Isolationin vitro ofStrongwellsea castrans [Fungi: Entomophthorales] a pathogen of adult cabbage root flies,Delia radicum [Dipt.: Anthomyiidae]

The entomogenous fungusStrongwellsea castrans was isolatedin vitro for the first time, by incubating conidia projected from infected cabbage root flies (Delia radicum) in a simple, semi-defined liquid medium comprising dextrose, yeast extract and lactalbumin hydrolysate buffered to pH 7. The fungus grew as long unitunicate hyphae. After transfer to a solid nutrient medium, multinucleate hyphal bodies were formed which developed a thick, laminated wall. Neither conidia nor resting spores developed in liquid or on solid media and the fungus survived successive sub-culturing only in liquid media. Using the API-ZYM system, tests on extracts on hyphae ofS. castrans were positive for 11 enzymes but there were no consistent differences in enzyme profiles betweenS. castrans and fungi of the related genusErynia.      

Molecular Detection and Pathogenicity Assay of Plasmodiophora brassicae in Chicken Manure

To determine the relationship between animal excreta and the occurrence of clubroot disease of cruciferous crops caused by Plasmodiophora brassicae, chickens were fed with resting spores of the pathogen. Their faeces were collected and used to inoculate crucifers. This study proved that both fresh and composted manures could induce clubroot and the presence of the pathogen in the manure was confirmed by PCR amplification. However, composting had detrimental effects on the virulence of the resting spores in the manure. When the temperature was over 32°C, the incidence and severity of clubroot declined with the increase in the exposure time of resting spores to high temperature and the pathogenicity was completely lost when the spores were kept at 48°C for 6 h. The control measures for the clubroot disease were discussed.     

Comparing two methods for quantifying soil-borne Entomophaga maimaiga resting spores

To improve usability of methods for quantifying environmentally persistent entomophthoralean resting spores in soil, we modified and tested two methods using resting spores (azygospores) of the gypsy moth pathogen Entomophaga maimaiga. Both methods were effective for recovering resting spores at concentrations >100 resting spores/g dry soil. While a modification of a method originally described by Weseloh and Andreadis (2002) recovered more resting spores than a modified method based on Percoll density gradients, the ability to estimate true densities from counts was similar for both methods. Regression equations are provided for predicting true resting spore densities from counts, with R² values for both methods ?0.90.     

Resting Spore Dormancy and Infectivity Characteristics of the Potato Powdery Scab Pathogen Spongospora subterranea

The soil‐borne potato pathogen Spongospora subterranea persists in soil as sporosori, which are aggregates of resting spores. Resting spores may germinate in the presence of plant or environmental stimuli, but direct evidence for resting spore dormancy is limited. A soilless tomato bait plant bioassay and microscopic examination were used to examine features of S. subterranea resting spore dormancy and infectivity. Dried sporosori inocula prepared from tuber lesions and root galls were infective after both short‐ and long‐term storage (1 week to 5 years for tuber lesions and 1 week to 1 year for root galls) with both young and mature root galls inocula showing infectivity. This demonstrated that a proportion of all S. subterranea resting spores regardless of maturity exhibit characteristics of stimuli‐responsive dormancy, germinating under the stimulatory conditions of the bait host plant bioassay. However, evidence for constitutive dormancy within the resting spore population was also provided as incubation of sporosorus inoculum in a germination‐stimulating environment did not fully exhaust germination potential even after 2.4 years. We conclude that S. subterranea sporosori contain both exogenous (stimuli‐responsive) and constitutively dormant resting spores, which enables successful host infection by germination in response to plant stimuli and long‐term persistence in the soil.     

Entomophthora parvispora sp. nov., a pathogen ofThrips tabaci

Entomophthora parvispora sp. nov., pathogenic forThrips species, (principallyT. tabaci Lind.), is described. The fungus is characterized by its very small conidia and resting spores, anadhesive spores with a characteristically-shaped fixture at their apex, and the mode of resting spore development. The records suggest that the pathogen is widespread and probably occurs throughout central and southern Europe.

