In vivo propagation of a microsporidan pathogenic to insects

Equivalent numbers of spores were produced when the microsporidan Nosema necatrix was propagated in either Trichoplusia ni or Heliothis zea. Maximum spore production was obtained at an inoculum level of 1 × 10⁵ spores/ml. Larvae inoculated 5 days post-hatching contained 1.6 × 10⁹ spores/gram larva after an incubation period of 21 days. Temperature optima for the parasite are 21–26°C in both hosts.     

Factors influencing biocontrol of jimsonweed (Datura stramonium L.) with the leaf-spotting fungus Alternaria crassa

In greenhouse tests, Alternaria crassa (Sacc.) Rands killed > 80% of inoculated jimsonweed (Datura stramonium L.) seedlings within 14 days following a 9-h dew period at 25°C with 1 × 10⁵ spores/ml, and after 8 h of dew at 1 × 10⁶ spores/ml. At least 10 h of dew with 1 × 10⁵ spores/ml and 9 h of dew with 1 × 10⁶ spores/ml were required to obtain 100% mortality of fungus-inoculated plants. Growth stage and inoculum concentration studies revealed that higher concentrations of inoculum were required to obtain 100% mortality of larger plants. Weed control was significantly reduced by day/night air temperatures of 35°C and 24°C, respectively, at all inoculum concentrations as compared to the controls at lower air and dew temperature regimes. The results of these studies indicate that A. crassa has potential as a biological herbicide for the control of jimsonweed.     

Physiological restriction of the L-lactic acid production by Rhizopus arrhizus

The influence of basic physiological factors on the quality of inocula and L(+)-lactic acid production by Rhizopus arrhizus CCM 81 09 were studied. The most effective preparation of the spores (5 × 10⁷ spores/ml) and subsequent good lactate production was achieved on the agar medium with soil extract and malt agar. The optimum initial amount of active spores for inoculation was 10³–10⁴ spores/ml. The preparation of inoculum required intensive stirring with lower aeration and pH maintained in the range from 4.8 to 6.0 by the addition of CaCO₃. The maximum yield of lactic acid production was achieved by using 5% (v/v) of 24-h-old inoculum. The intensity of lactic acid production in the inoculum was proportional to its production in the subsequent steps of fermentation and can be used as a fast control of the physiological state of the producers.     

Reproductive phenology of the introduced kelp <Emphasis Type="Italic">Undaria pinnatifida</Emphasis> (Phaeophyceae,Laminariales) in Port Phillip Bay (Victoria,Australia)

A thorough understanding of the reproductive phenology of introduced species is crucial for effective management and control. Undaria pinnatifida is an invasive macroalga from the Northwest Pacific which has been recently introduced into three countries in the Southern Hemisphere: Australia, New Zealand and Argentina. Reproductive phenological studies in Port Phillip Bay, Australia, were undertaken and compared with other populations in the Southern Hemisphere, especially with those from Tasmania which were suspected to be very different. The growth season began earlier in Port Phillip Bay than in Tasmanian populations, and abundance was higher. Growth rates were lower in Port Phillip Bay, but this might be due to the different morphology of both populations. The maximum spore release competency of U. pinnatifida in Port Phillip Bay was 12.1 × 10⁵ spores cm⁻² h⁻¹ which is 20 times the maximum obtained in Tasmania (0.6 × 10⁵ spores cm⁻² h⁻¹). For most of the growth season, spore release competency ranged between 2 and 3 × 10⁵ spores cm⁻² h⁻¹, 3–5 times more than in Tasmania. Undaria pinnatifida has not been established outside Port Phillip Bay in continental Australia, but a precautionary approach should be undertaken in order to avoid further spread. Monitoring for early detection and removal of immature sporophytes prior to spore release seem to be the best options. This monitoring should be continuous since new recruits may appear throughout the growth season (April–February) and it should be combined with informative programmes to reduce the chances of spread.     

