Development and ultrastructure of a microsporidian parasite in midgut cells of the larch sawfly, Pristiphora erichsonii (Hymenoptera: Tenthredinidae)

The developmental stages of a microsporidium from larvae of Pristiphora erichsonii were investigated. Meronts appeared to be the only stage containing a diplokaryon. Nuclei of sporonts in a parasitophorous vesicle underwent at least three divisions and uninucleate sporoblasts developed from these multinucleate sporonts. As many as 38 spores were observed with a vesicle. A thin pansporoblastic membrane limited the vesicle and was derived from rough endoplasmic reticulum of the host cell. The microsporidium was tentatively identified as Pleistophora sp. Infection levels of the microsporidium in natural populations of P. erichsonii reached 25%.     

Influence of Dimilin on a microsporidian infection in the gypsy moth Lymantria dispar (L.) (Lepidoptera: Lymantriidae)

Bioassay studies were conducted to investigate the influence of Dimilin (diflubenzuron), a chitinsynthetase inhibitor used for insecticidal control of the gypsy moth, Lymantria dispar, on the development and viability of a microsporidian pathogen of L. dispar. Before or after an infection with a Nosema species, L. dispar larvae were fed Dimilin in sublethal dosages. Dimilin fed to L. dispar larvae at 0.65 ng/cm² diet surface resulted in a total larval mortality of 53%. Although the microsporidian infection alone did not cause high mortality rates (9%), mortality increased to 96% when L. dispar larvae were inoculated with both Dimilin and Nosema spores. When Dimilin was fed to the larvae 24 h before or 6 days after inoculation with the microsporidium, the number of mature spores produced was significantly reduced. When Dimilin was fed to the larvae 24 h after microsporidian inoculation, the number of spores produced was not significantly reduced. Spores that were produced in larvae after Dimilin had been ingested with the diet were less infectious than spores produced in control larvae; the experimental infection rate decreased from 94% when spores obtained from control larvae were used, to 48 or 10% when spores obtained from larvae fed Dimilin 24 h or 6 days after Nosema inoculation, respectively, were used. Mature microsporidian spores washed in Dimilin solution prior to oral inoculation, however, were as infectious as spores stored in liquid nitrogen. We have shown that Dimilin interferes with the establishment of the parasite in its host. In addition, when Nosema sp. succeeds in infecting the L. dispar host despite treatment with Dimilin, the microsporidium does not develop optimally and spore production is reduced.     

GROWTH REGULATION BY ETHYLENE IN FERN GAMETOPHYTES. V. ETHYLENE AND THE EARLY EVENTS OF SPORE GERMINATION

Early events during the germination of spores of the fern Onoclea sensibilis were studied to determine the time during germination when ethylene had its greatest inhibiting effect. Water imbibition by dry spores was rapid and did not appear to be inhibited by ethylene. During normal germination DNA synthesis occurred about four hours before the nucleus moved from a central position to the spore periphery. Following nuclear movement, mitosis and cell division occurred, partitioning the spore into a small rhizoid cell and a large protonemal cell. Cell division was complete approximately six hours after nuclear movement. Ethylene treatment of the spores blocked DNA synthesis, nuclear movement, and cell division. The earliest DNA replication in uninhibited spores was observed after 14 hours of germination, and the maximal rate of spore labeling with ³H-thymidine was between 16 and 20 hours. Spores were most sensitive to ethylene, however, during the stages of germination prior to DNA synthesis, and it was concluded that ethylene did not directly inhibit DNA replication but blocked germination at some earlier fundamental step. The effects of ethylene were reversible. since complete recovery from inhibition of germination was possible if ethylene was released and the spores were kept in light. Recovery was much slower in darkness. It was hypothesized that light acted photosynthetically to overcome the ethylene inhibition of germination. Consistent with this, it was shown that spores exhibit net photosynthesis after only two hours of germination.     

