Barley net blotch (BNB) caused by Pyrenophora teres, and Septoria tritici blotch (STB) caused by Mycosphaerella graminicola, are destructive cereal diseases worldwide on barley and wheat respectively. Due to the lack of highly resistant cultivars, both diseases are widely controlled using fungicides. Systemic, site-specific modern fungicides have played an essential role in disease management in cereals. Triazole-based fungicides, which inhibit the C14 demethylation step in fungal ergosterol biosynthesis, known as demethylation inhibitors (DMIs) and strobilurins, known as quinine outside inhibitors (QoIs), which interfere with energy production in the fungal cell, by blocking electron transfer at site of quinone oxidation in the cytochrome bc1 complex, are two major site-specific systemic groups of fungicides, currently used to control cereal diseases. Multiple, consecutive and extensive use of these fungicides has led to the emergence of fungicide resistance in these plant pathogens. The existence of G143A and F129L mutations has been found to be associated with resistance of many plant pathogens to QoIs. However, in P. teres only F129L was found to confer insensitivity. The presence of an intron in several fungi (including rusts and P. teres) determines that it is impossible for the G143A mutation to survive and thus be selected for. Alterations in CYP51 gene in plant pathogens has also been found to be one of the major mechanisms resulting in reduced sensitivity towards DMIs. The aim of this research was to investigate the impact of the F129L mutation in isolates of P. teres, and mutations in the CYP51 gene in M. graminicola isolates on the activity of QoI and DMI fungicides respectively. Results revealed a high frequency of the F129L mutation within recent UK P. teres isolates. Furthermore, the common change (G143A) in cytochrome b was not found in P. teres strains. The results also showed a lack of any fitness penalty associated with the mutation. Bioassay tests indicated that inhibition of net blotch by QoIs was variable. Single QoI fungicides such as pyraclostrobin and picoxystrobin were found to be highly inhibitory whilst the efficacy of other QoIs was less pronounced. It has been found that efficacy of QoI fungicides varied amongst a population of isolates with the F129L mutation. This might suggest that some QoIs were compromised by the F129L mutation to some degree. However, the results obtained were in agreement with previous reports that the F129L mutation in the cytochrome b gene generates lower levels of resistance and was not as serious as that posed by the G143A mutation in other plant pathogens. In addition, fungicide mixtures, comprising QoIs and DMIs or the novel SDHI formulations, were found to have great efficacy in net blotch disease management. Sequence results of CYP51 gene fragment indicated existence of 15 alterations in recent UK and German isolates of M. graminicola. Some of these mutations, such as Y137F, were found to be rare whilst the I381V mutation was found to be increasing with time. However, investigations indicated a lack of phenotypic fitness penalties associated with these alterations. Apical germ tube growth measurement was found an effective method to assess in vitro activity of DMI fungicides against M. graminicola isolates. Based on bioassay studies, six categories within M. graminicola isolates were detected, showing different sensitivities to azole fungicides. In general, genotypes characterised S, R3+ and R4 were sensitive to most azole fungicides. The R3+ variant, however, showed less sensitivity to tebuconazole and prochloraz. In in vitro studies, the R5 variants, exhibited sensitivity to many DMIs but were less sensitive to prochloraz. This supporting the results obtained from in planta assays, where this genotype was found to be sensitive to tebuconazole but less sensitive to prochloraz. On the other hand, genotypes characterised R6a, R7 and R8, containing I381V mutation, were resistant to tebuconazole but sensitive to prochloraz. The latter variant, however, were more sensitive to prochloraz. It can be suggested from results obtained in this study that CYP51 alterations were differentially selected by different members of the azole class of fungicides. Q-PCR was also used to evaluate in planta fungicide activity on both diseases. The method indicated similar pattern to that observed in visual assessments. Detection of medium to high correlation values between both assessments confirmed the validity of q-PCR assessment. This suggests that q-PCR assays may serve as an alternative method for accurate assessment of the fungicide effects on cereal diseases. The method can be a valuable tool to evaluate disease occurrence in pathogens with a long latent period, such as M. graminicola, as q-PCR could readily detect the pathogen during the asymptomatic latent period.

