Ultrastructure and behaviour of the spindle pole body of the aphid-pathogenic fungusErynia neoaphidis

Summary A detailed account of the ultrastructure and behaviour of the spindle pole body (SPB) of the entomophthoraceous fungusErynia neoaphidis is presented for the first time.The SPB consists of extranuclear (ENC) and intranuclear (INC) components. The ENC is a saucepan-shaped structure which lies in a pocket of the nuclear envelope. It is composed of a forked, fibrillar handle and a shallow, cylindrical pan. The pan has a wall of two layers, both of which are thickened with a regular periodicity so that they appear to be beaded. It is postulated that the pan is formed from rough endoplasmic reticulum and that it synthesizes the amorphous, electron-dense material coating the ENC.The INC is a saucer-shaped, electron-dense plaque in which the ends of the spindle microtubules terminate. During metaphase, a clear zone separates the INC from the nuclear envelope and persists until telophase. The roles of the amorphous, electron-dense material and the clear zone as well as the method of SPB replication are discussed.     

Effects of 20 fungicides on the infectivity of conidia of the aphid entomopathogenic fungus Erynia neoaphidis

The effect of 20 fungicides on theinfectivity of conidia of the entomopathogenicfungus, Erynia neoaphidis, were assessedin the laboratory. After projection on broadbean leaves, conidia were treated withfungicides applied at their recommended fieldrate. Afterwards, the infectivity of theseinocula was assessed using an aphid bioassay.Four fungicides, carbendazim, kresoxym-methyl, nuarimol and thiophanate-methyl reduced the infectivity of the conidia by less than 25% and can be considered harmless for this aphid pathogen. Propiconazole was a little more toxic, with 37% reduction. Other products reducedinfectivity by between 50% and 100%. These are, from the least to the most toxic:flutriafol, prochloraz, epoxyconazole,iprodione, hexaconazole, triadimenol,azoxystrobine, cyproconazole, cyprodynil,flusilazole and tridemorph. Chlorothalonil,fenpropimorph, spiroxamine and tebuconazoletotally inhibited infectivity of the fungi. Analysis of the results according to chemicalclass showed that the benzimidazoles were theleast toxic for E. neoaphidis and themorpholines the most toxic. Effects oftriazoles and strobilurines were variable, withreduction ranging from 37% to 100% fortriazoles and from 17% to 68% forstrobilurines.     

The pathogenic fungi of the spittlebug in Mexico. III. Innocuity ofErynia neoaphidis andConidiobolus major in experimental animals

Previous research concerning theEntomophthorales has demonstrated their capabilities as biological control agents. To use them widely and at industrial levels, studies to demonstrate their innocuity in animals must be made. The present study concerns the intraperitoneal and subcutaneous inoculation of 274 animals (mice and guinea pigs) withErynia neoaphidis Remaudière & Hennebert andConidiobolus major (Thaxter) Remaudière & Keller, fungi pathogenic for the spittlebug [Aeneolamia postica (Walk) andProsapia simulans (Walk)] in Mexico. The alterations found were macroscopic tissue lesions consisting of nodules, abscesses and adhesions in the liver, spleen, kidneys and intraperitoneal cavity. No fungi could be recovered from organs at any time and only lysed filaments surrounded by polymorphonuclear cells could be seen at day 8 post-inoculation. The histopathological findings showed no important pathological alterations. These results suggest the innocuity ofErynia neoaphidis Remaudière & Hennebert andConidiobolus mamor (Thaxter) Remaudière & Keller in mice and guinea pigs.

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Résumé L'utilisation des Entomophthorales dans la lutte biologique contre les insectes n'est possible qu'après avoir vérifié leur innocuité vis-à-vis des vertébrés. Dans ce but, des injections sous-cutanées et intrapéritonéales ont été réalisées chez 274 animaux (cobayes et souris) à l'aide de mycélium et spores deErynia neoaphidis etConidiobolus major pathogènes de la “mosca pinta”. Les seules lésions tissulaires observées sont des nodules, des abcès et des adhérences au niveau de la rate, du foie, des reins et du péritoine. Les champignons n'ont jamais pu être réisolés à partir des divers organes. Les observations histopathologiques ne montrent aucune altération pathologique importante; seuls des filaments lysés entourés de polymorphonucléaires ont pu être retrouvés. Ces résultats indiquent l'innocuité deE. neoaphidis etC. major vis-à-vis des souris et des cobayes.

