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Beauveria bassiana as a pathogen of the Mexican fruit fly (Diptera: Tephritidae) under laboratory conditions.

Bioassays were carried out under controlled conditions (27 +/- 2 degrees C, 80 +/- 5% RH, and a photoperiod of 12:12 [L:D] h) to evaluate the effect of eight strains of the entomopathogenic fungus Beauveria bassiana upon larvae, pupae, and adult females of the Mexican fruit fly, Anastrepha ludens (Loew). Mortality of the immature stages was low, 2-8% in larvae and 0% in pupae. However, very high levels of mortality were obtained for adult flies, with values of 100, 98, and 98% for the strains Bb16, Bb24, and Bb26, respectively. LC50 values for these three strains ranged from 3.12 x 10(6) to 9.07 x 10(6) conidia/ml. Lethal time 50 (LT50) was 2.8, 3.7, and 4.2 d for Bb16, Bb26, and Bb24 strains, respectively, with an average LT50 of 4.4 d across all strains. The fungal mycelium emerged through the soft parts of the exoskeleton, such as the wing bases, mouth, intersegmental regions of the legs, and membranous regions of the abdomen, coxae, and neck. Maximum percentage sporulation ranged from 66.4 to 74.7% for the three most virulent strains.

Animals↗

Control of resistant pink bollworm (Pectinophora gossypiella) by transgenic cotton that produces Bacillus thuringiensis toxin Cry2Ab.

Crops genetically engineered to produce Bacillus thuringiensis toxins for insect control can reduce use of conventional insecticides, but insect resistance could limit the success of this technology. The first generation of transgenic cotton with B. thuringiensis produces a single toxin, Cry1Ac, that is highly effective against susceptible larvae of pink bollworm (Pectinophora gossypiella), a major cotton pest. To counter potential problems with resistance, second-generation transgenic cotton that produces B. thuringiensis toxin Cry2Ab alone or in combination with Cry1Ac has been developed. In greenhouse bioassays, a pink bollworm strain selected in the laboratory for resistance to Cry1Ac survived equally well on transgenic cotton with Cry1Ac and on cotton without Cry1Ac. In contrast, Cry1Ac-resistant pink bollworm had little or no survival on second-generation transgenic cotton with Cry2Ab alone or with Cry1Ac plus Cry2Ab. Artificial diet bioassays showed that resistance to Cry1Ac did not confer strong cross-resistance to Cry2Aa. Strains with >90% larval survival on diet with 10 microg of Cry1Ac per ml showed 0% survival on diet with 3.2 or 10 microg of Cry2Aa per ml. However, the average survival of larvae fed a diet with 1 microg of Cry2Aa per ml was higher for Cry1Ac-resistant strains (2 to 10%) than for susceptible strains (0%). If plants with Cry1Ac plus Cry2Ab are deployed while genes that confer resistance to each of these toxins are rare, and if the inheritance of resistance to both toxins is recessive, the efficacy of transgenic cotton might be greatly extended.

Animal Feed↗

Transgenic Indica rice breeding line IR58 expressing a synthetic cryIA(b) gene from Bacillus thuringiensis provides effective insect pest control.

The Indica rice breeding line IR58 was transformed by particle bombardment with a truncated version of a synthetic cryIA(b) gene from Bacillus thuringiensis. This gene is expressed under control of the CaMV 35S promoter and allows efficient production of the lepidopteran specific delta-endotoxin. R0, R1 and R2 generation plants displayed a significant insecticidal effect on several lepidopterous insect pests. Feeding studies showed mortality rates of up to 100% for two of the most destructive insect pests of rice in Asia, the yellow stem borer (Scirpophaga incertulas) and the striped stem borer (Chilo suppressalis), and feeding inhibition of the two leaffolder species Cnaphalocrocis medinalis and Marasmia patnalis. Introduction of stem borer resistance into the germplasm of an Indica rice breeding line now makes this agronomically important trait available for conventional rice breeding programs.

Animals↗

Enhancing biological control of basal stem rot disease (Ganoderma boninense) in oil palm plantations.