The mirids, Sahlbergella singularis Hagl and Distantiella theobroma (Dist) (Heteroptera: Miridae), are major insect pests of cocoa, a valuable crop in West Africa. Their control by the application of insecticides is problematic in terms of safety and cost. Therefore, this study was undertaken to determine the potential for use of mirid sex pheromone trapping as an alternative, environmentally-acceptable method of managing the mirids. Based on the behavioural responses of the mirids to pheromones in traps, parameters were standardised for efficient performance of the traps. A range of five blends of the synthetic pheromone, the diester, hexyl (R)-3- ((E)-2-butenoyl)-butyrate and the monoester, hexyl (R)-3-hydroxybutyrate, impregnated in polyethylene vials were assayed with a blank control. Blends of 1000:500 μg and 1000:1000 μg respectively attracted significantly higher numbers of male S. singularis than other blends and the lure attracted male mirids optimally for four weeks with minimal reduction in eight weeks. Field bioassays were conducted to determine the appropriate trap design for pheromone trapping from four models; 2.5 litre and 4.5 litre plastic water bottles, sticky plastic plates, cylinder and standard rectangular traps. All models were equally effective. A field experiment was conducted with sticky glue on the outside of the traps. Combined inside and outside surfaces caught more mirids than the inside surface alone which caught only about 23% of the male mirids. Three field experiments using two different experimental designs were conducted to determine optimal height for trap placement. Traps placed inside the canopy attracted significantly more mirids than below 2.7 m height from the ground. The potential for mass trapping of mirids as a method of control was studied through three mass trapping experiments on research plantations and smallholder farmers’ farms. Catches of male S. singularis in pheromone traps were significantly reduced in mass-trapped fields but pheromone trapping did not control mirid numbers or affect damage on cocoa. Densities of 150 and 230 traps/ha were found to be optimal for trapping S. sahlbergella and D. theobroma respectively. Catches of male S. singularis in pheromone traps, however, predicted the magnitude of total mirid populations, and also shoot and pod damage in cocoa farms, albeit inconsistently.

The aim was to develop a novel method to reduce infestations of the most common stored-product pest, Tribolium castaneum beetles, in bags of sorghum stored by small-scale farmers of Kebbi state. A survey of 240 farmers found greater quantities of sorghum than other grains (4,000 kilos/household, p<0.001) were stored and a majority in south Kebbi stored sorghum threshed (p<0.001), even though this form is more vulnerable to infestation. Inconsistencies in farmers’ perceptions of the efficacy of repellent plants were apparent. A more efficient and effective bioassay (Thigmotactic assay) was developed to identify a plant product highly repellent to T. castaneum, ‘Lem-ocimum,’ which is composed of Cymbopogon nardus (Lemongrass) plus Ocimum basilicum (Sweet basil), 0.5%w/w each (p<0.001). A paste of Lem-ocimum was applied between layers of 5kg double-bags to prevent contamination of grain within inner bag. Treated double-bags provided better protection from T. castaneum infestation than untreated single or double-bags (p<0.001) and were most effective when a high number (9-18) were placed on top of untreated bags (~1% weight loss after 5 months, p<0.01, n=150 store-rooms of 42 farmers). A survey indicated participants were satisfied with outcome of Lemocimum treatment for trials using high numbers of treated bags. Male and female farmers differed in plant species they collected and their plant-drying methods. Chemical analysis showed plant species and drying methods affected repellency; cultivated O. basilicum had higher repellent compound content and repelled more beetles (0.88±0.015) than O. africanum (0.62±0.020, p<0.001), and shade-drying repelled more beetles (0.76±0.039) than sun-drying (0.61±0.034, p<0.001). Therefore, it is recommended that double-bags treated with cultivated shade-dried Ocimum (as normally prepared by women) should be tested further in the field. Application of the Lem-ocimum treated double bags method should ensure farmers have a proportion of high quality grain to sell to the market, thereby increasing their financial status.

No description available.

