seem to alter the tumor-inhibitory activity of the antibodies in vivo and the same pattern of tumor development was observed during the first few weeks following administration. However, the stimulating effects of PEG were observed at later stages of tumor growth since tumor development was either slowed down or completely arrested. Furthermore, a second tumor implanted into the same mice during this later stage was significantly or completely inhibited, as compared to results in mice injected with the unmodified antibody. The Fab′-PEG2 monomeric derivative was also shown to be effective in inhibiting the growth of a second tumor. The extended and prolonged enhancing effect of PEG on the antitumor activity of antibodies or Fab′ fragments directed against ErbB2 may be of importance in the treatment of ErbB2-overexpressing neoplasms. Received: 2 September 1999 / Accepted: 19 February 2000     

荧光标记金针菇原生质体融合

以金针菇（Flmmulina velutipes）为材料,经异硫氨酸荧光素（FIC）标记的金针菇单核W19菌株的原生质体与未经标记的单核Y7菌株的原生质体在聚乙二醇（PEG）的诱导下进行融合,利用显微操纵仪直接挑取一个带有荧光而另一个不具荧光的原生质体对,培养后,根据融合形成的汉核菌丝具有“锁状连合”这一形态学特征选择、鉴定融合菌株,酯酶同工酶电泳分析结果表明,融合菌株具双亲互补酶带。经测定,多数融合菌株菌丝生长速度和生物量较亲株高,且出菇正常。     

The mode of interaction between Vitis and Plasmopara viticola Berk. & Curt. Ex de Bary depends on the host species

In order to obtain insight into host responses to grapevine downy mildew ( Plasmopara viticola ), we compared pathogen development on a panel of Vitis species from North America, Asia and Europe. Leaf discs from different host species were inoculated in parallel, and the colonisation of the mesophyll was visualised by aniline blue staining and quantified with respect to infection incidence and mycelial growth. In parallel, the morphology of guard cells was screened for the presence of an internal cuticular rim after staining with acridine orange and using low-temperature scanning electron microscopy. We observed three response patterns: (i) inhibition of pathogen development early after attachment of zoospores; (ii) successful colonisation of the mesophyll by the pathogen; and (iii) aberrant development, where the pathogen does not attach to guard cells, but produces hyphae on the leaf surface without formation of viable sporangiophores. Inhibition is observed in the North American and Siberian species, successful colonisation prevails in the European hosts, and surface hyphae are found on non-Siberian Asiatic species. We propose that the interaction between host and pathogen is under control of specific signals that have been subject to evolutionary diversification.     

Phosphorus effects on metabolic processes in monoxenic arbuscular mycorrhiza cultures

The influence of external phosphorus (P) on carbon (C) allocation and metabolism as well as processes related to P metabolism was studied in monoxenic arbuscular mycorrhiza cultures of carrot (Daucus carota). Fungal hyphae of Glomus intraradices proliferated from the solid minimal medium containing the colonized roots into C-free liquid minimal medium with different P treatments. The fungus formed around three times higher biomass in P-free liquid medium than in medium with 2.5 mM inorganic P (high-P). Mycelium in the second experiment was harvested at an earlier growth stage to study metabolic processes when the mycelium was actively growing. P treatment influenced the root P content and [(13)C]glucose administered to the roots 7 d before harvest gave a negative correlation between root P content and (13)C enrichment in arbuscular mycorrhiza fungal storage lipids in the extraradical hyphae. Eighteen percent of the enriched (13)C in extraradical hyphae was recovered in the fatty acid 16:1omega5 from neutral lipids. Polyphosphate accumulated in hyphae even in P-free medium. No influence of P treatment on fungal acid phosphatase activity was observed, whereas the proportion of alkaline-phosphatase-active hyphae was highest in high-P medium. We demonstrated the presence of a motile tubular vacuolar system in G. intraradices. This system was rarely seen in hyphae subjected to the highest P treatment. We concluded that the direct responses of the extraradical hyphae to the P concentration in the medium are limited. The effects found in hyphae seemed instead to be related to increased availability of P to the host root.     

A model for hyphal growth and branching.

A mathematical model for hyphal growth and branching is described which relates cytological events within hyphae to mycelial growth kinetics. Essentially the model quantifies qualitative theories of hyphal growth in which it is proposed that vesicles containing wall precursors and/or enzymes required for wall synthesis are generated at a constant rate throughout a mycelium and travel to the tips of hyphae where they fuse with the plasma membrane, liberating their contents into the wall and increasing the surface area of the hypha to give elongation. The hypothesis that there is a duplication cycle in hyphae which is equivalent to the cell cycle observed in unicellular micro-organisms is also included in the model. Predictions from the model are compared with experimentally observed growth kinetics of mycelia of Geotrichum candidum and Aspergillus nidulans. The finite difference model which was constructed is capable of predicting changes in hyphal length and in the number and positions of branches and septa on the basis of changes in vesicle and nuclear concentration. Predictions were obtained using the model which were in good agreement with experimentally observed data.     

Mycelial differentiation and spore formation by Streptomyces brasiliensis in submerged culture.

Streptomyces brasiliensis ATCC 23727 showed extensive sporulation when cultured in a liquid medium containing galactose and glutamic acid as carbon and nitrogen sources. Sporogenic hyphae formed under these conditions were morphologically similar and developmentally equivalent to aerial hyphae and metamorphosed into chains of spores by following a sequence of ultrastructural changes similar to that observed during growth on solid media. In addition, our electron microscopy study revealed two previously unrecognized aspects of hyphal development in streptomycetes: the formation of sporogenic hyphae was always preceded by changes in the structure of the nucleoid, and the sheath that characteristically covered these hyphae was not deposited coincidently with wall formation in the apical growing portion of the hypha.