The culture ofEntomophthora muscae (C) Fres. in carrot flies (Psila rosae F.) and the effect of temperature on the pathology of the fungus

A method for maintaining anin vivo culture ofEntomophthora muscae (C) Fres. on its original host, adult carrot flies (Psila rosae F.), is described. The lethal time for adult carrot flies was greatly influenced by temperature, both for infected and for uninfected flies. In the range 8.2°C–20.2°C the LT₅₀ for infected flies was about 5.4 times shorter than the estimated average life-span for uninfected flies. The discharge of primary spores was also strongly dependent on temperature. The total number of primary spores discharged per fly at 100% RH and in darkness ranged between 1.2×10⁴ and 9.6×10⁴ with a mean of 5.1×10⁴.      

Ecological investigations of the zooplankton community of Balsfjorden,northern Norway: Population structure and breeding biology of the chaetognath Sagitta elegans Verrill

The population dynamics of the chaetognath Sagitta elegans Verrill has been followed in Balsfjorden in 1976 and 1977. Seasonal variation in abundance, length-frequency distribution, growth in total length, and maturity stages are presented and discussed in relation to changes in hydrography.An annual generation of S. elegans was found, with a protracted and more or less continuous breeding season from May until October during 1977. The 1976 year-class consisted of two distinct length groups, both of which participated in the 1977 spawning. This spawning gave rise to possibly four sub-populations during 1977. The variation in numbers of sub-populations produced during the spawning season in 1976 and 1977 is discussed in relation to the hydrographical conditions in Balsfjorden. From November 1976 to March 1977 the abundance of S. elegans varied between 1 and 8 ind. · m^?3. The lowest value was recorded in May (0.9 ind. · m^?3). From September to December 1977 the population abundance was ≈2 ind. · m^?3.     

Studies on the Diagnosis of Bacterial Ring Rot of Potatoes

The eggplant test, described by Olsson (1976) and Lelliott and Sellar (1976) as a means of detecting Corynebacterium sepedonicum, the causal agent of bacterial ring rot, has been tested for its reliability. Eggplants inoculated with a pure suspension of bacterial cells developed typical chlorotic symptoms at a minimum concentration of 2 × 10³ cells/ml inoculum. Whereas with an inoculum which consisted of potato tissue equally mixed with the bacterial suspension, the first symptoms developed at a concentration of 10⁴ cells/ml. Reisolation of the pathogen by the IFAS-test or on YDC-medium was possible following innculation at a concentration of 2 × 10³ cells/ml. It was mostly possible to isolate the bacteria in pure culture. Further solanaceous host species tested in comparison with the eggplant did not develop typical symptoms following inoculation with C. sepedonicum. On this account, the eggplant test should currently be considered as the most suitable biological assay to detect C.sepedonicum.     

Laboratory studies on the fungus Verticillium lecanii, a larval pathogen of the large elm bark beetle (Scolytus scolytus)

From 1972 to 1974, estimates of the natural larval mortality (> second instar) of elm bark beetles caused by pathogenic organisms were always below 7'5 % of the beetle population. The pathogenic fungus Verticillium lecanii was frequently isolated from field-collected dead larvae, and in the laboratory all larvae were killed in 5 days when exposed to spore concentrations of 4·5 × 10⁶ spores/ml. V. lecanii begins to lose its pathogenicity after prolonged culture on artificial media. The time taken for V. lecanii to kill Scolytus scolytus larvae when exposed to a logarithmic series of spore dilutions from 9·1 × 10⁷/ml to 9·1 × 10³/ml increased with decreasing amounts of inoculum. Even at spore concentrations as low as 9·1 × 10³/ml the mortality of treated larvae was greater than that of untreated individuals. At 100% r.h. all treated larvae were killed over a temperature range of 5–30 °C; those maintained at 25 °C were killed most rapidly and those kept at 5 °C the slowest.     