A cotyledon double inoculation technique for evaluating resistance to anthracnose (Colletotrichum orbiculare) and scab (Cladosporium cucumerinum) in cucumber

A technique for simultaneous inoculation of cucumber cotyledons with Colletotrichum orbiculare race 1 and Cladosporium cucumerinum has been developed. The procedure permitted both resistant and susceptible plants to be recovered. Seedlings were grown at 20°C and inoculated 24 h after emergence with Colletotrichum orbiculare (200 spores in 2 μ1 of water) and Cladosporium cucumerinum (1000 spores in 5 μ1 of water) followed by 48 h of incubation in the dark at 20°C and 100% r.h., and 48 h in a 20°C lighted growth chamber. Seedlings were then moved to a growth chamber at 21°C at night and at 26°C during the day for 4 days and plants were rated as resistant or susceptible 8 days after inoculation. No interference in the expression of resistance or susceptibility of cultivars to either pathogen was detected in simultaneous inoculations.     

Effects of microtubule-inhibitors on nuclear migration and rhizoid differentiation in germinating fern spores (Onoclea sensibilis)

Summary Germinating spores of the sensitive fern,Onoclea sensibilis L., undergo premitotic nuclear migration before a highly asymmetric cell division partitions each spore into a large protonemal cell and a small rhizoid initial. Nuclear movement and subsequent rhizoid formation were inhibited by the microtubule (MT) inhibitors, colchicine, isopropyl-N-3-chlorophenyl carbamate (CIPC) and griseofulvin. Colchicine prevented polar nuclear movement and cell division so that spores developed into enlarged, uninucleate single cells. CIPC and griseofulvin prevented nuclear migration, but not cell division, so that spores divided into daughter cells of approximately equal size. In colchicine-treated spores, MT were not observed at any time during germination. CIPC prevented MT formation at a time coincident with nuclear movement in the control and caused a disorientation of the spindle MT. Both colchicine and CIPC appeared to act at a time prior to the onset of normal nuclear movement. The effects of colchicine were reversible but those of CIPC were not. Cytochalasin b had no effect upon nuclear movement or rhizoid differentiation. These results suggests that MT mediate nuclear movement and that a highly asymmetric cell division is essential for rhizoid differentiation.     

Development,Ultrastructure, and Mode of Transmission of Amblyospora sp. (Microspora) in the Mosquito*

SYNOPSIS. Amblyospora sp. in Culex salinarius (Coquillett) is transovarially transmitted and has 2 developmental sequences, one in each host sex. In females, the entire life cycle is restricted to oenocytes which become greatly hypertrophied due to the multiplication of diplokaryotic cells during merogony and come to lie next to ovaries. Sporulation occurs only after a blood meal is taken and is shortly followed by infection of the oocytes and subsequent transmission to the next host generation. In the male host, infections spread from oenocytes to adipose tissue where diplokaryotic cells undergo a 2nd merogony. During this merognic cylce, the number of diplokaryotic cells greatly increases and the infection is spread throughout the body of the larval host. Sporulation is initiated with the physical separation of the 2 members of the diplokaryon and the simulatneous secretion of a pansporoblastic membrane. Subsequent meiotic division and morphogenesis result in the formation of 8 haploid spores enclosed with a pansporoblastic membrane. Buildup of spores and subsequent destruction of host adipose tissue prove fatal to the male host during the 4th larval stage.     

Observations on Purpureofilum apyrenoidigerum gen. et sp. nov. from Australia and Bangiopsis subsimplex from India (Stylonematales, Bangiophyceae, Rhodophyta)