Seed extracts from 18 varieties of legumes were tested for virus inhibitory activity against TMV. Unheated seed extracts fall into two categories. Those extracts which give 73-95% inhibition and includes G. max (soybean), and extracts in which inhibition is between 0-60%, for example P. vulgaris (French bean). Inhibition was decreased by heating some extracts such as G. max, whilst in other extracts, such as P. vulgaris, the percentage inhibition was increases by heating. P. vulgaris and G. max seed extracts were studied in detail. P. vulgaris was also inhibitory to TMV, whilst G. max was inhibitory to TMV and PVX. Dilution experiments confirmed the presence of inhibitors and not inactivators in both extracts. None of the inhibitors was nucleic acid. However, dialysis precipitation with alcohol or ammonium sulphate and disc electro-phoresis experiments suggested that the inhibitors are composed of proteins and glycoproteins stable to a wide range of ph. Sephadex G-100 gel filtration showed that G. max seed extracts inhibitors have molecular weights of about 158,500 and 17,780. On the other hand, P. vulgaris inhibitors have molecular weights of about 177,800 and 12,590. Seven fractions were obtained from DEAE chromatography of each of G. max (soybean) and P. vulgaris (French bean) seed extracts. Soybean contains three virus inhibitor fractions, one basic in nature and two acidic. None of the virus inhibitors agglutinated erythrocytes) however, the acidic inhibitors showed inhibition activity. French bean contains also three virus inhibitors as well as confounds reducing the effects of the inhibitors. These are termed masking compounds. Such masking compounds were agglutinins. The basic inhibitor also showed agglutination of erythrocytes whilst only one of the two acidic virus inhibitors showed trypsin inhibition. Plant lectins were also tested for virus inhibition and agglutination activity, and it was found that the situation is complex and although soybean and French bean seed extracts seemed to have surface effect on the susceptibility of the host, the mode of action of lectins and the virus inhibitors are different. The virus inhibitors seem either to affect the attachment of the virus to the infective centres or perhaps allow attachment but prevent entry of virus into the cells.

Groundnut, Arachis hypogaea (L.), can be attacked by a range of pathogens, including Aspergillus species, which can cause accumulation of the mycotoxin aflatoxin. Although some success in controlling this pathogen has been achieved with application of fungicides, their use is not always feasible in developing nations like Nigeria. The aim of this study was, therefore, to evaluate naturally-occurring plant oils and BCAs with a past history of efficacy as alternatives to fungicides for reduction of Aspergillus infection and aflatoxin accumulation in groundnut. Aspergillus strains and thirteen different plant essential oils were tested. The oils were derived from clove, camphor, vanilla, garlic, galangal, green oregano, lemon grass, neem, ginger, basil, tea tree, thyme and onion. The biocontrol agents used were fungi Trichoderma harzianum strain T-22, T. asperellum and T. viride from a commercial biocontrol product, TUSAL, and bacteria Pseudomonas chlororaphis ssp. aureofaciens and Bacillus amyloliquefaciens (strains MBI600, 62P, and 66P). The identities of a strain of A. niger, isolated from Nigerian groundnut samples, and of T. asperellum and T. viride were confirmed by PCR amplification of DNA and sequence comparison to reference isolates in the GenBank database. Some of the plant oils (clove, camphor and vanilla) and biocontrol agents (Trichoderma strains) tested proved effective in inhibiting the A. flavus and A. niger strains used in the research, in both in vitro and in planta experiments. Improved seedling emergence in pathogen-contaminated compost and reduced post-harvest pod infection were observed. Combinations of the most active BCAs and EOs also provided disease suppression. ELISA analysis of aflatoxin B1 in treated, A. flavus-inoculated groundnut pods showed a reduction in toxin concentrations, to a level below that recommended by the European Commission of 15 ppb. Of the control agents tested, the most effective were T. harzianum T-22 as a BCA and probably clove oil as a plant extract. Commercial products based on Trichoderma are used world-wide. EOs, have, to date, had little use in control of Aspergillus infection of groundnut. It was also demonstrated that detection of asymptomatic A. flavus pod infection could be achieved by the traditional method of surface sterilisation and plating out, and by use of a LAMP assay to detect pathogen DNA. The latter could provide a rapid, portable method for A. flavus detection in harvested groundnut pods and could have application in both developed and developing nations. Since low resource growers in nations like Nigeria need alternative, low-cost methods for protecting groundnut from Aspergillus infection, to produce a nutritionally-valuable, high protein foodstuff low in toxin contamination, such alternative methods of disease control may have a future role to play in global food security. It may prove possible to extract antifungal components from appropriate, locally-sourced plant material in a cost-effective manner. However, whether the level of disease control and suppression of aflatoxin accumulation reported here was adequate for possible commercial application is unclear. Further evaluation, including field experiments, is required.