Effect of spore concentration on germination and autotropism inTrichoderma hamatum

Summary The effect of spore concentration on spore germination and germtube growth ofTrichoderma hamatum on water agar and on potato dextrose agar (PDA) was studied. Increasing inoculum size up to 10⁹ spores/plate on PDA and up to 10⁷ spores/plate on water agar shortened the incubation period required for germtubes emergence and increased germination rate. However, on water agar germination was inhibited at 10⁸ and was completely arrested at 10⁹ spores/plate. Inhibition in germination of 10⁷ spores/plate was observed on water agar when the plates were preincubated with 10⁹ spores/plate for 5 h or more. Addition of glucose and ammonium nitrate to the water agar medium allowed only 25% of the spores to germinate at 10⁹ as compared to 78% at 10⁷ spores/plate after 8 h of incubation. Addition of polysaccharides to the C+N supplemented medium, significantly increased germination up to 84% as compared to 100% on PDA, after 8 h of incubation. Germlings ofTrichoderma hamatum phialospores exhibited positive autotropism and anastamosis on both media. The phenomenon was positively related to inoculum size, being most pronounced at 10⁷ spores/plate.     

Production of Metarhizium anisopliae spores using nutrient‐impregnated membranes and its economic analysis

Metarhizium anisopliae spores were produced on nutrient‐impregnated membranes (NIMs). The NIM system involved wetting the membrane with a spore and nutrient suspension, followed by harvesting the spores produced after incubation. The cost efficiency of spore production was assessed for a range of nutrient sources and membrane types. Skim milk powder (20 g l^‐1) was found to be the most cost‐effective nutrient source of the nine nutrients examined. Yield was 5.7 × 10⁶ spores/cm² after 28 days incubation on a paper membrane. Supplementation of the skim milk with either sucrose (2 g l^‐1) or dextrose + KNO₃ maximized yield. Superwipe, an absorbent fibrous material, was the most efficient of 16 membranes tested which ranged from fibreglass mesh to paper and cloth. A series of small pilot plants were built, but the cost efficiency of spore production decreased as the size of the membrane increased from 24 × 24 cm to 270 × 15 cm and up to 100 × 80 cm. Yield on the two smaller pilot plants was over 10⁷spores/cm², but the cost (nutrient and membrane only) of producing 10¹³ spores (standard dose required per hectare) was around $A37 and was found not to be competitive with spore production on grain.     

Virulence of entomopathogenic Fungi (Fungi imperfecti) for the adults of Acanthoscelides obtectus (Coleoptera: Bruchidae)

Tests with the bean weevil, Acanthoscelides obtectus, in which the hosts were exposed indirectly to various dilutions of conidia of four entomopathogenic fungi showed that mortality was a function of the concentration of the inoculum. In these tests a given spore suspension was sprayed on the internal surfaces of a Petri dish. Adult weevils of a known age were placed in the dish, held there for 24 hr, then removed and kept at 20°C. After 20 days, the host mortality was determined. From the data obtained, it was possible to trace a probit regression line of the mortality in relation to the increasing spore concentration. Infection was observed in hosts exposed to a concentration of approximately 5 × 10⁶ spores/ml up to a maximum of about 1 × 10⁹ spores/ml. The A. obtectus was susceptible to infection by spores of Beauveria bassiana, B. tenella, Metarrhizium anisopliae, and Paecilomyces fumoso-roseus.     

