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.     

Interactions between Vairimorpha necatrix,Vairimorpha sp., and a nuclear polyhedrosis virus from Rachiplusia ou in Agrotis ipsilon larvae

Vairimorpha sp. and V. necatrix were assayed in combination with one another, and independently, with a nuclear polyhedrosis virus (RoMNPV) from a mint looper, Rachiplusia ou, against neonate and third-stage black cutworm larvae, Agrotis ipsilon. Initially the effect of Vairimorpha sp. was subadditive, additive, or slightly inhibiting to the V. necatrix in the dual microsporidian assays; later, V. necatrix antagonized the effect of the Vairimorpha sp. In combination with Vairimorpha sp. gradients, V. necatrix significantly (P < 0.05) reduced, in most instances, the LT₅₀ values in both neonate and third-stage larval assays. RoMNPV assayed against neonate and third instars, in combination with either Vairimorpha sp. or V. necatrix gradients, usually significantly reduced the LT₅₀ values (P < 0.05). RoMNPV, when combined with either Vairimorpha species, had varying effects on its pathology. In all assays, the particular relationship that was expressed seemed to be a function of the concentration of each pathogen, which may indicate that the two microorganisms compete for entry and/or infective sites within the larval host. Larval size, which was significantly reduced (P < 0.05) by both microsporidia, would be involved in such competition because it would limit the tissue mass available for infection. Histological examinations of larvae with dual infections revealed pathogens in the tissues that they normally infect. Vairimorpha sp. primarily infected epithelial, fat body, and Malpighian tubule tissue and, occasionally, muscle tissue. Viral polyhedral inclusion bodies were found in the same fat body tissues as the Vairimorpha spores.     

Dissemination of the biocontrol agent Vairimorpha necatrix by the spined soldier bug, Podisus maculiventris

The ability of the spined soldier bug, Podisus maculiventris (Say) (Heteroptera: Pentatomidae), to disseminate infective forms of two lepidopteran pathogens, Vairimorpha necatrix (Kramer) (Microspora: Microsporidia) and Lacanobia oleracea granulovirus (LoGV) was investigated. Individual female P. maculiventris that had fed on Lacanobia oleracea L. (Lepidoptera: Noctuidae) larvae, infected with V. necatrix, excreted approximately 6 × 10⁸V. necatrix spores during the subsequent 7 days. Excreted spores were fed to L. oleracea larvae, causing 100% mortality, indicating that the spores remained viable after passing through the gut of the predator. Podisus maculiventris that had fed on V. necatrix or LoGV‐infected larvae were allowed to defecate on the foliage of tomato plants, prior to the infestation of the plants with L. oleracea or Spodoptera littoralis (Boisduval) (Lepidoptera: Noctuidae) larvae. This proved to be an effective way of infecting the pest larvae with the pathogens, particularly when five predatory bugs were used per plant. After 20 days, the number of S. littoralis and L. oleracea surviving on the plants was reduced by 75% and 61%, respectively. Female P. maculiventris maintained on V. necatrix‐infected prey showed reduced egg production and longevity, whilst those fed on LoGV‐infected prey showed only reduced egg production. The potential for P. maculiventris to disseminate insect pathogens is discussed in the context of improved biological control of lepidopteran pests.     

Eimeria acervulina,E. brunetti,E. maxima, and E. necatrix: Low doses of oocysts to immunize young chickens