Purpureofilum apyrenoidigerum gen et sp. nov. was obtained from a mangrove habitat in New South Wales, Australia. It had unbranched uniseriate to multiseriate filaments less than 1 mm tall, with a unicellular base. Each cell had a single multilobed parietal chloroplast without a pyrenoid. During reproduction vegetative cells were discharged directly as monospores that remained motile for several hours after release. Spores with long tails moved more slowly (0.053–0.195 μm sχ) than spores without tails (0.43–1.76 μm s′¹). Phylo‐genetic analysis of sequences of the small subunit of the nuclear‐encoded rRNA and plastid‐encoded ribu‐lose bisphosphate carboxylase/oxygenase genes revealed that Purpureofilum is a member of the Stylonematales and is most closely related to the filamentous genus Bangiopsis. Bangiopsis differs from Purpureofilum by having longer (to 5 mm) multiseriate filaments, cells containing a stellate chloroplast, a conspicuous central pyrenoid, and monospores often formed in packets. Monospores of Bangiopsis were also motile. Transmission electron microscopy investigation of Purpureofilum and Bangiopsis revealed that the Golgi complexes are associated only with rough endo‐plasmic reticulum and that the plastid contains a peripheral thylakoid; this combination of features being the same as in all other multicellular members of the Stylonematales. The low molecular weight carbohydrates of Purpureofilum and Bangiopsis were digenea‐side and sorbitol, which were present in most other Stylonematales.     

Biological control of postharvest blue mold of oranges by <Emphasis Type="Italic">Cryptococcus laurentii </Emphasis>(Kufferath) Skinner

Cryptococcus laurentii (Kufferath) Skinner was evaluated for its activity in reducing postharvest blue mold decay of oranges caused by Penicillium italicum in vitro and in vivo. The results showed that washed cell suspensions of yeast provided control of blue mold decay better than yeast in culture broth. Autoclaved cell culture and cell-free culture filtrate failed to provide protection against the pathogen. The concentrations of antagonist had significant effects on biocontrol effectiveness. When the washed yeast cell suspension reached the concentration of 1 × 10⁹ CFU/ml, challenged with pathogen spore suspension at 1 × 10⁴ spores/ml, the blue mold decay was completely inhibited during 5 days of incubation at 20 °C. No complete control was obtained when oranges were stored at 4 °C for 30 days, but the decay was distinctly prevented. Efficacy of C. laurentii was maintained when applied simultaneously or prior to inoculation with P. italicum. Efficacy was reduced when C. laurentii was applied after inoculation. In drop-inoculated wounds of oranges, the populations of C. laurentii increased by approximately 50-fold during the first 24 h at 20 °C. The maximum yeast populations, approximately 250-fold over the initial populations, were reached 15 days after inoculation at 4 °C.     

Glugea gasti sp. n., a Microspridan Pathogen of the Boll Weevil Anthonomus grandis1,2,3

SYNOPSIS. Glugea gasti sp. n., a microsporidan pathogen of Anthonomus grandis Boheman (the boll weevil), is described and a probable life cycle presented. The alimentary canal, and probably the mesenteron 1st, is the initial site of infection, altho the disease later becomes generalized thruout most body tissues. Binucleate sporoplasms initiate the 1st schizogonic phase, characterized by mono- and bi-nucleate schizonts. The 2nd schizogonic phase is characterized by mono-, bi- and quadrinucleate schizonts, by prolific multiplication, by the dense compact nuclei early in this phase, and late in this phase by larger schizonts with less dense vesicular nuclei. This phase terminates in formation of diplokarya. The sporogonic phase is characterized by combination of the 2 nuclei in the diplokaryon followed by nuclear divisions in a sequence closely resembling meiosis. Two sporoblasts are produced from each sporont. Mature spores in wet mounts by phase contrast were 4.3 ± 0.3 μ long by 2.3 ± 0.2 μ wide. The polar filament averaged 76 μ long. Mature spores were present about 24 hours after infection. Some observations are presented on an external filament extending from one pole of the spore to host tissue and other events during the process of spore morphogenesis.     