Basal Stem Rot (BSR) disease caused by Ganoderma boninense is the most destructive disease in oil palm, especially in Indonesia and Malaysia. The available control measures for BSR disease such as cultural practices and mechanical and chemical treatment have not proved satisfactory due to the fact that Ganoderma has various resting stages such as melanised mycelium, basidiospores and pseudosclerotia. Alternative control measures to overcome the Ganoderma problem are focused on the use of biological control agents and planting resistant material. Present studies conducted at Indonesian Oil Palm Research Institute (IOPRI) are focused on enhancing the use of biological control agents for Ganoderma. These activities include screening biological agents from the oil palm rhizosphere in order to evaluate their effectiveness as biological agents in glasshouse and field trials, testing their antagonistic activities in large scale experiments and eradicating potential disease inoculum with biological agents. Several promising biological agents have been isolated, mainly Trichoderma harzianum, T. viride, Gliocladium viride, Pseudomonas fluorescens, and Bacillus sp. A glasshouse and field trial for Ganoderma control indicated that treatment with T. harzianum and G. viride was superior to Bacillus sp. A large scale trial showed that the disease incidence was lower in a field treated with biological agents than in untreated fields. In a short term programme, research activities at IOPRI are currently focusing on selecting fungi that can completely degrade plant material in order to eradicate inoculum. Digging holes around the palm bole and adding empty fruit bunches have been investigated as ways to stimulate biological agents.

Arecaceae↗

In vitro entomopathogenic activity of Beauveria bassiana against Psoroptes spp. (Acari: Psoroptidae).

An indigenous strain (IHEM 18747) of Beauveria bassiana (Balsamo) Vuillemin (Deuteromycetes) was evaluated for its in vitro entomopathogenic activity against the parasitic mite Psoroptes ovis Hering (Acari: Psoroptidae) from rabbits. The following aspects were evaluated: (1) effects of conidial concentration on the viability of adult females; (2) influence of the infection on the fertility, and on the hatchability of eggs; (3) and transmission of infection between mites, and from contaminated surface. Adult females immersed into increasing concentrations of conidia (10(4)-10(9)conidia ml(-1)) showed a dose-related susceptibility. At the highest concentration of conidia, LT50 was 1.6 days while LT50 of the controls reached 5.8 days. The fungus was able to sporulate on the body surface and 100% of the mites were covered with mycelium after immersion in solutions containing 10(7)-10(9)conidia ml(-1). One hundred percent of healthy mites exposed to infected cadavers or surfaces acquired the infection (LT50 reached 1.9 and 1.73 days, respectively, versus 6.1 and 5.1 days in controls, respectively). Egg laying was not reduced by the fungal infection but both the hatchability of the eggs and the life span of the emerging larvae were significantly reduced. Eggs directly infected with the fungus did not show reduced hatchability but the life span of the larvae was shortened. It is concluded that B. bassiana has a high entomopathogenic activity against Psoroptes spp. The in vivo use of this biocontrol agent against Psoroptes spp. in rabbit, sheep and cattle deserves further attention.

Animals↗

Paecilomyces fumosoroseus (Deuteromycotina: Hyphomycetes) as a potential mycoparasite on Sphaerotheca fuliginea (Ascomycotina: Erysiphales).

Hyphomycete Paecilomyces fumosoroseus that is well known as saprophytic and entomopatogenic fungus was investigated for its mycoparasitism on the cucumber powdery mildew pathogen. Mycoparasitism was documented by using standard bioassay and SEM. Effects of mycoparasitism were evaluated in three types of experiments. Paecilomyces fumosoroseus was applied in the form of graded suspensions into a colony of powdery mildew on a leaf segment. Interaction between both fungi was observed as the percentage of colonized area vs. experimental time. In the second experiment, young cucumber plants were sprayed with a suspension of Paecilomyces fumosoroseus 24 h before inoculation of Sphaerotheca fuliginea. Pre-treatment with P. fumosoroseus reduced development and spreading of powdery mildew infection significantly 15 days post-inoculation in contrast to pre-treatments with sulfur fungicide and distilled water. The development of pure culture powdery mildew under determined experimental conditions was observed and compared with treated variants. In the third experiment, mildewed plants were treated with a suspension of P. fumosoroseus. The control treatments with sulfur fungicide and distilled water were tested. Effects of P. fumosoroseus on the dispersion of powdery mildew during a 21-day period were observed. P. fumosoroseus suppressed the development and spread of cucumber powdery mildew significantly during the time of the experiment. The mechanical and physical damages and disruptions of vegetative and fruiting structures of powdery mildew were recorded under light microscopy and S.E.M. Results were concluded in pursuance to differences between the natural behaviour and development of S. fuliginea on cucumber plants treated with P. fumosoroseus and non-treated plants.

Ascomycota↗

Virus-derived genes for insect-resistant transgenic plants.