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Résumé Entomophthora parvispora sp. nov. pathogène pour des espèces deThrips, notammentT. tabaci Lind., est décrit. Ce champignon est caractérisé par ses conidies et spores durables très petites, par des spores non adhérentes présentant à leur apex une excroissance de forme caractéristique, ainsi que par le mode de développement des spores durables. Les observations montrent que cet agent pathogène est largement répandu et se trouve probablement dans toute l'Europe centrale et méridionale.

     

Population dynamics of an ice-associated diatom, Thalassiosira australis Peragallo, under fast ice near Syowa Station, East Antarctica, during austral summer

A clear shift from vegetative cells to auxospores and resting spores in Thalassiosira australis was observed in the water column and sinking fluxes under the fast ice near Syowa Station in the austral summer of 2005/2006. This is the first report of the auxosporulation by T. australis in situ. Resting spores were also observed in the sediment even before new spore formation, suggesting that T. australis can overwinter in the sediment. Heterotrophic dinoflagellates ingested and digested vegetative cells and auxospores but did not digest resting spores, suggesting a high tolerance of resting spores to grazing by heterotrophic dinoflagellates. We discuss the possible life history and overwintering strategy that T. australis uses in an Antarctic coastal area to cope with the unpredictable timing of sea ice growth and decay.     

First record on epizootics of Entomophthora grylli on grasshopper in Indian subcontinent: pathogen city and bio control potential on Oxya velox

A naturally-occurring fungus called Entomophthora grylli was for the first time isolated from two species of grasshopper Oxya velox and Oxya vicinia in Jammu and Kashmir, India. The epizootic was confined along Indo–Pak border between 74 degrees 24′ and 75 degrees 18′, East longitude and 32 degrees 50′ and 33 degrees 30′ North latitude. The fungus proved to be highly pathogenic and the natural mortality was significantly influenced by the population density which increased from 26.00 to 73.60 over the period of epizootics. On the basis of the available literature this appears to be the first record from the Indian sub-continent. While many infected grasshoppers apparently produced neither conidia nor resting spores, the cadavers were found to be full of hyphal bodies and resting spores towards the end of epizootics. These resting spores or their germ tubes were not invasive as such but if provided a saturated environment for a week, they start germinating, resulting in germ conidia which were able to induce dermal pathogencity. Further, it was observed that the disease could not be transmitted to healthy individuals by ingestion. However, the intra-haemocoel infectivity of fresh resting spores, germinated resting spores, and germ conidia proved to be highly pathogenic as they resulted in 81.4% grasshopper mortality. Although E. grylli is fastidious, it is possible to multiply it on a large scale as protoplasts which are infective upon injection in their hosts. However, the lack of a cell wall renders them very fragile, and they are neither infective upon application to the insect's cuticle nor upon ingestion. In the present study, a method on delivery of pathogen through “sticky molasses pan trap” was developed for inducing infection in grasshoppers in a paddy nursery which would facilitate its use as a bioinsecticide, analogous to other entomopathogenic fungi.     

Life Cycle of <Emphasis Type="Italic">Plasmodiophora brassicae</Emphasis>

Plasmodiphora brassicae is a soil-borne obligate parasite. The pathogen has three stages in its life cycle: survival in soil, root hair infection, and cortical infection. Resting spores of P. brassicae have a great ability to survive in soil. These resting spores release primary zoospores. When a zoospore reaches the surface of a root hair, it penetrates through the cell wall. This stage is termed the root hair infection stage. Inside root hairs the pathogen forms primary plasmodia. A number of nuclear divisions occur synchronously in the plasmodia, followed by cleavage into zoosporangia. Later, 4–16 secondary zoospores are formed in each zoosporangium and released into the soil. Secondary zoospores penetrate the cortical tissues of the main roots, a process called cortical infection. Inside invaded roots cells, the pathogen develops into secondary plasmodia which are associated with cellular hypertrophy, followed by gall formation in the tissues. The plasmodia finally develop into a new generation of resting spores, followed by their release back into soil as survival structures. In vitro dual cultures of P. brassicae with hairy root culture and suspension cultures have been developed to provide a way to nondestructively observe the growth of this pathogen within host cells. The development of P. brassicae in the hairy roots was similar to that found in intact plants. The observations of the cortical infection stage suggest that swelling of P. brassicae-infected cells and abnormal cell division of P. brassicae-infected and adjacent cells will induce hypertrophy and that movement of plasmodia by cytoplasmic streaming increases the number of P. brassicae-infected cells during cell division.     