Resistance to reinfection varied with the species of Eimeria and with the number of oocysts in the inoculum. Chickens immunized with doses of 20,000 and 80,000 oocysts of E. acervulina, 312 and 1250 oocysts of E. brunetti or E. necatrix, or 1250 and 5000 oocysts of E. maxima at 2 and 4 weeks of age, respectively, were almost completely immune to a challenge dose at 6 weeks of age. Resistance was slightly less in chickens immunized with 1250 and 5000 oocysts of E. acervulina or 312 and 1250 oocysts of E. maxima. Birds given three immunizing infections of 1250, 5000, and 20,000 oocysts of E. maxima were completely immune 8 weeks after the last dose. Resistance was slightly less in birds immunized with similar doses of E. brunetti or E. necatrix. Doses of 20,000, 80,000, and 320,000 oocysts appeared necessary to confer a high level of immunity to E. acervulina. More than three low doses of oocysts appear necessary to induce a complete and enduring immunity against a high challenge for E. acervulina, E. brunetti, and E. necatrix. Higher immunizing doses would not be satisfactory due to the pathogenic effects of the coccidia after the initial infection.     

Detection of Autographa californica and Heliothis zea baculovirus proteins by enzyme-linked immunosorbent assay (ELISA)

The structural proteins of Autographa californica (AcMNPV) and Heliothis zea (HzSNPV) nuclear polyhedrosis viruses were detected by indirect enzyme-linked immunosorbent assay (ELISA). The immunoassay detected less than 1 ng of AcMNPV protein. The extent of immunological relatedness between AcMNPV-occluded virus and AcMNPV polyhedral protein, AcMNPV-nonoccluded virus, Estigmene acrea granulosis virus, Amsacta moorei entomopoxvirus Heliothis zea NPV, and Lymantria dispar NPV was determined. No immunological relatedless was detected between HzSNPV, AcMNPV, and a persistent rod-shaped virus isolated from the Heliothis zea cell line (IMC-Hz-1). The polyhedral proteins of HzSNPV and AcMNPV were found to be immunologically identical.     

Cross immunity of DNA vaccine pVAX1-cSZ2-IL-2 to Eimeria tenella, E. necatrix and E. maxima

The study describes cross protection experiments with chimeric DNA vaccine pVAX1-cSZ2-IL-2 to determine its efficacy against four important Eimeria species. Seven-day-old chickens were randomly divided into nine groups; group 1 negative control, groups 2, 3, 4, 5 positive controls; and groups 6, 7, 8 and 9 experimental groups. On days 7 and 14, groups 1-5 were injected with TE buffer, and groups 6-9 with the vaccine. At 21 days of age, all chickens were inoculated with 5 × 10⁴ sporulated oocysts except for the negative control. Groups 2 and 6 were inoculated with Eimeria tenella, groups 3 and 7 with Eimerianecatrix, groups 4 and 8 with Eimeria acervulina and groups 5 and 9 with Eimeria maxima. Seven days later, all chickens were weighed and slaughtered to obtain intestinal samples. Efficacy of immunization was evaluated on the basis of oocyst decrease ratio, lesion score, body-weight gain and anti-coccidial index. The results indicated that the recombinant plasmid can induce host immune responses by alleviating intestinal lesions, body weight loss and oocyst ratio and imparting good protection against E. tenella and E.acervulina, medium protection against E. necatrix but little effect against E. maxima. It is concluded that the conserved antigen can provide cross protection and should be explored further.     

Relative pathogenicity of nuclear polyhedrosis viruses from Heliothis punctigera and Heliothis zea for larvae of Heliothis armigera and Heliothis punctigera

Laboratory bioassays indicated that the potency of a nuclear polyhedrosis virus from Heliothis zea derived from a commercial American formulation was similar to that of a naturally occurring nuclear polyhedrosis virus from H. punctigera in Australia. Both viruses exhibited high virulence for neonate larvae of H. armigera and H. punctigera, the major pest species in this genus in Australia. Hence evaluation of the virus in Australia can proceed employing virus from either H. punctigera or H. zea.     