Insect viruses have evolved to counter physiological barriers to infection presented by the host insect. For the Lepidoptera (butterflies and moths), these barriers include (1) the peritrophic membrane (PM) lining the gut, which presents a physical barrier to virus infection of the midgut epithelial cells, (2) the basement membrane (BM) that overlies the gut thereby restricting secondary infection of other tissues, and (3) the immune system of the host insect. Hence, insect viruses provide a resource for genes that disrupt host physiology in a specific manner, and these genes in turn serve as a resource both for the study of physiological processes, and for disruption of these processes for pest management purposes. There are several examples of the application of genes used by an insect virus to overcome the PM barrier for production of insect-resistant transgenic plants. There are other examples of intrahemocoelic effectors, such as BM-degrading proteases that can only be used with an appropriate system for delivery of the agent from the gut into the hemocoel (body cavity) of the insect pest. In this chapter, we describe (1) baculovirus- and entomopoxvirus-derived genes that alter the physiology of the host insect, (2) use of these and homologous genes for production of insect-resistant transgenic plants, (3) other viral genes that have potential for use in development of insect-resistant transgenic plants, and (4) the use of plant lectins for delivery of intrahemocoelic toxins from transgenic plants. Plant expression of polydnavirus-derived genes is described by Gill et al. (this volume, pp. 393-426).

Animals↗

Proline-based modulation of 2,4-diacetylphloroglucinol and viable cell yields in cultures of Pseudomonas fluorescens wild-type and over-producing strains.

The antifungal compound 2,4-diacetylphloroglucinol (DAPG) is produced in the rhizosphere of wheat by pseudomonad populations responsible for the natural biological control phenomenon known as "take-all decline." Studies were conducted to elucidate the impact of DAPG and its co-product 2,4,6-trihydroxyacetophenone (THA) on the production of Pseudomonas fluorescens for biological control. Increasing DAPG from 0.1 g/l to 0.5 g/l and THA from 0.05 g/l to 0.5 g/l significantly inhibited the growth and lowered the yield of viable bacteria in liquid cultures. On further examination of these metabolites applied in seed coatings, levels of DAPG and THA exceeding 0.05 mg/g seed significantly reduced wheat germination percentages. The three-way interaction of DAPG, THA, and culture medium ingredients was significant, and greatest seed germination loss (40-50%) was observed when 0.5 mg DAPG and 0.25 mg THA were combined in a coating of 0.5 ml culture medium per gram of seed. Based on the results of Biolog GN microplate, flask, and fermentor screens of C sources, proline was found to optimize the viable cell yields of the P. fluorescens strains tested. The combination of proline with glucose and urea as C and N sources in growth media could be optimized to minimize DAPG production and maximize the vitality of P. fluorescens Q8R1-96 and Q69c-80:miniTn5:phl20 (DAPG over-producer). In production cultures, the proline supply rate offers a potentially useful means to optimize the biological control agent yield and quality.

Antifungal Agents↗

Bacillus thuringiensis toxin (Cry1Ab) has no direct effect on larvae of the green lacewing Chrysoperla carnea (Stephens) (Neuroptera: Chrysopidae).

Earlier studies have shown that larvae of the green lacewing predator Chrysoperla carnea are negatively affected when preying on lepidopteran larvae that had been fed with transgenic maize expressing the cry1Ab gene from Bacillus thuringiensis. To test whether the observed effects were directly caused by the Cry1Ab toxin, we have developed a bioassay which allows us to feed high concentrations of the toxin directly to the predator. The results of these feeding studies show no direct toxic effect of Cry1Ab on C. carnea larvae. The amount of toxin ingested by first instar C. carnea in the present study was found to be a factor 10,000 higher than the concentration ingested when feeding on Bt-reared lepidopteran larvae, a treatment that was previously shown to have a negative impact on the predator. In addition, feeding first instar C. carnea with the Cry1Ab toxin did not affect the utilisation of subsequently provided prey. Furthermore, the quality of the prey provided to first instars did not affect the sensitivity of second and third instar C. carnea to the Bt-toxin. The presented results strongly suggest that C. carnea larvae are not sensitive to Cry1Ab and that earlier reported negative effects of Bt-maize were prey-quality mediated rather than direct toxic effects. These results, together with the fact that lepidopteran larvae are not regarded as an important prey for C. carnea in the field, led us to conclude that transgenic maize expressing Cry1Ab poses a negligible risk for this predator.

Administration, Oral↗

The prospects for biological control of the free-living stages of nematode parasites of livestock.