Evaluation of Trichoderma spp. from central and northern regions of Turkey for suppression of Polymyxa betae as a vector of rhizomania disease

In the current study, 18 Trichoderma spp. isolates were obtained from different provinces in central and northern regions of Turkey. The ability of nine selected isolates to suppress the colonisation of roots by P. betae and the multiplication of BNYVV in sugar beet roots under controlled conditions were tested. Roots of seedlings growing in the P. betae-BNYVV-infested soil were analysed by enzyme-linked immunosorbent assay to test for the presence of BNYVV and checked microscopically for the density of cystosori of P. betae. The numbers of P. betae resting spores in cystosori for each treatment were counted using a light microscope. Except for isolates Tr-1 and Tr-5, the effect of selected Trichoderma isolates on suppressing multiplication of BNYVV varied between 4 and 53%. The total number of resting spores in the roots varied between 14.4 and 25.1 for the different Trichoderma spp. treatments. The lowest number of resting spores in clusters was recorded in T. harzianum Tr-8. In addition, the shapes of resting spores were not normal in the Tr-8 treatments. The cystosori from this treatment were also abnormally dark in colour and had deformed walls.     

MORPHOLOGY AND THERMAL DEATH POINT OF OLPIDIUM BRASSICAE

The morphology of single-sporangial isolates of lettuce, tomato, mustard, and oat Olpidium brassicae (Wor.) Dang. growing in their respective hosts as well as in cowpea were compared in situ and after extraction from the roots. The sporangia, zoospores, and resting spores of all isolates were within the established limits of the species. Single exit tubes or pores predominated which means that these isolates should not be transferred to the genus Pleotrachelus. A satisfactory assay for the presence of resting spores was developed by air-drying of the roots for a week or longer. This treatment killed zoospores and vegetative sporangia, but not resting spores. Factors affecting resting spore formation were investigated unsuccessfully. The thermal death point of zoospores of mustard isolates that did not form resting spores was between 40 and 45 C for 10 min.     

STUDIES ON MARINE PLANKTON DIATOMS. II. RESTING SPORE MORPHOLOGY1

A morphological study of resting spores in five marine planktonic diatom species using electron microscopy indicates that Bacteriastrum delicatulum Cleve and Leptocylindrus danicus Cleve spores bear little resemblance, to their vegetative cells. Detonula confervacea (Cleve) Gran and Stephanopyxis turris (Grev. & Arn.) Ralfs spores have several features in common with their vegetative cells, and Rhizosolenia setigera Brightwell lies between the two extremes. The function of resting spores in relation to diatom life cycles is briefly discussed. Spore formation may be a primitive characteristic in the life cycle and may no longer have significant survival value for the species.     

Potato Root Exudation and Release of Spongospora subterranea Resting Spore Germination Stimulants are Affected by Plant and Environmental Conditions

Variation in plant and environmental conditions were studied to determine the effect thereof on the exudation of low‐molecular‐weight organic compounds by potato roots. The results of the phytochemical analyses showed that among the conditions investigated, root vigour, potato cultivar, nutrients in incubation solution and temperature influenced the number and the type of primary metabolites released. Moreover, these conditions influenced our detection of compounds known to stimulate germination of resting spores of the pathogen Spongospora subterranea, causal agent of powdery scab and root diseases of potato. We conclude that changes in plant and environmental conditions can affect the release of specific compounds that stimulate germination of S. subterranea resting spores. The impact of the factors affecting potato root exudation on subsequent disease development is discussed.     

