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.     

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.     

Persistence of spores of Pleistophora schubergi (Cnidospora: Microsporida) in the field,and their application in microbial control

Spores of Pleistophora schubergi, when applied to oak trees in the field at 2 × 10⁸ spores/ml with a uv protectant, “Shade,” infected 88% of Anisota senatoria larvae at 4 days after spray application. Spores without the uv protectant infected only 10% of the larvae at 4 days after application. When the spores were applied at the rate of 2 × 10⁸ and 2 × 10⁷ spores/ml in the field, 96 and 72% of the A. senatoria larvae and 100 and 100% of the Symmerista canicosta larvae were infected 14 days after spray application.     

Reduction of consumption by grasshoppers (Orthoptera: Acrididae) infected with Nosema locustae canning (Microsporida: Nosematidae)

The effect of ingestion of Nosema locustae Canning spores on feeding by grasshoppers was measured in simultaneous laboratory and field experiments. After 21 days, fourth-instar Melanoplus sanguinipes (F.) nymphs, administered spores at the rates of 0, 2.0 × 10⁴, 2.0 × 10⁵, and 2.0 × 10⁶ per grasshopper, showed dry matter consumption of 102, 87, 64, and 26 mg in 48 hr, respectively. Rate of inoculation was a significant factor in suppression of feeding after correction for the effects of developmental stage, sex, and body weight. The quantity of dry matter consumed decreased linearly with increasing rate of spore ingestion. Experiments on 50 caged 1-m² plots on pasture grass yielded similar trends in per capita consumption independent of the effects of mortality. Field consumption per integrated grasshopper-day was 108, 77, 31, and 27 mg dry wt at the four inoculation rates, over 20 days.     

The use of a microinjection procedure for large-scale production of the microsporidian Nosema eurytremae in Pieris brassicae

Nosema eurytremae, a microsporidian parasite of Malaysian trematodes, was injected at the rate of 1 × 10⁴ spores/larva into Pieris brassicae. The larvae, which subsequently pupated, were incubated at 25 to 26°C and on harvesting 19 days later yielded an average of 6 × 10⁸ spores/pupa. This was equivalent to 60,000 times the initial dose. Purity of filtered, washed spore suspensions ranged from 80 to 99% with up to 20% host debris.     

The preservation of infective spores of Octosporea muscaedomesticae in Phormia regina,of Nosema algerae in Anopheles stephensi,and of Nosema whitei in Tribolium castaneum by lyophilization

Infective spores of three species of microsporidia were subjected to the lyophilization process by employing varying media as cryoprotectants. The infectivity of the lyophilized spores was then tested against a standard fresh spore preparation in the appropriate host insect. Spores of Octosporea muscaedomesticae served as an experimental model and were rendered noninfective in host Phormia regina (Calliphoridae: Diptera) after lyophilization with the following cryoprotective agents: skim milk (12%), ascorbic acid (5%) combined with thiourea (5%), glycerol (10%), mesoinositol (5%), and equine serum. Spores of O. muscaedomesticae lyophilized or vacuum-dried in 50% sucrose as well as in the hosts' tissues remained highly infective for as long as 2 years at a dose of 10⁶ spores/fly and a trial length of 12 days. At a dose of 5 × 10⁴ spores/fly there was a slight decrease in infectivity of the spores which had been lyophilized in the host's abdomen after a 2-year storage period compared with that of fresh, nonlyophilized spores. Naked spores of Nosema algerae suspended in 50% sucrose and lyophilized produced infection in 50% of the host population of Anopheles stephensi (Culicidae: Diptera) compared with 70% infection produced by fresh non-lyophilized spores. Spores of Nosema whitei lyophilized within its host larva Tribolium castaneum (Tenebrionidae: Coleoptera) remained 100% infective at a dose of 5 × 10⁵ spores/gram diet. It is concluded that an aqueous solution of 50% sucrose and/or the host's tissues are excellent protectants for the cryogenic or vacuum-drying process of the above-named spores, and their protective function may apply also to other microsporidian species.     