Functional analysis of a melanin biosynthetic gene using RNAi-mediated gene silencing in Rosellinia necatrix

Rosellinia necatrix causes white root rot in a wide range of fruit trees and persists for extended periods as pseudosclerotia on root debris. However, the pathogenesis of this disease has yet to be clarified. The functions of endogeneous target genes have not been determined because of the inefficiency in genetic transformation. In this study, the function of a melanin biosynthetic gene was determined to examine its role in morphology and virulence. A polyketide synthase gene (termed as RnPKS1) in the R. necatrix genome is homologous to the 1,8-dihydroxynaphthalene (DHN) melanin biosynthetic gene of Colletotrichum lagenarium. Melanin-deficient strains of R. necatrix were obtained by RNA interference-mediated knockdown of RnPKS1. The virulence of these strains was not significantly reduced compared with the parental melanin-producing strain. However, knockdown strains failed to develop pseudosclerotia and were degraded sooner in soil than the parental strain. Microscopic observations of albino conidiomata produced by knockdown strains revealed that melanization is involved in synnema integrity. These results suggest that melanin is not necessary for R. necatrix pathogenesis but is involved in survival through morphogenesis. This is the first report on the functional analysis of an endogenous target gene in R. necatrix.     

Replication and infectivity of the single-embedded nuclear polyhedrosis virus, Baculovirus heliothis, in homologous cell lines

Three cell lines of Heliothis zea and one cell line of Heliothis virescens replicated the singleembedded, nuclear polyhedrosis virus (NPV) of H. Zea, (i.e., Baculovirus heliothis) with concomitant production of polyhedral inclusion bodies (PIB). Between 20 and 60% of the H. zea cells produced PIB, whereas only 3% of H. virescens cells were found to produce PIB. The H. zea cell lines produced 10 to 20 times more PIB than did the H. virescens cell line. The PIB from all cell lines produced typical symptoms of an NPV infection when bioassayed against larvae of H. zea. More than 99% of the total viral activity of the final whole culture was due to the PIB.     

Does Bacillus thuringiensis have adverse effects on the host egg location by parasitoid wasps?

This study investigated the interaction between two pest biological control agents, the parasitoid wasp Trichogramma pretiosum (Hymenoptera: Trichogrammatidae) and the entomopathogen Bacillus thuringiensis (Bacillales: Bacillacea) (Bt). The aim of this study was to evaluate if the presence of Bt (formulated products Agree^®, Dipel^® and HD1 and HD11 strains) interferes in the oviposition preference of T. pretiosum to eggs of Helicoverpa zea (Lepidoptera: Noctuidae). Using an olfactometry test, the eggs of H. zea were bathed with the commercial formulations, with the Bt suspensions or distilled water, and offered to the parasitoid wasps in order to evaluate parasitism. The results showed that H. zea eggs sprayed with commercial formulations and Bt strains did not interfere in the choice made by the parasitoid. The parasitoid wasp is not able to distinguish between eggs with or without B. thuringiensis treatment, independently of strains suspension or commercial formulations. Therefore, these two control agents may be used together without negative interaction.     

Interaction between the entomopathogenic bacterium Bacillus thuringiensis subsp. kurstaki and two entomopathogenic fungi in bio-control of Sesamia nonagrioides (Lefebvre) (Lepidoptera: Noctuidae)

The interactions between the entomopathogenic bacterium Bacillus thuringiensis ssp. kurstaki and two entomopathogenic fungi Beauveria bassiana Balsamo (Vuillemin) (Hypocreales: Cordycipitaceae) and Metarhizium robertsii (Metchnikoff) Sorokin (Hypocreales: Clavicipitaceae) were examined on larvae of Sesamia nonagrioides (Lefebvre) (Lepidoptera: Noctuidae) in 8, 13 and 16 days post-treatment intervals. An overall positive interaction between the pathogens was observed and the larval mortality at 16 days was 56–100 % exposed to M. robertsii combined with B. thuringiensis subsp. kurstaki, whereas B. bassiana combined with B. thuringiensis ssp. kurstaki killed 54–100 % of exposed larvae. After 8 days, in 6 of the combinations, we found an additive relationship between the pathogens, whereas, a negative interaction was observed in 10 of them. In contrast, after 13 days, in 2 of the combinations the positive interaction could be considered as synergistic between pathogens, in 10 as additive, and in only 4 as negative. Finally, after 16 days, in 11 of the combinations we found an additive connection between the pathogens, wheras a negative interaction was seen in 5. Applying both pathogens simultaneously offers a method of Sesamia nonagrioides control that could be more effective than using each pathogen separately.     