Control of nematode parasites of livestock is focused almost entirely on the parasitic stages within the host. Current methods rely on anthelmintic drugs, but these are under increasing threat with the development of resistance covering the whole spectrum of anthelmintics amongst the important nematode species of a range of livestock. However, invariably the greatest proportion of the parasite biomass resides not within the animal hosts, but in the external environment-commonly on pasture. It is in this environment that the free-living stages are vulnerable to a range of abiotic factors (extremes in temperature and desiccation) and biotic factors (macro- and micro-organisms) that may decimate their numbers. Of the latter, there are organisms, which exert their effects either indirectly by rendering faecal deposits inimical for the development of nematode eggs through to infective larvae, or directly by acting as pathogens or by exploiting the free-living stages as a food source. Within this vast assemblage of organisms, which include microarthropods, protozoa, viruses, bacteria and fungi, could well emerge a variety of biological control agents of nematode parasites. At present, greatest interest lies with the nematode-destroying fungi. Work has progressed from Petri dishes, to plots, to paddocks with several species of the genus Arthrobotrys and Duddingtonia flagrans. These studies indicate that the voracious nematophagous capabilities of these fungi, clearly demonstrated in vitro, translate to reductions in the number of infective larvae on pasture and indicate that levels of control, comparable to conventional schemes using anthelmintics, can be achieved. The challenge now lies in developing methods of administration of fungi to animals which can be applied under practical farm conditions. However, the pursuit of candidates for biological control of nematode parasites of livestock should not involve just a few species of nematophagous fungi. More than 100 species of fungi have been identified as possessing nematode destroying capabilities. These need to be more specifically investigated for their effects on free-living stages of nematode parasites of livestock, together with other classes of organisms, particularly bacteria, which have proved successful as biological control agents of arthropods.

Animals↗

Pathogenicity of mollicutes for insects: possible use in biological control.

Acholeplasmas, spiroplasmas and other non-helical sterol-requiring mycoplasmas of unknown phylogenetic affinity inhabit insects. Of these, only spiroplasmas are known to be pathogenic. Group I-2 spiroplasmas, or Spiroplasma apis, especially in combination with other organisms, reduce honey-bee longevity. Plant pathogenic mycoplasma-like organisms are often found intracellularly in insects. Spiroplasmas are found predominantly in the gut lumen or haemolymph (or both) of their insect hosts. Pathogenicity of mycoplasmas is usually altered by extended passage in unusual hosts, in only one of two alternate hosts, or in culture media. Enhancement of experimental pathogenicity may occur with extended cultural passages, but maintenance of natural pathogenicity must be accomplished by continuous exposure to the usual host. Recent data provide new information on the ecology of pathogenicity. Spiroplasmas from unique habitats also tend to be unique. Spiroplasmas isolated from flowers appear to be adapted to insect species that frequent floral surfaces. Group IV spiroplasmas have been isolated from members of 4 holometabolous insect orders (including Lepidoptera), all of which visit flowers. Social or predatory insects, or insects with an "aggregation" phase in their life histories, also appear to be prone to spiroplasma infection. Some insect species which harbor spiroplasmas also carry infections of other mollicutes, some of which involve the haemolymph. Appearance of spiroplasmas in adult insects in nature is strongly affected by seasonality. Extensive tests of the host ranges of the new insect mollicutes will be required before their suitability for biological control can be evaluated.

Acholeplasma↗

The mustard trypsin inhibitor 2 affects the fertility of Spodoptera littoralis larvae fed on transgenic plants.

The effects of mustard trypsin inhibitor MTI-2 expressed at different levels in transgenic tobacco lines have been evaluated by feeding the lepidopteran Spodoptera littoralis throughout its larval life. Specific conditions were selected to study the long-term effects of feeding larvae on transgenic plants expressing the inhibitor at various levels. The data obtained led to the establishment of three relevant parameters to be considered during the experimentation: (i) the PI content of the plant lines to be used; (ii) the developmental stage of larvae sensitive to that PI content; (iii) the ratio of MTI-2/proteases sufficient to inhibit gut proteases. The experimental data obtained from feeding S. littoralis larvae using these conditions led to two main results. First, when L2 S. littoralis larvae were fed on high MTI-2 expressing tobacco plants, no effects on larval development were detected but there was a significantly reduced fertility. When the same larvae were fed on low expressing MTI-2 tobacco plants, only a less marked lowering of fertility was observed. Second, after the first generation, no differences in protease activity were observed in insects derived from larvae fed on high or low MTI-2 expressing tobacco lines, suggesting that genetic traits observed in previous studies were not inherited.

Animals↗