     

A PCR-based tool for the cultivation-independent monitoring of Pandora neoaphidis

Pandora neoaphidis is one of the most important fungal pathogens of aphids and has a great potential for use in biocontrol. Little is known on how this fungus persists in an area and in particular on its overwintering strategies. It is hypothesized that natural areas play an important role for survival and that soil may serve as a source of inoculum for new aphid populations in spring. To test these hypotheses, a cultivation-independent PCR-based diagnostic tool was developed, that allows the detection of P. neoaphidis in the environment. Two P. neoaphidis specific PCR primer pairs were designed, targeting sequences in the ribosomal RNA gene cluster. Specificity of both primer pairs was demonstrated with P. neoaphidis and non-target close entomophthoralean relatives. Moreover, single amplicons of expected sizes were obtained with both primer pairs from various environmental sample types, including aphid cadavers, plant material, and soil. The PCR-based diagnostic tool was applied to investigate the persistence of P. neoaphidis in soil samples obtained in 2004/2005 from a nettle field harboring infected aphids in fall 2004. P. neoaphidis was detected in every sample collected in November 2004 and March 2005, suggesting an overwintering stage of P. neoaphidis in top soil layers. The developed cultivation-independent PCR-based tool will be valuable for further investigation of the ecology of P. neoaphidis and for the development and future implementation of management strategies against aphids involving conservation biocontrol.     

Ultrastructure of mitosis inAmoeba proteus

Summary The fine structure ofAmoeba proteus nuclei has been studied during interphase and mitosis. The interphase nucleus is discoidal, the nuclear envelope is provided with a honeycomb layer on the inside. There are numerous nucleoli at the periphery and many chromatin filaments and nuclear helices in the central part of nucleus.In prophase the nucleus becomes spherical, the numerous chromosomes are condensed, and the number of nucleoli decreases. The mitotic apparatus forms inside the nucleus in form of an acentric spindle. In metaphase the nuclear envelope loses its pore complexes and transforms into a system of rough endoplasmic reticulum cisternae (ERC) which separates the mitotic apparatus from the surrounding cytoplasm; the nucleoli and the honeycomb layer disappear completely. In anaphase the half-spindles become conical, and the system of ERC around the mitotic spindle persists. Electron dense material (possibly microtubule organizing centers—MTOCs) appears at the spindle pole regions during this stage. The spindle includes kinetochore microtubules attached to the chromosomes, and non-kinetochore ones which pierce the anaphase plate. In telophase the spindle disappears, the chromosomes decondense, and the nuclear envelope becomes reconstructed from the ERC. At this stage, nucleoli can already be revealed with the light microscope by silver staining; they are visible in ultrathin sections as numerous electron dense bodies at the periphery of the nucleus.The mitotic chromosomes consist of 10 nm fibers and have threelayered kinetochores. Single nuclear helices still occur at early stages of mitosis in the spindle region.     

Effect of oleic acid on vegetative growth of the aphid-pathogenic fungus Erynia neoaphidis

Abstract The aphid-pathogenic fungus Erynia neophidis grew on a semi-defined medium containing 16 g·1⁻¹ glucose, 3 g·1⁻¹ yeast extract and 5 g·1⁻ mycological peptone only when the medium was supplemented with low concentrations of certain fatty acids. Of these, oleic acid fulfilled the growth requirement at a concentration of 0.02% (v/v), but higher concentrations were toxic, causing complete loss of viability of cultures at a concentration of 0.2% (v/v) in liquid medium and at 4% (v/v) on solid medium. The reduced viability of the fungus in liquid culture compared to that on equivalent solid medium, and at low inoculum density compared to high inoculum density in liquid medium, is explicable in terms of this toxicity.     

The effect of introducing the aphid-pathogenic fungus Erynia neoaphidis into populations of cereal aphids

The aphid-pathogenic fungus Erynia neoaphidis, as dried fungus-infected aphids, was applied to caged plots of winter wheat infested with cereal aphids at two sites, one in Hertfordshire and the other in Hampshire, in 1983. In each trial, the fungus became established in the aphid populations in the treated plots even though conditions were drier than average and therefore sub-optimal for fungus spread. Treatment applied in the third week of June increased the proportion of infected aphids more than that applied two weeks later at one site, and the early application was the only treatment to have an obvious effect at the other. In spite of the observed effect of treatments on the proportion of infected aphids, the fungus failed to reduce the numbers of aphids relative to those in untreated plots, chiefly because in these plots many aphids were killed by fungi of the same species as that introduced and other related species from natural sources. Artificial introduction of E. neoaphidis acts too slowly and unpredictably to be likely to form a practical alternative to conventional insecticides for cereal aphid control.     