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.     

Infection of the corn earworm, Heliothis zea, with Nosema acridophagus and Nosema cuneatum from grasshoppers: Relative virulence and production of spores

Per os inoculations of 4- to 6-day-old larvae of the corn earworm, Heliothis zea, with suspensions containing 10⁶ spores of Nosema acridophagus or 10⁴, 10⁵, and 10⁶ spores of Nosema cuneatum retarded the growth and development of the larvae. Migratory grasshoppers, Melanoplus sanguinipes, inoculated with N. acridophagus produced fewer spores than similarly inoculated corn earworms, but spore production was similar in these insects when they were inoculated with N. cuneatum. Standard bioassay procedures showed that spores of both microsporidians were some-what more virulent when they were produced in corn earworms than when they were produced in grasshoppers. Spores of these microsporidians might be produced more efficiently in corn earworm larvae than in grasshoppers.     

Selection for resistance to a nucleopolyhedrosis virus in laboratory populations of the cotton bollworm, Heliothis zea

Resistance to a nucleopolyhedrosis virus (Baculovirus heliothis) did not develop in laboratory populations of the cotton bollworm, Heliothis zea. A selection pressure of LD₅₀ to 70 was maintained throughout 20 to 25 generations of selection. No significant changes in LD₅₀, slope, or intercept of dose-mortality lines were detected. Laboratory populations under selection were as susceptible to the virus as nonselected or wild populations of H. zea. The resistance ratio (LD₅₀ of selected generation/initial generation) ranged from 0.5 to 1.2.     

Acanthamoeba polyphaga is a possible host for Vibrio cholerae in aquatic environments

Acanthamoeba is a genus of free-living amoebae found to be able to host many bacterial species living in the environment. Acanthamoebae and Vibrio cholerae are found in the aquatic environments of cholera endemic areas. Previously it has been shown that V. cholerae O1 and O139 can survive and grow in Acanthamoeba castellanii. The aim of this study was to examine the ability of Acanthamoeba polyphaga to host V. cholerae O1 and O139. The interaction between A. polyphaga and V. cholerae strains was studied by means of viable amoeba cell counts and viable count of the bacteria in the absence and presence of amoebae. The viable count of intracellularly growing bacteria was estimated by utilizing gentamicin assay. Electron microscopy was used to determine the localization of V. cholerae inside A. polyphaga. The results showed that A. polyphaga enhanced growth and survival of V. cholerae, which grew and survived inside the amoeba cells for 2 weeks. The electron microscopy showed that A. polyphaga hosted intracellular V. cholerae localized in the vacuoles of amoeba cell. Neither the presence of V. cholerae together with A. polyphaga nor the intracellular localization of the bacteria inhibited growth and survival of A. polyphaga. The outcome of the interaction between these microorganisms may support strongly the role of A. polyphaga as host for V. cholerae O1 and O139.     

Validating the in vitro antimicrobial activity of Artemisia afra in polyherbal combinations to treat respiratory infections

Artemisia afra is one of the most widely used medicinal plants in African traditional medicine and is commonly administered in polyherbal combinations to treat respiratory infections. Focussing on plant volatiles, the aim of this study was to provide scientific evidence for the antimicrobial activity of A. afra (principle plant) in combination with essential oils from three medicinal aromatic plants; Agathosma betulina, Eucalyptus globulus and Osmitopsis asteriscoides. In vitro minimum inhibitory concentration (MIC) assays were undertaken on four pathogens (Enterococcus faecalis ATCC 29212, Moraxella catarrhalis ATCC 23246, Klebsiella pneumoniae NCTC 9633 and Cryptococcus neoformans ATCC 90112) to determine antimicrobial efficacy of the oils and their combinations. The fractional inhibitory concentration (FIC) and isobolograms were used to interpret pharmacodynamic interactions such as synergy, antagonism or additive profiles. The antimicrobial activity of the individual oils mostly displayed moderate activity. Predominantly, additive interactions were noted. The most prominent synergistic interaction (FIC value of 0.5) was observed when A. afra was combined with O. asteriscoides in the 8:2 ratio (eight parts A. afra with two parts O. asteriscoides) against C. neoformans. No antagonistic interactions were evident.     