On Chytridium braun,Diplochytridium N. G., and Canteria N. G. (Chytridiales)

Summary The operculate genus Chytridium Braun is emended and restricted to approximately 25 known species and several incompletely known members which lack an apophysis and develop much like species of Rhizophydium, insofar as the zoosporangium and absorbing or rhizoidal system are concerned, and form endobiotic or intramatrical resting spores. This interpretation corresponds fairly closely to Braun's diagnosis of the type species.A new genus, Diplochytridium, is established to segregate the former Chytridium species with an endobiotic or intramatrical apophysis or prosporangium and resting spores, which may develop sexually or asexually. Some of these species have a well-defined endo-exogenous alternation of growth and development in which the apophysis appears to function as a prosporangium. As interpreted here, Diplochytridium includes approximately 20 of the known species.Another new genus, Canteria, is created for a parasite of Mougeotia which Canter first described as a species of Phlyctidium but later found to develop endobiotic resting spores or zygospores by the fusion of conjugation tubes.     

Developmental Regulation of Susceptibility and Tolerance to Fusarium Root Rot in Beans

The soil-borne fungus, Fusarium solani f. sp. phaseoli, attacks roots and hypocotyls of bean (Phaseolus vulgaris) plants causing a devastating disease called root and foot rot. In a study of the host-pathogen relationship it was found that young bean roots, with the radicle just emerging, were highly tolerant to the pathogen, whereas older bean seedlings, with a fully developed root system, were completely susceptible. Investigations by low-temperature scanning electron microscopy demonstrated that significantly fewer spores and hyphae were present on the root surface of young bean seedlings as compared to older ones. A similar pattern of attachment was found when bean roots were inoculated with spores of F. solani f. sp. pisi, a related pathogen causing disease on peas but not on beans. Light microscopic studies showed that F. solani f. sp. pisi did not penetrate the root but rapidly formed thick-walled resting spores on the root surface. F. solani f. sp. phaseoli on the other hand quickly penetrated the root and formed an extensive network of fungal hyphae. These results demonstrate that the ability of fungal propagules to adhere to and to penetrate host tissues are two distinct processes. Furthermore, the data indicate that young bean roots lack a surface component necessary for attachment of fungal spores which may help explain their tolerance to Fusarium root rot.     

OBSERVATIONS ON CHYTRIDIACEOUS PARASITES OF PHANEROGAMS. XIV. PHYSODERMA CALAMI

Physoderma calami, a chytrid (Phycomycetes), has not been studied or apparently even collected in Europe since 1895 and has never been recorded in this hemisphere. Material widely distributed in herbaria indicated it was a Physoderma but no details save color, shape, and size of resting spores were known. A study of living material from Vermont indicates resting spore germination occurs by dehiscence of a lid and production of a sporangium with zoospores. The latter may form either an epibiotic or “ephemeral” sporangial stage and zoospores, or an endobiotic one, with extensive polycentric thallus bearing turbinate cells along the rhizoids, and, as outgrowths of these turbinate cells, straw-colored, thick-walled resting spores. The latter stage produces dark-brown spots on infected parts of the host.     

INTERACTIVE EFFECTS OF COPPER AND SILICIC ACID ON RESTING SPORE FORMATION AND VIABILITY IN A MARINE DIATOM1

The toxic effects of copper on resting spore formation and viability in the marine diatom Chaetoceros protuberans Lauder were determined both with and without silicic acid added to the medium. With silicic acid available, partial inhibition of resting spore formation occurred only at the highest cupric ion activity (pCu 8.6), while the percentage of cells forming spores at pCu's 10.2 and 11.3 was nearly the same as in the controls. Without silicic acid added to the medium, sporulation was completely inhibited at pCu 8.6 and greatly inhibited, at pCu 10.2. At pCu 11.3 and in the controls, the rate of spore formation was less than 50%. The results indicate that the inhibition of resting spore formation by copper is related to the concentration of silicic acid available to cells of C protuberans. This is consistent with previous studies which show that copper toxicity during vegetative growth involves interference with silicification in diatoms and is a Junction of the silicic acid concentration of the medium. Viable resting spores of C. protuberans were still present in cultures following exposure to elevated copper concentrations during a 100-day incubation period. This indicates that resting spores can serve to enhance diatom survival in areas polluted by heavy metals.     