Selection of Antonospora locustae (Protozoa: Microsporidae) with higher virulence against Locusta migratoria manilensis (Orthoptera:Acrididae)

Antonospora locustae is a microsporidian parasite of grasshopper insects that is used as a biological control agent. We report on laboratory selection of isolates from different regions with increased virulence. Bioassays were conducted against third instar nymphs of Locusta migratoria manilensis. AL2008L01 was originally imported from the USA in 1986, AL2008M01 was isolated from Melanoplus differentialis in USA and AL2008F01 was isolated from infected Fruhstorferiola tonkinensi collected in Guangdong, China. The results showed that all three isolates can infect the locust and that pathogenicity increased gradually with increased dose. The LD₅₀ values of the original isolates at the highest dose (5×10⁶ spores/nymph) were 19, 23 and 22 days and LD₅₀ values were 3.2×10⁵, 3.4×10⁶ and 0.7×10⁶ spores/g, respectively. After selecting for three generations, the virulence of all isolates increased significantly. LT_50s were reduced to 17, 20 and 21 days at the highest dose (5×10⁶ spores/nymph) and LD_50s were reduced to 1.4×10⁵, 2.5×10⁵ and 1.7×10⁵ spores/g.     

Accelerated Biodegradation of High and Low Concentrations of p-Nitrophenol (PNP) by Bacterial Inoculation in Industrial Wastewater: The Role of Inoculum Size on Acclimation Period

The effect of inoculum size on the acclimation period and rate and extent of p-nitrophenol (PNP) degradation at high (1–10 mg/L) and low (26 μg/L) concentrations for two bacteria was determined in defined media as well as industrial wastewater. Increased inoculum size did not affect the acclimation period of either bacterium at high (1–10 mg/L) PNP concentrations. At low PNP concentrations (26 μg/L), the two bacteria behaved differently. The acclimation period was shortened and both the rate and extent of mineralization of PNP were enhanced by increasing the Corynebacterium sp. inoculum size from 3 × 10⁵ to 3 × 10⁶ cells/ml. Addition of phosphate or elimination of predators also reduced the acclimation period. Conversely, increasing the inoculum size from 3 × 10⁵ to 5 × 10⁶ cells/ml of Pseudomonas putida lengthened the acclimation period and reduced both the rate and extent of mineralization. It is suggested that, in a given environment, the success of an introduced species to enhance the degradation of a chemical depends upon (i) concentration of the chemical, (ii) selection of an appropriate microorganism, and (iii) utilization of a suitable inoculum size. Received: 1 April 1996 / Accepted: 6 May 1996     

Effect of plant stage, <Emphasis Type="Italic">Colletotrichum truncatum</Emphasis> dose,and use of herbicide on control of <Emphasis Type="Italic">Matricaria perforata</Emphasis>

Colletotrichum truncatum (Ct) was examined in a tank mix with the herbicide 2,4-D, clopyralid plus MCPA (Caurtail M^®), or metribuzin (Sencor^®) for control of scentless chamomile at 8- (younger) and 11-leaf stages (older) under controlled conditions. In initial trials, Ct at 7 × 10⁶ spores/ml (200 l/ha) reduced the fresh weight of scentless chamomile only slightly. However, its combinations with herbicides improved the efficacy variably depending on the herbicide used and stage of the weeds. Ct plus 2,4-D reduced the fresh weight by about 50% at both leaf stages of scentless chamomile when compared to untreated controls but no plants were killed. The fungus plus Curtail M consistently killed younger but not older plants, and the efficacy was substantially greater than that of the herbicide alone. The herbicide Sencor was highly effective on younger plants, and adding Ct did not achieve additional benefits. On older plants, however, Ct plus Sencor was substantially more effective than the herbicide alone, causing 76% fresh-weight reduction when compared to controls and killing 9 out of 16 older plants in four trials. Sencor applied alone reduced the fresh weight of older plants by 65%, but no plants were killed. Tested at doses ranging from 2 × 10⁶ to 20 × 10⁶ spores/ml, Ct plus Curtail M was most effective at the highest fungal inoculum dose, consistently killing younger but not older plants. In comparison, Ct at a medium dose (7 × 10⁶ spores/ml) plus Sencor killed the majority of older chamomile plants (7 out of 12), whereas the herbicide alone did not cause plant mortality. Further increasing fungal inoculum dose from this medium level did not enhance the weed control by Ct plus Sencor.     