Role of hydrogen peroxide and antioxidant enzymes in the interaction between a hemibiotrophic fungal pathogen, Leptosphaeria maculans, and oilseed rape

Reactive oxygen species play a dual role in host-pathogen interaction. They impede the spread of biotrophic pathogens via stimulating cell death and hypersensitive response (HR), and, on the other hand, they provide access to nutrients for necrotrophic pathogens feeding on dead tissues and facilitate their colonizing the host. The participation of ROS in defending plants from pathogens with a combined lifestyle (hemibiotrophs) is not yet understood, and it varies in its dependence on the particular host-pathogen combination. In the present study, we inoculated rapeseed plants (Brassica napus) with a hemibiotrophic fungus, Leptosphaeria maculans, and manipulated the H₂O₂ content in cotyledons by infiltrating catalase and/or H₂O₂ into tissues. The action of catalase resulted in a significant decrease in lesions development, but when H₂O₂ was applied instead, lesion formation was only moderately stimulated compared to the untreated control. When H₂O₂ toxicity to L. maculans was tested in vitro, concentrations above 5 mM and 10 mM H₂O₂ were lethal for germinating conidia and growing mycelia of L. maculans, respectively. We can assume that L. maculans behaves as a necrotroph during this early stage of infection even though its resistance to H₂O₂ does not exceed standard concentrations. To investigate antioxidant mechanisms implicated in the response of B. napus to L. maculans, the cotyledons were both inoculated with conidia and treated with L. maculans elicitor. Increased activities of guaiacol peroxidase, ascorbate peroxidase, glutathione reductase and superoxide dismutase were recorded both in L. maculans-infected and elicitor-treated cotyledons. The results indicate the importance of these enzymes for ROS scavenging in B. napus-L. maculans interaction.     

The preservation of infective spores of Nosema necatrix (Protozoa: Microsporida) in Spodoptera exempta (Lepidoptera: Noctuidae) by lyophilization

Cadavers of Spodoptera exempta infected with Nosema necatrix were lyophilized. The lyophilized material was tested against a standard spore preparation 1 day after lyophilization and retested 2 yr later. There was some loss of spore viability. The feasibility of using dose-mortality experiments to estimate the loss in viability is discussed.     

Mapping QTL for Biomass Yield and Its Components in Rice (Oryza sativa L.)

Addicive effects, additive by additive epistatic effects, and their environmental interactions of QTLs are important genetic components of quantitative traits. Genetic architecture underlying rice biomass yield and its two component traits (straw yield and grain yield) were analyzed for a population of 125 DH lines from an inter-subspecific cross of IR64/Azucena. The mixed-model based composite interval mapping approach (MCIM) was used to detect QTLs, There were 12 QTLs detected with additive main effects, 27 QTLs involved in digenic interaction with aa and/or aae effects, and 18 QTLs affected by environments with ae and/or aae effects. It was revealed that epistatic effects and QE interaction effects existed on biomass yield and its component traits in rice. In addition, the genetic basis of relationships among these traits were investigated. Four QTLs and one pair of epistatic QTLs were detected to be responsible for the positive correlation between biomass yield and straw yield. Three QTLs might be responsible for the negative correlation between straw yield and grain yield. This result could partially explain the genetic basis of correlation among the three traits, and provide useful information for genetic improvement of these traits by marker-assisted selection.