Soil borne fungi isolated from forest areas and nurseries in North east of Scotland using baiting techniques, were identified using classical taxonomy and molecular methods (PCR amplification of ITS regions; restriction digestion; sequencing of PCR products) as Fusarium lateritium, F. tricinctum, F. sambucinum, Phytophthora cinnamomi, Pythium ultimum var. ultimum and Rhizoctonia binucleate (Ceratobasidium sp.). Virulence was tested in vitro on young seedlings of Pinus sylvestris and Alnus glutinosa, and Koch's postulates fulfilled through reisolation of the pathogens and confirmation of fungal penetration into host tissues. Root growth was measured using the Winrhizo program, and dry weights recorded. Symptoms on aerial parts were assessed using a categorical scale from 0 (healthy) to 5 (damage > 76%). Fusarium spp. caused significant different (P 0.01) symptom intensity on both host plants. However, no significant difference in root growth was found between treatments and control (P 0.05). The effects of different compost treatments on disease development in seedlings of both hosts inoculated with the same fine root pathogens was tested in the glasshouse confirming the virulence of the fungal pathogens on P. sylvestris and A. glutinosa seedlings. Although mean dry weights of P. sylvestris and A. glutinosa varied between compost treatments, differences were not significantly different. Isolation, characterization and identification of bacterial isolates, Bacillus subtilis B1, fluorescent pseudomonads B4 and B5 with antagonistic action against pathogens were also carried out. These isolates along with the known bacterial antagonists Bacillus subtilis MB600, MB205 and Pseudomonas corrugata R117 were used for biological control in vitro and in planta experiments using Alnus glutinosa or Pinus sylvestris seedlings. All bacterial isolates colonized root systems of both tree species. Higher numbers of bacterial cells were observed on roots of A. glutinosa than on P. sylvestris roots. High bacterial cell numbers were observed in plants of both tree species inoculated with fluorescent pseudomonads B4 or B5. In vitro antagonism on agar plates, indicated by inhibition in fungal colony diameter growth, was recorded for F. tricinctum, F. lateritium and F. sambucinum, Pythium ultimum var. ultimum and Phythophthora cinnamomi with all bacterial isolates tested (P 0.05). Biological control of the fine root pathogens on Pinus sylvestris and Alnus glutinosa seedlings by bacteria semi in vivo in test tubes was carried out with various responses in both tree hosts. All bacterial treatments resulted in a lower sporangium germination rate for P. ultimum var. ultimum than was found in controls (P 0.05). Effect of the bacterial isolates separately on growth and disease development in Pinus sylvestris and Alnus glutinosa seedlings inoculated with the pathogens under glasshouse conditions using autoclaved compost was tested. The bacterial isolates had various effects against the pathogens, although in most cases no significant differences were observed relative to controls. Further soil-based trials were carried out in the glasshouse to achieve control of root disease development on Pinus sylvestris and Alnus glutinosa using a combination of different antagonists, based on a mixture of the bacterial isolates used previously and Trichoderma koningii (TC6-Colombia). None of the antagonistic treatments showed a clear antagonistic effect in Pinus sylvestris against the fungal infections compared to control plants inoculated with the pathogens alone. In contrast, in Alnus glutinosa plants T. koningii co-inoculation improved plant growth in several of the growth parameter measured.

Two Tobravirus expression vectors were evaluated for the use as a rapid screening method for anti-nutritional proteins against plant pathogens. Accumulation of green fluorescent protein (GFP) and snowdrop lectin gene (Galanthus nivalis agglutinin, LECGNA2, M55556) in Nicotiana benthamiana by Tobacco rattle virus expression vectors was characterized. Virally expressed proteins were detected in leaves (3-14 days post-inoculatiion) and roots (6-24 dpi) by UV (GFP), western blotting and tissue printing. 25 -50 ng of GNA was detected in root extracts. Cross protection was induced by TRV-GFP. Foreign genes inserted in place of TRV RNA2 non-structural genes (2b and 2c) were stably maintained over serial passages. But recombination at remaining 'cross-over' sites may occur. 2D iso-electricfocusing detected a 50-kDa GNA molecule in root and leaf extract. GNA did not confer resistance to root-knot nematodes, although gall by root-knot nematodes (mixed Meloidogyne spp. and M.javanica Crete line 17) were significantly reduced by 22% in roots infected by TRV-GNA (3.83 sqrt galls and 4.5 sqrt galls respectively) compared to virus-free roots treatment (4.94 sqrt galls, sed 0.398; p<.025 and 5.273 sqrt galls, sed 0.2403; <.003 respectively). Effects of GNA on Aulacorthum solani was delayed to the second nymph generation (N2). Mean N2 weights feeding on TRV-GNA (0.246 mg ±0.0159; p <05) and TRV-fsGNA (0.212 mg ±0.018; p<.001) infected plants were significantly smaller by 15.2% and 26.6% respectively, compared to virus-free treatments (0.290 mg ±0.014). Similar trends were detected for total nymph weights. Low toxicity was related to high quality phloem and ingestion of smaller volumes for normal development (i.e. concentration effect). Decrease in gall by the mixed Meloidogyne population and an unexpected toxicity to A solani indicated that truncated GNA was a protein with merolectin properties. The viability of this system as a rapid 'm planta' expression system is discussed.

