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The amount of biopesticides currently used in pest control is still below 1% of the global pesticide market, with environmentally damaging products constituting the majority of all commercial insecticides. There is thus an increased need for biopesticides, including those from invertebrate venoms, which are often highly specific. One example of this is to use a fusion protein approach where a peptide-toxin is fused to a carrier protein, in this instance GNA, which has the capability of crossing the insect gut epithelium allowing inhibitory molecules of the neuromuscular system to be delivered to these remote sites of action via oral ingestion. In this study, five variants of spider Segestria florentina toxin (SFI) fused to snowdrop lectin (Galanthus nivalis agglutinin; GNA) were successfully expressed in Pichia pastoris X33 and subsequently purified. To improve the level of expression of the intact recombinant protein SFI1/GNA, an expression vector construct containing two gene copies was assembled. Insecticidal activities of all these novel fusion proteins were demonstrated by oral feeding to Myzus persicae. SFI1, SFI3, SFI5, SFI6, and SFI8 GNA-based fusion proteins (0.1 mg/ml), which target voltage-gated ion channels in the insect CNS, caused significant mortality to M. persicae compared to GNA alone. LC50 values for the variants 2XSFI1/GNA, SFI5/GNA and SFI8/GNA were 0.006 mg/ml, 0.038 mg/ml, and 0.08 mg/ml respectively. The GNA-based fusion proteins expressing -conotoxin E1 from cone snails, which target nicotinic acetylcholine receptors, was also successfully expressed in P. pastoris; in these, GNA was at the N-terminus and the toxin at the C-terminus. The LC 50 values for the GNA/-conotoxin E1 was 8 μg/μl. The results demonstrate that these candidate molecules show promise for future development as bio-pesticides.

The effects of virus disease upon certain aspects of the nitrogen metabolism of the cultivated mushroom Agaricus bisporus (Lange) Sing, were of host metabolism. The significance of these observations is discussed. Relationship shown by diseased cultures of vegetative mycelium on agar were studied in particular. Disturbances of sporophore metabolism which could be used for diagnostic techniques were also sought. Healthy myceilal cultures and two diseased cultures of different growth rates were isolated from sporophores and used as inoculums for submerged culture. Growth of the various isolates in submerged culture was studied in detail. Rates of protein synthesis and of uptake of the amino acids glycine and proline were measured by the use of radioactive isotopes; qualitative aspects of protein metabolism were studied by polyacrylamide gel electrophoresis. Glycine uptake by healthy mycelium was found to vary with the age of the mycelium, to be affected by pH and metabolic poisons and to show saturation kinetics. Diseased cells showed reduced rates of protein responsible for the observed reductions in uptake is presented. Protein patterns and isoenzymes of diseased mycelium were different to those of healthy. A special feature established by this study was that cultures of intermediate growth rate also showed intermediate degrees of disturbance of host metabolism. The significance of these observations is discussed. Diseased mycelium was rendered non-infectious by heat treatment; such treated cultures resembled normal healthy mycelium in every respect. Isoenzyme patterns of healthy sporophores were studied by disc electrophoresis and found to be very reproducible; diseased tissue gave different enzymes. The use of disc electrophoresis as a diagnostic tool for mushroom virus disease is indicated. An increase in O-diphenoloxidase activity in extracts from diseased mushrooms was also found.

The pathogenicity of two strains of the fungus Rhizoctonia solani Kuhn, and a strain of a related fungus,Corticinm praticola Kotila, to throe hosts, was studied, to determine which factors affect the specificity of parasitism. One R.solani strain parasitized lettuce, the other, cabbage, and the third fungus C.praticola parasitized both these hosts. Wheat was not severely parasitized by any of these fungi. Pathogenicity was unrelated to the stimulation of fungus growth by host exudates, which also had little effect on the formation of infection cushions, by which these fungi normally penetrate their hosts. The surface configuration of the host, and the contact stimulus were important in penetration, and attachment of the fungi. Adhesive structures were formed on non-living surfaces by each fungus, but not on damaged host surfaces. Each fungus penetrated only some tissues of each host. Pathogenicity was found to be unrelated to the inhibitory effect of host tissue on fungus growth, but was correlated to the speed of invasion and direction of advance of the hyphae inside the host tissues. A hypersensitive reaction took place, but this could not account for the specificity of parasitism. Pectolytic and cellulolytic enzymes were produced in culture by each fungus when grown on synthetic substrates. Enzyme activity was unrelated ti the growth of the fungi, and to pathogenicity. Extracts of diseased plants contained pectolytic and cellulolytic enzymes, but their activity was unrelated to that of culture filtrates from synthetic media, and also unrelated to pathogenicity. These extracts were toxic to young seedlings, but toxicity too, was unrelated to the specificity of parasitism. The toxic principle was thermolabile and may have been enzymic, and was able to macerate young seedling tissues.

Cacao swollen shoot virus (CSSV) causes the Cacao swollen shoot virus disease (CSSVD) and significantly reduces production in West African cacao. This study characterised the current status of the disease in the major cacao growing States in Nigeria and attempted a clarification on the manner of CSSV transmission. Two separate field surveys and sample collections were conducted in Nigeria in summer 2012 and spring 2013. PCR-based screening of cacao leaf samples and subsequent DNA sequencing showed that the disease continues to persist in Ondo and Oyo States and in new cacao sites in Abia, Akwa Ibom, Cross River and Edo States. Mealybug samples collected were identified using a robust approach involving environmental scanning electron microscopy, histology and DNA barcoding, which highlighted the importance of integrative taxonomy in the study. The results show that the genus Planococcus (Planococcus citri (Risso) and/or Planococcus minor (Maskell)) was the most abundant vector (73.5%) at the sites examined followed by Formicococcus njalensis (Laing) (19.0 %). In a laboratory study, the feeding behaviour of Pl. citri, Pseudococcus longispinus (Targioni-Tozzetti) and Pseudococcus viburni (Signoret) on cacao were investigated using electrical penetration graph (EPG) analysis. EPG waveforms reflecting intercellular stylet penetration (C), extracellular salivation (E1e), salivation in sieve elements (E1), phloem ingestion (E2), derailed stylet mechanics (F), xylem ingestion (G) and non-probing phase (Np) were analysed. Individual mealybugs exhibited marked variation within species and significantly differed (p ≤ .05) between species for E1e and E1. PCR-based assessments of the retention time for CSSV in viruliferous Pl. citri, Ps. longispinus and Ps. viburni fed on a non-cacao diet showed that CSSV was still detectable after 144 hours. These unusually long durations for a pathogen currently classified as a semi-persistent virus have implications for the design of non-malvaceous barrier crops currently being considered for the protection of new cacao plantings.