In vivo development and pathogenicity of Ascosphaera proliperda (Ascosphaeraceae) to the alfalfa leafcutting bee, Megachile rotundata

First instar larvae of the leafcutting bee, Megachile rotundata, were fed on either artificial or natural provisions containing spores of Ascosphaera proliperda. Two isolates were used as a source of inocula: one originated from in vitro isolates obtained while culturing what was thought to be pure spores of A. aggregata, the second originated from in vitro cultures from Denmark. Histological and scanning electron microscopy studies revealed that the spores germinated in the gut lumen and the developing hyphae invaded all tissues, after which they penetrated through larval integument and began the sexual phase of the life cycle aerially. Virtually all fungus-exposed larvae developed symptoms of disease regardless of source of inoculum, type of provision, and spore dose (1.5 × 10³ to 3 × 10⁶) per insect. It was concluded that the fungus was pathogenic to the alfalfa leafcutting bee under laboratory conditions and future studies should be conducted to determine its etiology, cross infectivity, and natural distribution in other bee taxa.     

Postharvest biological control of Penicillium digitatum decay on citrus fruit by Bacillus pumilus

A new antagonist/pathogen combination is reported, with Bacillus pumilus showing strong antagonistic activity against Penicillium digitatum. B. pumilus (1.6 × 10¹⁰ to 1.6 × 10¹²*cfu ml^-1) gave significant control of P. digitatum infection in Valencia orange, which was as effective as imazalil (500 μg ml^-1) and was significantly better than benomyl treatment (500 μg ml^-1). When lower concentrations of B. pumilus (1.9 × 10⁷ to 1.9 × 10⁹ cfu ml^-1) were tested on Washington Navel orange and Lisbon lemon fruit, the antagonist caused significant control of P. digitatum infection at two inoculum levels (6.5 × 10⁴ and 6.5 × 10⁵ spores ml^-5). Concentrations of both the pathogen and the antagonist affected the biocontrol effect.     

Pathologie d’une microsporidiose de l’arpenteuse du chou,Trichoplusia ni [Lep.: Noctuidae], parVairimorpha necatrix

Résumé Les larves deTrichoplusia ni (Hübner) infectées parVairimorpha (=Nosema) necatrix (Kramer) présentent des sympt?mes chroniques, semi-chroniques et aigus selon les doses de spores deV. necatrix (5 à 500, 5.10³ à 5.10⁵ et 5.10⁶ par larve, respectivement) ingérées par larve.V. necatrix parasite principalement le corps adipeux et les tissus des muscles quand les larves ont des sympt?mes chroniques, tandis que les larves manifestent les sympt?mes aigus lorsque les microsporidies attaquent principalement l’intestin moyen. L’histopathologie de la maladie est étudiée.

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Summary Infection of larvae ofTrichoplusia ni (Hübner) byVairimorpha, (=Nosema) necatrix was classified as chronic, semi-chronic and acute symptoms depending on the quantity of spores (5 to 500, 5×10³ to 5×10⁵, and 5×10⁶ per larva, respectively) ingested per larva.V. necatrix infects mainly the fat body and some muscle tissue of larvae with chronic symptoms and mainly midgut tissue of larvae with acute symptoms.V. necatrix caused death in 3 to 4.5 days when a larva ingested 5×10⁶ spores. The histopathology of the disease was studied.

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Ce travail a fait l’objet d’une thèse de 3^e cycle, présentée à l’Université de Paris VI en mars 1977, parWei Hsuang Chu.     

Quantitative and qualitative aspects in the production of a nuclear polyhedrosis virus in Spodoptera exigua larvae

Spodoptera exigua nuclear polyhedrosis virus was produced in late fourth instar S. exigua larvae, reared on semi-artificial diet. A maximum amount of virus, 1–2 × 10⁹ polyhedra/larva, was produced in individually-reared larvae after 7 days of incubation, with an inoculum of 7–5 × 10⁴ polyhedra/cm² diet surface. Virus yield was slightly reduced to 9 × 10⁸ polyhedra/larva when production was carried out in groups of 400 and 600 larvae per container. Biological activity of virus harvested from living larvae and from dead cadavers was similar. Microbial contaminants, predominantly bacteria, in the virus product numbered 1–6% of the number of polyhedra. Tests for the presence of vertebrate-pathogenic bacteria in the virus product were all negative.     