Ultrastructural Study and Description of Ovavesicula popilliae N. G., N. Sp. (Microsporida: Pleistophoridae) from the Japanese Beetle,Popillia japonica (Coleoptera: Scarabaeidae)1

A new genus and species of microsporidia, Ovavesicula popilliae n. g., n. sp., is described from the Japanese beetle, Popillia japonica, on the basis of studies by light and electron microscopy. Parasite development primarily occurs within the Malpighian tubules of larvae, and spores are formed in a sporophorous vesicle. Meronts have diplokaryotic nuclei, develop in direct contact with the host cell cytoplasm, and divide by binary fission. Sporonts have unpaired nuclei, develop within a thick sporophorous vesicle, and undergo synchronous nuclear divisions producing plasmodia with 2, 4, 8, 16, and 32 nuclei. Cytokinesis of sporogonial plasmodia does not occur until karyokinesis is complete with 32 nuclei. Intact sporophorous vesicles are ovoid, containing numerous secretory products, and are surrounded by a persistent two-layered wall. The uninucleate spores are regularly formed in groups of 32, and the polar tube in each has six coils.     

The ultrastructure of spores ofClaviceps purpurea (Fr.) Tul.

The ultrastructure of saprophytic and parasitic spores of the AscomyceteClaviceps purpurea (Fr.) Tul. was studied. Considerable differences were found to exist between the saprophytic and parasitic spores as to morphology and fine structure. The reason for the different ultrastructural morphology is probably connected with the intensity of cell metabolism. Whereas the parasitic spores obtained from the honeydew possess the character of a resting cell with a thick electron-dense cytoplasm, abundant lipid bodies, few mitochondria, an underdeveloped and hence little active endoplasmic reticulum and with a homogenous thick cell wall, the saprophytic spores appear as cells with higher metabolic rate, containing more numerous mitochondria, a thinner cytoplasm, a highly developed endoplasmic reticulum, fewer lipid bodies and abundant large vacuoles as well as frequently a new wall layer.     

Changes of the cytoplasmic ultrastructure during development of sclerotia in Claviceps purpurea (Fr.) Tul.

The development of sclerotia of Claviceps purpurea was investigated by light and electron microscopy. During the first days after infection sterigma and conidiospores are formed. The spores show a moderately developed vacuolar system, they are thick walled and contain about 20% lipid (related to the cell volume) embedded in glycogen. The sterigma are cylindrical unicellular hyphae with electron dense cytoplasm and isolated strongly contrasted lipid droplets. In maturing sclerotia the hyphae become septated with increasingly thick cell walls and a large lipid content. The lipid forms small droplets in young cells, while in the mature sclerotium it occurs in the form of very large drops, occupying the major part of the cell. Simultaneously the composition of the lipid is changed. The mature cells have several nuclei. They are partially connected by osmiophilic substances, forming a network of intercellular spaces.Abbreviations HEPES N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid - DMSO Dimethylsulfoxide     

Ultrastructural changes in the spore and mycelia of Ascosphaera apis after treatment with benomyl (Benlate 50 W)

In Ascosphaera apis, after 8 days growth in darkness at 28° C, numerous sporocysts were observed, within which mature spores were seen aggregated into a spore ball. The mature spore of A. apis had a thick spore wall with an electron-opaque outer layer, a spore membrane with many depressions, and sporoplasm containing numerous ribosomes and mitochondria. In the cytoplasm of the mycelium, mitochondria with well-defined cristae and numerous ribosomes were observed. At a concentration of 1 g/ml of culture medium, benomyl appeared to inhibit colony growth of A. apis, but some sporocysts containing deformed spores were found. Deformed spores possessed a thick spore wall with a grainy matrix, and depressions were no longer detected in the spore membrane. Ribosomes were lacking in the sporoplasm and mitochondria appeared degenerate. The mycelium from the treated culture contained mitochondria with an electron-lucid matrix and no well defined cristae, while ribosomes were completely depleted. The significance of these observations in relation to the use of benomyl to control chalkbrood disease in the honey bee is discussed.     