Ultrastructural basis of mitosis in the fungusNectria haematococca (sexual stage ofFusarium solani)

Summary Microtubules (MTs) in the mitotic asters of the fungusNectria haematococca (teleomorph ofFusarium solani f. sp.pisi) pull on the spindle pole bodies (SPBs) during anaphase. To elucidate the structural basis of astral forces, we conducted an ultrastructural study using primarily freeze-substitution, three-dimensional reconstruction, and computerized numerical data acquisition and analysis. The asters were composed of numerous (68–171), mostly short (<0.5 m) MTs and varied widely in total MT length (34–83 m). Both the number and total length of MTs varied up to twofold or more among asters, even between the two asters of the same mitotic apparatus (MA). Surprisingly, less than one half (38%) of the MTs in each aster were attached to the SPB. Both the number and total length of these polar MTs varied up to twofold between the two asters of the same MA. Some asters included MTs oriented back toward the opposite SPB, whereas others did not, and the number and total length of such MTs varied among asters. These results are best interpreted by assuming that astral MTs inN. haematococca have a rapid rate of turnover and exhibit dynamic instability. Any of these parameters of astral architecture could vary during mitosis and thereby give rise to the oscillations of the mitotic apparatus that occur during anaphase B by generating unequal and fluctuating forces in the two sister asters. Astral MTs were arranged asymmetrically around the astral axis, and this asymmetry could produce the lateral movements of the SPB that occur during anaphase B. An apparently extensive system of 10nm filaments occurred in these cells, and some astral MTs were associated either terminally (at the plasma membrane) or laterally with these filaments. Such associations could be involved in the development and maintenance of astral forces.Abbreviations fMT free microtubule - MA mitotic apparatus - MT microtubule - pMT polar microtubule - SPB spindle pole body     

Ultrastructural features of mitosis inLeishmania adleri

Summary The interphase nucleus ofLeishmania adleri has clumps of chromatin associated with the nuclear envelope and a large centrally located nucleolus. Prior to mitosis the basal bodies replicate at the cell anterior. Subsequently, dense plaques appear in the equatorial region of the nucleus at the time of spindle development. Microtubules appear in the nucleus adjacent to the nuclear envelope and embedded in the matrix of the plaques. A central spindle composed of a single bundle of microtubules develops and spans the nucleus. Plaques and nucleolar components laterally associate with the spindle and migrate towards the poles. The central spindle elongates to three to four times its original length separating the forming daughter nuclei and producing an interzonal spindle. A remnant of the interzonal spindle remains attached to each of the daughter nuclei until late into cytokinesis. The kinetoplast does not divide until after the completion of mitosis.     

Ultrastructure of mitosis and cytokinesis inZygnema sp. (Zygnematales,Chlorophyta)

Summary Mitosis and cytokinesis have been studied in the green algaZygnema C. A. Agardh using interference-contrast light and transmission electron microscopy. At prophase, the nucleolus disintegrates and numerous extranuclear microtubules near the nuclear periphery penetrate into the nucleoplasm. When aligned in the equatorial plane of the open metaphase spindle the chromosomes are coated with persistent nucleolar fragments. At anaphase, vacuoles intrude into the interzonal spindle region and seemingly contribute to the anaphase movement of the chromosomes. At telophase, the spindle is persistent and the reforming nuclei are separated by cytoplasmic strands containing microtubules, interspersed with vacuoles. Extensive bundles of microtubules, dictyosomes and parallel, slightly inflated ER-profiles extend from the poles of the telophase nucleus along the longitudinal side of the chloroplast. Conceivably, these microtubules guide the nucleus during its post-mitotic migration towards its central interphase position between the two halves of the dividing chloroplast. Throughout the mitotic cycle, ubiquitous dictyosomes, positioned near the chloroplast core, seem very active. Arrays of microtubules run towards these dictyosomes and may conduct the dictyosome-vesicles to the cleavage plane. At metaphase, septum growth becomes visible as an annular ingrowth of the plasmalemma. At late telophase or at entering interphase, an extensive clump of vesicles, associated with longitudinal bundles of microtubules, appears between the leading edges of the advanced furrow. Apparent fusion of these vesicles with the head of the centripetally-growing furrow results in its completion. The pattern of mitosis and cytokinesis inZygnema is compared with that of closely related green algae.     