A laboratory evaluation of the microsporidian Vavraia culicis as an agent for mosquito control

The susceptibility of the mosquitoes Aedes aegypti, Aedes taeniorhynchus, Anopheles albimanus, Culex pipiens quinquefasciatus, Culex salinarius, and Culex tarsalis to infection by the microsporidian Vavraia culicis was determined. Using 18-hr exposures to 5 × 10³, 1 × 10⁴, 5 × 10⁴, and 1 × 10⁵ spores/ml, C. salinarius, C. tarsalis, and A. albimanus were found to be significantly more susceptible than A. aegypti. The most severe infections were observed in C. salinarius and C. tarsalis, although heavy infections of approximately 1 million spores per adult were recorded at the higher dosages in all species tested except A. aegypti. Production trials indicated that up to 5.4 × 10⁸ spores could be routinely produced in individual corn earworms, Heliothis zea. Inactivation of the spores by sunlight was measured by observing the subsequent incidence of infection and spore production in A. albimanus. These two measurements provided an LT₉₀ of 5.5 and 3.3 hr, respectively.     

Effect of entomopathogenic Aspergillus strains against the pea aphid,Acyrthosiphon pisum (Hemiptera: Aphididae)

Aphids (Homoptera: Aphididae) are sap-sucking insect pests that feed on several plants of agronomical importance. Entomopathogenic fungi are valuable tools for potential aphid control. As part of a selection process, laboratory bioassays were carried with five different concentrations of Aspergillus clavatus (Desmazières), Aspergillus flavus (Link) and Metarhizium anisopliae ((Metschnikoff) Sorokin) spores against the pea aphid, Acyrthosiphon pisum (Harris). Aspergillus isolates induced higher mortalities than M. anisopliae, which is a well-known entomopathogen in the literature. Lethal concentrations (LC₅₀ and LC₉₀) were 1.23 × 10³ and 1.34 × 10⁷ spores/ml for A. flavus, 4.95 × 10² and 5.65 × 10⁷ spores/ml for A. clavatus, and 3.67 × 10³ and 9.71 × 10⁷ spores/ml for M. anisopliae 5 days after treatment. Mycelia development and sporulation on adult cadavers were observed 48 h after incubation. The intrinsic growth rate of A. pisum decreased with increased spore concentration for all fungal strains, suggesting an increase in pathogen fitness related to a consumption of host resources. In conclusion, Aspergillus species could be useful in aphid control as pest control agents despite their saprophytic lifestyle. This is also to our knowledge the first report of A. clavatus and A. flavus strains pathogenic to aphids.     

Some characteristics of a new isolate of Helicosporidium and its effect upon mosquitoes

A Helicosporidium sp. was isolated by feeding spores concentrated by continuous flow centrifugation from ditch water to starved Heliothis zea larvae. This Helicosporidium sp was infectious to Anopheles quadrimaculatus, Culex salinarius, and Aedes aegypti with IC₅₀'s of 4.4 × 10², 2.6 × 10⁴, and 2.4 × 10⁴ spores/ml, respectively. Larval mortality was dosage dependent with LC₅₀ values 72 hr postexposure of 6.8 × 10⁴ for An. quadrimaculatus, 9.4 × 10³ for Cx. salinarius, and 1.5 × 10⁵ for Ae. aegypti. The spores of this Helicosporidium were also tolerant of freezing and desiccation. Because of these traits and the melanization response they provoked in host tissues, this is probably not naturally a mosquito pathogen and is most likely from a terrestrial insect.     

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.     