THE EFFECT OF METHANOL ON SPORE GERMINATION AND RHIZOID DIFFERENTIATION IN ONOCLEA SENSIBILIS

In germinating spores of Onoclea sensibilis, the nucleus migrates to one end prior to an asymmetric cell division that partitions each spore into two daughter cells of unequal size. The larger cell develops into a protonema, whereas the smaller cell immediately differentiates into a rhizoid. When spores were germinated in the presence of methanol, nuclear migration was inhibited and most nuclei moved only to the raphe on the proximal side of the spores. Subsequent cell division partitioned each spore into daughter cells of equal size of which both developed into a protonema and neither into a rhizoid. Spores became sensitive to methanol at a time just prior to or coincident with nuclear migration and the effects of the alcohol were rapidly reversible as long as the spores were removed from methanol prior to the completion of cell division. Exposure to methanol prior to, but not during, nuclear migration or after mitosis had no effect upon rhizoid differentiation. The alcohol disrupted the formation of crosswalls after mitosis and they were often convoluted and irregularly branched. These results are consistent with the interpretation that methanol may disrupt a membrane site that plays an essential role in nuclear movement and rhizoid differentiation.     

ATTACHMENT AND FUSION OF GAMETES DURING FERTILIZATION OF PALMARIA SP. (RHODOPHYTA)1

Fertilization of cultured microscopic female gametophytes by spermatia from field-collected male gametophytes of Palmaria sp. was observed by light and transmission electron microscopy. Liberated spermatia had a prophase-arrested nucleus with a pair of polar rings. The protoplast of spermatia was covered with ca. a 3-μm-thick hyaline covering. After spermatium inoculation, the spermatial covering was attached specifically to the coat surrounding the cell wall of the trichogyne. The spermatial covering was eliminated only at the site of gamete attachment, resulting in direct attachment of the spermatial plasma membrane to the trichogyne within 5 min after spermatium inoculation. This direct attachment was followed by completion of spermatial nuclear division and cell wall formation. The polar rings disappeared before prometaphase. The cytoplasm of the binucleate spermatium invaded the trichogyne cell wall and subsequently fused with the trichogyne cytoplasm. The trichogyne could fuse with many spermatia, and many male nuclei (the derivative nuclei of spermatial nuclear division) could enter the trichogyne cytoplasm.     

Effects of Leaf Age, Inoculum Dose and Freezing on Development of Chocolate Spot (Botrytis fabae) Lesions on Field Bean (Vicia faba) Leaves

Botrytis fabae spore suspensions containing c. 1, 10, 10², 10³, 10⁴, 10⁵, or 10⁶ spores/ml were used to inoculate 5, 17 or 30-day-old field bean leaves. The percentages of the leaf areas covered by, chocolate spot lesions and the percentages of the leaf areas bearing conidiophores were assessed 1, 6, 12, 14, and 19 days after inoculation. The percentage of the area covered by lesions and the percentage of the area bearing conidiophores (logit-transformed) increased linearly with increasing spore concentration (log₁₀-transformed). The proportions of leaf areas covered by lesions and bearing conidiophores were both greater on 17 and 30-day-old leaves than on 5-day-old leaves. The rate of lesion growth increased with both increasing inoculum dose and increasing leaf age. Generally there was no interaction between the effects of leaf age and the effects of inoculum dose on either lesion growth or sporulation. Two days after inoculation with suspensions of either 10⁴ or 10⁶ spores/ml, 7-day-old leaves grown at 15°C were transferred to –16°C or 2.5°C or kept at 15°C for 4 days. Two days later more spores had been produced on leaves which had been frozen (–16°C) than on, leaves kept at 2.5°C.     