Importance of ambient saturation deficits in an epizootic of the fungus Neozygites floridana in cassava green mites (Mononychellus tanajoa)

The mite-pathogenic fungus Neozygites floridana Fisher (Entomophthorales: Neozygitaceae) is considered to have potential for the biological control of the cassava green mite, Mononychellus tanajoa (Bondar). However, its activity is sporadic and laboratory data suggest a strong dependence on night-time saturation deficits for transmission. We report on an epizootic of this fungus in a mite population in northeastern Brazil. During the epizootic, host populations appeared to be limited by a combination of the pathogen and a predatory mite Neoseiulus idaeus (Acari: Phytoseiidae). When temperatures increased, the epizootic finished and the host population began to grow. Abiotic conditions could not explain the variation in host mortality following pickup of infective propagules in this epizootic. However, night-time saturation did help to explain the variation in transmission from infective cadavers to newly killed hosts. This supports laboratory observations that horizontal transmission between hosts is determined mainly by saturation deficits, while the process of infection is little affected by abiotic conditions. A further field observation was the near-absence of resting spores in dead mites (ca. 0.1% of cadavers), suggesting that the pathogen population was unsuccessful in producing inoculum to infect future M. tanajoa populations. The implications are that this pathogen will only be effective as a biological control agent in periods of high relative humidity, and establishment in new areas may be limited by resting spore formation. This revised version was published online in July 2006 with corrections to the Cover Date.     

In vitro formation of resting spores by the insect pathogenic fungus Entomophaga maimaiga

Field-collected resting spores (azygospores) of the fungal pathogen of Lymantria dispar (gypsy moth), Entomophaga maimaiga, have been used to release this biological control agent in areas where this pathogen is not established. We have found that E. maimaiga can produce resting spores in vitro using Grace's insect tissue culture medium (95%) plus fetal bovine serum (5%). The majority of spores become mature between 7 and 21 days after cultures are initiated. Spore production varies by fungal isolate; of 38 isolates tested, 10 produced no resting spores while 7 produced >1000 resting spores/ml. Resting spore production was not affected when isolates were mixed. Glycerol (used for fungal storage), trehalose, and selected amino acids each inhibited resting spore formation. Fetal bovine serum was required for spore production but the presence of >5% yielded lower resting spore densities. A large surface area:volume ratio (12.5 cm(2):ml versus 4.2 cm(2):ml) was required for abundant formation of resting spores. At present, resting spores have only been produced in small volumes with a maximum of 3 x 10(4) resting spores/ml.     

Modeling the Influence of Rainfall and Temperature on the Phenology of Infection of Gypsy Moth, Lymantria dispar, Larvae by the Fungus Entomophaga maimaiga

A computer model driven by daily maximum-minimum temperature and rainfall records was developed to investigate the influence of weather on times when gypsy moth larvae, Lymantria dispar, can become infected by the fungal pathogen Entomophaga maimaiga. In the model, gypsy moth eggs are hatched and neonates are exposed primarily to germinating resting spores in the soil during the spring. Risk of infection is related to forest floor moisture. Larval and fungal development follows a degree-day model. When larvae become 4th instars, they can again become infected by resting spores because they hide in the litter during daylight hours. If rain falls when infected caterpillars die, the fungus sporulates, producing conidia. The number of conidia produced is assumed to directly influence the probability of infection of other larvae. The model was run using weather records from 1990 through 1992. Predicted times of infection were compatible with estimated fungal recruitment rates and changes in field disease prevalence rates. Assumptions about infection mechanisms in the model are discussed as they relate to the real world.