Foliar application ofNosema pyrausta for suppression of populations of European corn borer

In 1974, an application of the microsporidan,Nosema pyrausta (Paillot, 1927)Kotlan, 1928, with a back-pack type sprayer (22.5×10⁷ spores/plant) to whorl stage maize infested with European corn borers,Ostrinia nubilalis (Hübner) reduced the number of larvae/plant by 48.1% and produced an infection of 15.3×10⁴ spores/mg of larval weight in 62.1% of the collected larvae. In 1975, applications of 24.3×10⁷ spores/plant to similar maize, in 2 separate tests, reduced the number of larvae/plant by 18.8 and 43.8% and caused an infection of 14.3 and 19.1×10⁴ spores/mg of larval weight in 65.9 and 63.3% of the collected larvae. Also, in 1975, applications of 24.3×10⁷ spores/plant to pollen shedding maize in 2 separate tests reduced the number of larvae/plant by 17.2 and 14.1% and caused an infection of 24.3 and 27.2×10⁴ spores/mg of larval weight in 99.2 and 95.2% of the collected larvae.     

Some factors affecting spore replication of Nosema algerae (Microspora,Nosematidae) in Pieris brassicae (Lepidoptera)

The production of Nosema algerae spores was examined in Pieris brassicae. Spore replication in the insect host followed a logistic pattern of development. The factors studied which affected spore production and replication were dose level (5 × 10², 5 × 10³, and 5 × 10⁴ spores per insect), larval instar (fourth and fifth), and cool pretreatment of the insects at 20°C prior to inoculation compared with a constant temperature of 26°C. A three-way analysis showed the interactions between these factors. The logistic pattern of spore replication was used to explain the results.     

Spore production in solid-state fermentation of rice by Clonostachys rosea,a biopesticide for gray mold of strawberries

Gray mold caused by Botrytis cinerea is an important disease of strawberry. Clonostachys rosea is a mycoparasite of B. cinerea that reduces fruit losses when used as a biocontrol agent. Since spore production by C. rosea has not been optimized, we investigated factors affecting sporulation under aseptic conditions on white rice grains. The greatest spore production in glass flasks, 3.4 × 10⁹ spores/g-dry-matter (gDM), occurred with an initial moisture content of 46% (w/w wet basis), inoculated with 1 × 10⁶ spores/gDM and hand shaken every 15 days. However, a lower inoculum density (9 × 10³ spores/gDM) and no shaking also gave acceptable sporulation. In plastic bags 1.1 × 10⁸ spores/gDM were produced in 15 days, suggesting that larger scale production may be feasible: with this spore content, 24 m² of incubator space would produce sufficient spores for the continued treatment of 1 ha of strawberry plants.     

A rapid method to estimate the resistance of cold stored carrot roots to Botrytis cinerea

Inoculating whole carrot roots at 4°C with mycelial/agar discs of the grey mould fungus Botrytis cinerea gives a measure of their resistance and hence storage potential to this pathogen, but results are not obtained for at least 33 days. In the present investigation a more rapid method was used which involved inoculating root slices with spore suspensions containing 5 × 10^3–5 × 10⁶ spores/cm³ at 24°C. Resistance was assessed visually and by a chitin estimation 48 h after inoculation. Both methods were used to measure the resistance of cold stored carrot roots during two storage seasons, 1976/77 and 1977/78. In each season there was a particular inoculum level which most clearly recorded the increasing susceptibility of roots with time in store. In 1976/77 this was 1 × 10⁵ spores/cm³ whereas in 1977/78 it was the lower inoculum concentration of 5 × 10⁴ spores/cm³, suggesting the roots were generally more susceptible in 1977/78 than the previous season. This was in accord with the results from the whole root method of assessment. A chitin estimation proved to be the more sensitive method of assessment for inoculum potential experiments.     