Application of Beneficial Microorganisms to Seeds during Drum Priming

Five microbial plant growth promoters or biocontrol agents (Pseudomonas fluorescens CHA0, Pseudomonas sp. AB842, Bacillus subtilis MBI600, Trichoderma harzianum T22 and T. virens G20) were assessed for ability to proliferate on seeds of carrot, parsnip and leek. In small-scale priming systems, both pseudomonads and MBI600 (when applied as cells) at levels between 10⁵ and 10⁶ cfu g^?1 seed were able to colonise all seeds at the end of priming (240 h) despite initial poor recovery after addition of the cells in some cases. Pf CHA0 was a particularly aggressive seed coloniser often comprising the total pseudomonad population at the end of priming. Drying the seed after priming resulted in &lt;1 log₁₀ cfu g^?1 seed loss for the pseudomonads but greater losses for MBI600 on carrot and leek seed. Application of spores of MBI600 resulted in little loss in cfu g^?1 seed following addition of the cells and these levels were maintained throughout the priming period and after drying back. Both T22 and G20 were recovered from carrot and parsnip at the end of priming and in general reflected survival of the inoculum rather than proliferation. T22 and G20 could not be recovered from leek seed following priming. Comparisons were made between proliferation and survival in large-scale drum priming with post-priming application of microorganisms. Pf CHA0 proliferated on carrot and parsnip seed during drum priming and survived the drying back process whereas there was no recovery when applied as a post-priming treatment. In contrast, MBI600 could not be recovered when applied during priming as cells, whereas recovery was good when applied post-priming as spores. T22 spores could be applied in either manner. Post-priming application of metalaxyl and thiabendazole had variable effects on microorganism recovery. The significance of the application of microorganisms to seeds during priming is discussed.     

Differences in the response of several cell types to inhibition of surface receptor mobility by local concanavalina binding

Concanavalin A (conA) modulates the lateral mobility of cell surface receptors differently on different cell types. This was demonstrated by using fluorescence photobleaching recovery (FPR) to measure the inhibition of the lateral mobility of conA receptors by localized binding of conA on lymphocytes, fibroblasts, and macrophages. On mouse spleen lymphocytes, binding of conA platelets above a threshold coverage (about 12% of the upper cell-surface area) reduced the diffusion coefficient of mobile TMR-SconA-receptor complexes from 3.0×10^?10 cm²/sec to 0.6× 10^?10 cm²/sec (a 5-fold decrease), and the fraction of mobile receptors was concomitantly reduced from 0.4 to 0.11. Below the threshold occupancy, no effect on either parameter was detected. On 3T3 cells, a qualitatively similar threshold phenomenon was observed: coverage of over 9% of the upper cell surface by conA platelets induced a 3-fold reduction in the diffusion coefficient of TMR-SconA-receptor complexes from 5×10^?10 cm²/sec to 1.7× 10^?10 cm²/sec. However, no effect on the mobile fraction (about 0.4) was observed. In contrast, neither the diffusion coefficient nor the mobile fraction of TMR-SconA-receptor complexes on mouse peritoneal macrophages (both resident and thioglycolate-stimulated) or on the mouse macrophage cell line P388D₁ were affected by the binding of conA platelets in amounts covering over 50% of the upper cell surface (approx. 4.6× 10^?10 cm²/sec and 0.5 for the diffusion coefficient and mobile fraction, respectively). These differences are correlated to the different cytoskeletal functions of the various cell types studied, and are discussed regarding the mechanism of the conA-induced modulation.