Distribution in time and space of resistance to the pathogenic fungus Erynia neoaphidis in the pea aphid Acyrthosiphon pisum

Two biotypes of pea aphid, Acyrthosiphon pisum, have been recognized in Australia, by their susceptibility (S) or resistance (R) to certain isolates of the fungal pathogen Erynia neoaphidis. The responses of these two biotypes to 40 isolates of the fungus have shown that the R form is largely confined to two States, New South Wales and Victoria, but appears to have recently spread into Queensland and Tasmania. There is no evidence to suggest it occurs outside Australia. Sequential sampling of two field populations of pea aphids during 1981 and 1982 showed that the proportion of R form remained stable at 10.7±3.0 and 14.6±2.6% (mean±standard error) for the two populations. Glasshouse competition experiments run at the comparatively high temperature of 25°C resulted in the R form becoming dominant even when the initial ratio was 4:1 in favour of the S form. The ecological and genetical implications are discussed.

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Résumé Deux types biologiques d'Acyrthosiphon pisum ont été définis, en Australie, suivant leur sensibilité (S) ou leur résistance (R) à certains isolats du champignon pathogène Erynia neoaphidis. Les réponses des deux biotypes à 40 isolats du champignon ont montré que la forme R est essentiellement cantonnée à deux états: Nouvelle Galle due Sud et Victoria, mais a récemment gagné le Queensland et la Tasmanie. Aucun élément ne fait dire qu'elle existe hors d'Australie. Des échantillonnages séquentiels des deux populations de pucerons dans la nature en 1981 et 1982 ont montré que la proportion de la forme R est restée stable à 10,7 et 14,62 dans les deux populations. Des expériences de compétition en serre à la température relativement haute de 25°C ont rendu la forme R dominante, même quand le rapport initial était de 4/1 en faveur de la forme S. La discussion porte sur les conséquences écologiques et génétiques.

     

Ultrastructure of polymorphicMucor as observed by means of freeze-etching

Dormant sporangiospore ofMucor was observed by means of freezeetching. There was considerable inter- and intraspecies variation in spore size. Large spores were clearly multinucleate. The spore wall was covered with two thin layers, each about 10 nm thick, which may correspond to ordinary spore sheath. However, fracture never occurred along the spore surface. The cell membrane did not have invaginations like those of higher fungi. Instead, there were numerous round depressions about 50 nm in diameter. They revealed a small hollow when crossfractured. Occasionally they combined to form a structure resembling a rod-like invagination. This, presumably, is one step towards generation of the rod-like invagination of conidiospore. Mitochondria became much larger than those found in the vegetative forms, and showed wide and deep invaginations of membrane. Cristae became indistinct. Lipid droplets had multilayered shells and were much more highly developed than those found in mycelial cells, yeast or arthrospores of this organism. No endoplasmic reticulum or vacuoles were found.     

Ultrastructure of cell division and reproductive differentiation of male plants in theFlorideophyceae (Rhodophyta). Mitosis inDasya baillouviana

Summary Mitosis in the marine red algaDasya baillouviana (Ceramiales, Florideophyceae) was observed with the electron microscope. Most details of the process are quite similar to those observed in the other macroscopic red algae studied to date. However, some minor variations were noted. At late prophase a very small nuclear envelope protrusion (NEP) is formed at each division pole subjacent to the nucleus associated organelle known as the polar ring (PR) and 2–3 cisternae of perinuclear endoplasmic reticulum (PER) are commonly present during metaphase-anaphase. In contrast, in the other florideophycean algae where mitosis has been reported, a prominent NEP is present at late prophase (McDonald personal communication,Scott et al. 1980) and only a single cisterna of PER is observed. Additionally, during mid-late interphase and in mitotically-quiescent cells ofDasya, a single cisterna of smooth-surfaced ER is always juxtaposed with each PR. The possible significance of PER in theFlorideophyceae and other multinucleate organisms is discussed as well as the likely functions of spindle-associated smooth ER. It is suggested that ultrastructural features of mitosis should be useful as criteria to aid in the interpretation of the phylogeny of red algae.     