Influence ofNosema bordati (Microsporida: Nosematidae) on the biological cycle ofChilo partellus (Lep: Pyralidae), a stem borer of cultivated graminae

The biological cycle ofChilo partellus (Swinhoe) was described on artificial diet. From egg to adult, it lasted 32 to 49 days with an average of 36.6 days. About 2,000 larvae from the 2^nd to the 5^th instars were artificially infected by ingestion with doses ofNosema bordati Goudegnon, varying from 2×10² to 2×10⁷ spores per ml. Only 72 survived (7.66 %) of these infected larvae.N. bordati, when present in the larvae, continued to multiply in the resulting pupae. The parasite affected the adults of this Pyralid reducing in a proportion of 5 the productivity of infected females and increasing the production of sterile eggs in the proportion of 8.     

Mass production and storage of Vairimorpha necatrix (protozoa: Microsporida)

Mass production and storage methods were evaluated for maximization of spores of Vairimorpha necatrix, a promising protozoan for microbial control due to its virulence and prolificity in lepidopterous pests. In vivo spore production was at a maximum when 3rd instar Heliothis zea were exposed to 6.6 spores/mm² of artificial diet surface and reared for 15 days. Approximately 1.67 × 10¹⁰ spores/larva were produced, or ca. 1 × 10¹⁰ spores/larva after partial purification of the spores by homogenization of the larvae in water, filtration, and centrifugation. The spores were inactivated by relatively short exposures to several chemicals which were tested to counteract contamination of the diet surface by fungi in the spore inoculum. Spores of V. necatrix were stored at refrigerated and freezing temperatures for up to 2 years and bioassayed periodically with 2nd instar H. zea. Spores lost little infectivity after 23 months at 6°C if they were stored in a purified water suspension plus antibiotic, but they were noninfective after 18 months at 6°C if stored in host tissue. Storage at ?15°C caused little loss of infectivity whether the spores were stored in water and glycerine, in host tissue, or after lyophilization. The spores withstood lyophilization in host cadavers better than in purified water suspension. Samples of a dry V. necatrix-corn meal formulation, which was prepared for field efficacy tests and stored at ?15° and 6°C, were highly infective after 9 months. Large numbers of V. necatrix spores can thus be produced and later made available for microbial control field trials with little loss of infectivity.     

Patterns of primary spore discharge of Entomophthora spp from the blue green aphid,Acyrthosiphon kondoi

Experiments were conducted to study the effects of time, temperature, and light regime on primary spore formation at 100% RH for the three major pathogens of Acyrothosiphon kondoi. Only small differences were detected between the continuous light and continuous dark regimes. Entomophthora obscura produced between 6 and 10 × 10³ primary spores mostly during the first 48 hr. Total primary spore production was similar at the five temperatures tested from 5° to 25°C. Entomophthora planchoniana produced large numbers of primary spores (about 5 × 10⁴ per aphid) only at temperatures between 10° and 20°C. The majority of primary spores were formed during the first 24 hr. Primary spore production with Entomophthora nr. exitialis ranged from about 10⁵ per aphid at 5° and 10°C to 3 or 4 × 10⁵ at 15° to 25°C, with most spores being formed during the first 48 hr. It is suggested that rainfall is more likely to be important for transmission of E. obscura and E. nr. exitialis than for transmission of E. planchoniana, and that E. obscura is likely to be the most important pathogen during cool or cold weather.     

Nosema whitei,a microsporidan pathogen of some species ofTribolium

The pathogenicity ofNosema whitei was studied using a dose-mortality technique; larvae ofTribolium castaneum were reared for the duration of each experiment in flour mixed with known numbers of spores. The susceptibility of each of the first 5 larval instars was compared. The LD₅₀ (for mortality after 20 days) increased consistently from the first instar (1.8×10⁶ spores/g) to the fifth instar (1.0×10¹⁰ spores/g). The slopes of the probit lines increased consistently as age increased (from b=1.1 to b=3.9). Two factors which reduce the development time ofT. castaneum, high temperature and high humidity, both reduced the pathogenicity ofN. whitei. Thus pathogenicity decreased as the temperature was increased fram 25°C (LD₅₀=4.2×10⁶) through 30°C (LD₅₀=1.3×10⁷) to 35°C (LD₅₀=3.2×10⁶), also pathogenicity decreased consistently as humidity was increased fram 10%, through 30, 50, 70% to 90% R.H. Adults, emerging fromNosema free larvae, became infected only when exposed to a very high dose (2×10¹⁰ spores/g for 14 days from the day of emergence). Infected larvae were treated for 1 hr. at 45°C in an attempt to cure the infection. The infected larvae were not cured, rather the treatment had an adverse alfect on their survival.