Stemphylium botryosum f. lactucae- its developmentjn vifro and its pathogenicity to lettuce leaves

Stemphylium botryosum f. lactucae, incitant of a leaf-spot disease of stored lettuce, was found to be relatively restricted in its host range. Cross-inoculations with spore suspension of this fungus failed to induce symptoms in any of the host plants tested, except carrot. Among isolates of S. botryosum from various hosts, only the isolate from carrot induced slight symptoms on lettuce. While mycelial growth of the lettuce isolate was confined to the range 13–37 ^oC spores germinated at more extreme temperatures. The optimum temperature for germination and for radial growth on PDA was found to be between 25 and 30 ^oC. Wet spores were quickly inactivated at 50 ^oC, whereas more than 40 % of dry spores withstood a 24 h exposure to that temperature. Only the outer leaves of lettuce responded readily to inoculation with a spore suspension, the required incubation period being 3 days at 25 ^oC. Symptoms developed less readily on bruised leaves. Relative humidity approaching saturation was necessary for prompt and typical infection, notably during the 24 h following inoculation. Short dry periods (60 % r.h.) interposed at a later stage, while somewhat inhibitory, did not prevent infection.     

Paranucleospora theridion n. gen., n. sp. (Microsporidia,Enterocytozoonidae) with a Life Cycle in the Salmon Louse (Lepeophtheirus salmonis,Copepoda) and Atlantic Salmon (Salmo salar)

ABSTRACT. Paranucleospora theridion n. gen, n. sp., infecting both Atlantic salmon (Salmo salar) and its copepod parasite Lepeophtheirus salmonis is described. The microsporidian exhibits nuclei in diplokaryotic arrangement during all known life‐cycle stages in salmon, but only in the merogonal stages and early sporogonal stage in salmon lice. All developmental stages of P. theridion are in direct contact with the host cell cytoplasm or nucleoplasm. In salmon, two developmental cycles were observed, producing spores in the cytoplasm of phagocytes or epidermal cells (Cycle‐I) and in the nuclei of epidermal cells (Cycle‐II), respectively. Cycle‐I spores are small and thin walled with a short polar tube, and are believed to be autoinfective. The larger oval intranuclear Cycle‐II spores have a thick endospore and a longer polar tube, and are probably responsible for transmission from salmon to L. salmonis. Parasite development in the salmon louse occurs in several different cell types that may be extremely hypertrophied due to P. theridion proliferation. Diplokaryotic merogony precedes monokaryotic sporogony. The rounded spores produced are comparable to the intranuclear spores in the salmon in most aspects, and likely transmit the infection to salmon. Phylogenetic analysis of P. theridion partial rDNA sequences place the parasite in a position between Nucleospora salmonis and Enterocytozoon bieneusi. Based on characteristics of the morphology, unique development involving a vertebrate fish as well as a crustacean ectoparasite host, and the results of the phylogenetic analyses it is suggested that P. theridion should be given status as a new species in a new genus.     

Fine Structure of Encephalitozoon cuniculi from Rabbits,Mice and Hamsters*

SYNOPSIS. Fine structure and development of Encephalitozoon cuniculi from rabbits were studied in rabbit choroid plexus (CP) cell cultures and were compared to hamster and mouse microsporida. Sporoplasms had a single limiting membrane and contained a large nucleus. Proliferative forms (schizonts) had double outer membranes, the outermost being associated with the formation of the limiting membrane of vacuoles formed within the host cell cytoplasm. These organisms were often binucleate and divided to form sporonts. Sporonts divided once to form 2 sporoblasts which developed into electron-dense spores. Spores had a thick, 3-layered wall and contained a polar filament. The developmental cycle of E. cuniculi in rabbit CP cultures progressed rapidly. Sporoplasms were observed in host cells at 3 hr postinoculation (PI). By 24 hr PI proliferative forms were associated with host cell cytoplasmic vacuoles which contained developing organisms. Mature spores were present in vacuoles by 2 days PI, indicating that the life cycle in the CP system is ∼ 48 hr. The fine structure and the sequential developmental cycle of the mouse and hamster isolates were observed to be identical to those of the rabbit isolate and different from those of the genus Nosema. It is proposed, therefore, that the 3 organisms represent the same species, Encephalitozoon cuniculi.