Phytoplasmas are wall-less, non-culturable, phloem-limited bacterial pathogens that belong to the Mollicutes. They cause many diseases in lots of plant species (wild and cultivated) belonging to many different plant families, resulting in significant losses in important crops, and economically damaging epidemics worldwide. Phytoplasmas infect major cultivated crops such as many annual crops, fruit trees, grapevines and palms, which makes control of these diseases a priority, and the first important step for management is efficient and effective phytoplasma diagnosis. Detection of phytoplasmas is difficult because of their irregular distribution within the diseased plants and low concentration inside infected plants. In the last two decades most research toward the detection of phytoplasmas has used nucleic acid-based techniques such as PCR, which is used to amplify regions of phytoplasma genomes existing inside infected plants. However, most routine diagnostics has moved from general PCR to real-time PCR, due to the improved sensitivity and reduced risk of contamination due to the use of a closed system for product detection. Also the method can be developed into a semi-quantitative method. In this project the aim has been to improve the specificity and reliability of phytoplasma diagnostic techniques by using primers that detect specific genes in phytoplasma genomes, and designing new universal primers for conventional and real-time PCR. This has involved developing new assays for 16Sr groups II, III, V, VI, XI, and XII, to facilitate analysis of changes in levels of different phytoplasmas in mixed infections. In addition, this work has involved the evaluation of LAMP (loop mediated isothermal amplification) diagnostic assays for different phytoplasma groups (I, II, III, V, VI, X, and XII) and also validating a rapid DNA extraction method and whether this is effective for all plant species (i.e. Madagascan periwinkle versus Napier grass and other grasses). The second main objective was to investigate the rate of evolution of phytoplasma genomes. For this, infected plants (of phytoplasma groups 16SrI, II, III, VI, and X) were grafted onto fresh plants at 3-4 month intervals throughout the project. Once the phytoplasma had re-established, DNA was extracted and a range of genes including 16S rRNA, secA, tuf, and rp were amplified and sequenced. The aim was to determine whether there was any evidence of genome evolution over time. However, the results suggested that in these genes at least, the rate of change due to point mutations and/or insertion of potential mobile elements (PMUs) was slow, with no sequence changes being detected over the three years of study.

The major aim of this research was to investigate interactions between the bacterial pathogen Xanthomonas campestris pv. raphani (Xcr) and Arabidopsis thaliana which are largely unexplored as a model pathosystem, and to identify genetic loci conferring resistance to this pathogen. Xcr is genetically close to X. campestris pv. campestris (Xcc) and both pathogens infect common Brassicaceae species including A. thaliana, but cause distinct diseases - leaf spot and black rot, respectively. Phenotypic variation was identified among interactions between 22 A. thaliana accessions of wide geographic origin and Xcr and Xcc strains representing known host genotype specific races. Accessions were identified showing broad resistance and broad susceptibility to multiple Xcr and Xcc strains as well as accessions that differentiate between Xcr races. Genetic mapping of resistance to a strain of Xcr race 2 in a multi-parent recombinant inbred population revealed two major effect loci: RXCR1 at the bottom arm of chromosome 3, and RXCR2 at the bottom arm of chromosome 5. RXCR1 was confirmed by fine-mapping and loss- and gain-of-function experiments, as a single gene encoding a kinase-like protein conferring resistance in the accession Columbia. Columbia is resistant to strains of all three known Xcr races, but RXCR1 is insufficient on its own to explain the broad resistance. A summary of phenotypic and genetic analyses of interactions between Xcr and A. thaliana is presented in a gene-for-gene model. X. campestris strains associated with outbreaks of a leaf spot and blight disease of brassica crops in Mauritius, were characterized. These strains were similar to reference Xcc strains in pathogenicity tests and molecular analyses. The presence of Xcr in these outbreaks was not confirmed. Whole-genome sequencing data was used to identify genes that may contribute to the distinct modes of pathogenesis of Xcr and Xcc as well as variation in host specific races within each pathovar. Genes differentially present/absent between nine Xcr and 23 Xcc strains were identified and include genes predicted/known to encode type III effectors some of which have been previously described to have an effect on Xcc pathogenicity. Candidate avirulence determinants of Xcr and Xcc races were also identified.