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Résumé La pathogénicité deNosema whitei a été étudiée en élevant des larves deT. castaneum dans de la farine mélangée à des quantités connues de spores. La sensibilité des larves diminue uniformément en fonction de l'age; La DL₅₀ varie de 1,8×10⁶/g (1^er stade) à 1,0×10¹⁰ spores/g (5^e stade). Deux facteurs, qui accélèrent le développement deT. castaneum, des températures et des humidités élevées, réduisent tous les deux la pathogénicité deN. whitei. Les adultes ne peuvent être infectés qu'en les exposant à la dose extrêmement élevée de 2×10¹⁰ spores/g. Un traitement par la chaleur (45°C pendant une heure) n'a pas réussi à guérir les larves.

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This work financed by a Science Research Council (U.K.) studentship is based on a thesis submitted for a degree of Ph. D. at the University of Newcastle-upon-Tyne.     

Soybean molasses-based bioindicator system for monitoring sterilization process: Designing and performance evaluation

A novel cost-effective Bacillus atrophaeus Sterilization Bioindicator System (BIS) with a high quality and performance was developed from a soybean byproduct and compared with the commercial BIS. It was composed of recovery medium and dry-fermented spores with sand as the support. The BIS was developed and optimized using a sequential experimental design strategy. The recovery medium contained soluble starch (1.0 g/L), soybean molasses (30.0 g/L), tryptone (40.0 g/L), and bromothymol blue (0.02 g/L) at pH 8.5. The solid-state fermentation conditions of the bioreactor and environmental humidity had no significant effects on the spore yield and dry-heat resistance. The only substrate mineral that showed a positive effect was Mn²⁺, allowing Mg²⁺, K⁺, and Ca²⁺ to be eliminated from the formulation. Validation of optimized medium indicated D _160°C = 6.8±1.0 min (3.6 min more than the minimum) and spore yield = 2.3 ± 0.5 × 10⁹ CFU/g dry sand (10,000 × initial values). BIS performance resulted in D _160°C = 6.6 ± 0.1 min. Sporulation and germination kinetics allowed the sporulation process to be reduced to three days, and the growth of heat-damaged spores was sufficient to achieve visual identification of a non-sterile BIS within 21 h. Process economics was a minimum of 23.9%, and process cycle time was reduced from 29 to 15 days. The new BIS parameters demonstrated compliance to all regulatory requirements. No studies have yet described a BIS production from soybean molasses.     

A novel milky disease organism from Australian scarabaeids: Field occurrence,isolation, and infectivity

A novel milky disease organism has been found causing disease in Aphodius tasmaniae and other scarabaeid larvae in the field in Australia. The sporangium is exceptionally long, measuring 10.5 × 1.5 μm, with a small central spore, measuring 1.0 × 0.6 μm. The vegetative cell is about half the size of the sporangium. The disease was easily transmitted by injection of spores into the hemocoel, with typically milky symptoms developing in 2–4 weeks. Spores will form in vivo at temperatures down to 12°C. For A. tasmaniae third-instar larvae, the ID₅₀ by injection was 3 × 10² spores/larva, yet no infection resulted when larvae were reared in peat containing up to 10⁸ spores/g, i.e., the disease was not successfully transmitted per os. All 10 species of scarabaeids tested were susceptible to the disease when spores were injected; however, all attempts to infect larvae per os were unsuccessful. In vitro culture was also unsuccessful.