Pandora neoaphidis transmission and aphid foraging behaviour

Pandora neoaphidis is an aphid-specific entomopathogen that produces infective conidia. As aphid movement increases, so does the likelihood of contact with conidia. Volatile distress signals released in response to aphid infestation as an indirect defence against herbivory may affect aphid foraging and, therefore, the fungus-aphid interaction. In this study, two different methods were used to investigate the effect of plant volatiles and P. neoaphidis-sporulating cadavers on (1) the colonisation of Vicia faba plants by Acyrthosiphon pisum and (2) P. neoaphidis transmission. This study indicates that A. pisum does not avoid bean plants containing P. neoaphidis and that transmission of conidia occurs during plant colonisation and, to a lesser extent, during in situ feeding. Although significantly more aphids were recovered from damaged plants compared to undamaged plants, the likelihood of infection was not affected by previous infestation by aphids.     

Transmission of Pandora neoaphidis in the presence of co-occurring arthropods

Transmission of the entomopathogenic fungus Pandora neoaphidis to the nettle aphid Microlophium carnosum was assessed in the presence of arthropods that co-exist with the fungus within the habitat but do not compete for aphid hosts. The presence of a parasitoid significantly enhanced transmission, and transmission rates were similar for both enemy and non-enemy parasitoids. Although herbivory of nettle leaves by Peacock butterfly (Inchis io) caterpillars indirectly reduced the number of M. carnosum by >30% due to a reduction in leaf area for feeding, the addition of I. io significantly increased transmission of P. neoaphidis in the remaining aphids. It is likely that enhanced transmission in the presence of A. rhopalosiphii and I. io is due to disturbance and subsequent movement of the aphid, resulting in contact with conidia deposited on the leaf surface. The presence and impact of co-occurring arthropods should be taken into consideration when assessing the transmission of fungal entomopathogens.     

Effects of constant and fluctuating temperatures on sporulation and infection by the aphid-pathogenic fungus Pandora neoaphidis

Laboratory studies were performed to assess the importance of temperature on sporulation and infection by the aphid-pathogenic fungus Pandora neoaphidis (Remaudière and Hennebert) Humber. Numbers of primary conidia discharged from mycelium formulated as alginate granules and unformulated mycelial mats were assessed, as well as infection of the potato aphid, Macrosiphum euphorbiae (Thomas) (Homoptera, Hemiptera, Aphididae), using culture plugs as inoculum sources. Sporulation from experiments at constant temperatures indicated the optimum temperature range was 10–20°C for both mycelial preparations and there was no or very little sporulation at 30°C. Infection of aphids kept at 15°C was 34–50%, while infection at 25°C was 11–44%. At 20°C, 77–79% of aphids were infected. Under fluctuating temperature cycles, conidia numbers did not differ when mycelial preparations were maintained at 18–25°C compared with 18–20°C, but fewer conidia were recorded when preparations were exposed continuously to 18–30°C. Infections of inoculated aphids kept for varying numbers of days at 18–25°C varied between 24–47%, but only 3–32% of aphids were infected when exposed to a cycle of 18–30°C for various times. Unformulated mycelial mats of P. neoaphidis appear to be superior to forumlated alginate granules for use in experimental greenhouse and field trials, since temperature stability is similar for both materials but mycelial mats are much easier to produce.     

Time course of hormonal control of the first mitosis in tobacco mesophyll protoplasts cultivated in vitro

The presence of auxin and cytokinin is necessary for the induction of mitosis in tobacco mesophyll protoplasts cultivated in vitro. In their absence, protoplasts firstly accumulate inhibitors of mitosis in the culture medium, possibly because of non-coordinated cell-wall synthesis, and secondly evolve a nonmitotic and degenerative metabolism. By changing the intoxified medium, it is possible to show that auxin is necessary from the beginning of culture, while cytokinin is only required later to allow a step in the development of the mitotic apparatus.Abbreviations 2.4-D 2.4 dichlorophenoxyacetic acid - 6-BA 6-benzyladenine - NAA naphthaleneacetic acid - IAA indoleacetic acid     

Fine structure of plasmodial nuclei in the slime moldPhysarum polycephalum I. Comparison of diploid and haploid vegetative mitosis

Summary The same basic ultrastructural features of interphase and mitotic nuclei were found for both the haploid Colonia and the diploid wild type strains of the myxomycete,Physarum polycephalum. Differences in nuclear size and chromocenter numbers were observed, but the nucleolar cycle and the intranuclear and acentriolar type of mitosis characteristic of the plasmodial stage of the diploid is present in haploid plasmodia, ruling out any relation between ploidy level and type of mitotic figure.