Electron-vibration-vibration two-dimensional infrared spectroscopy as a structural probe of interactions in proteins and DNA

Modern structural biology has a number of powerful tools but despite this there are a number of problems in structural biology that these methods are unable to address. Some of these pertain to the need for large number for precise comparative structures for the drug discovery process. EVV 2DIR has the potential to fill some of these gaps, having the potential to determine molecular binding geometry. This thesis presents the first steps in exploring the potential of EVV 2DIR to be applied to the analysis of the structure of inhibitor- protein binding and presents the first EVV spectra of an inhibitor-protein complex. For the inhibitor-protein complex studied, six vibrational couplings between seven vibrational modes were identified exclusively upon complex formation due to interactions between the two molecules. Experimental spectra were compared with ab initio calculations to assign these vibrations to specific motions on both the inhibitor and protein molecules. EVV 2DIR cross peaks can be sensitive to the geometry of the interacting groups which produce them. By measuring the spectra of the inhibitor-protein complex using two different polarisation schemes, quantitative comparison between calculated and experimental spectra was made possible. This allowed for the prospect of using calculation aided EVV 2DIR to determine the structure of protein-ligand complexes to be explored. This thesis also presents the first EVV spectra of DNA. EVV 2DIR spectra were measured of duplex and G-quadruplex structures and compared with those of unstructured controls. In the absence of calculated spectra, assignments were made to some of the spectral features observed. EVV 2DIR was shown to be sensitive to the structural form of the DNA samples, containing cross peaks indicative of WatsonCrick base pairing, G-quadruplex formation and glycosidic bond conformation. The DNA spectra contained many unassigned peaks leaving open the possibility to assign many more structural indicators.Open Acces

Persistent, symptomless, systemic, and seed-borne infection of lettuce by Botrytis cinerea

Experiments are presented which show that Botrytis cinerea, the cause of gray mould disease, is often present in symptomless lettuce plants as a systemic, endophytic, infection which may arise from seed. The fungus was isolated on selective media from surface sterilized sections of roots, stem pieces and leaf discs from symptomless plants grown in a conventional glasshouse and in a spore-free air-flow provided by an isolation propagator. The presence of B. cinerea was confirmed by immuno-labelling the tissues with the Botrytis-specific monoclonal antibody BC-12.CA4. As plants grew, infection spread from the roots to stems and leaves. Surface sterilization of seeds reduced the number of infected symptomless plants. Artificial infection of seedlings with dry conidia increased the rate of infection in some experiments. Selected isolates were genetically finger-printed using microsatellite loci. This confirmed systemic spread of the inoculating isolates but showed that other isolates were also present and that single plants hosted multiple isolates. This shows that B. cinerea commonly grows in lettuce plants as an endophyte, as has already been shown for Primula. If true for other hosts, the endophytic phase may be as important a component of the species population as the aggressive necrotrophic phase

Harnessing the Therapeutic Properties of Ginger (<em>Zingiber officinale</em> Roscoe) for the Management of Plant Diseases

Ginger (Zingiber officinale Roscoe) is one of the most widely used spices in the world. The therapeutic benefits of ginger are mainly due to the presence of volatile oils, phenols, alkaloid, and high oleoresin content. Ginger extracts have been extensively studied for a broad range of biological activities including antibacterial, antifungal, antiviral, anticonvulsant, analgesic, antiulcer, gastric antisecretory, and antitumor. This is all the more necessary because ginger is of plant origin, specifically more biodegradable, readily available, cheaper, and environmentally friendlier than synthetic chemicals. Since, some farmers in developing countries use ginger extracts as traditional medicine in the treatment of human diseases, it will be easy for them to adopt these extracts as biopesticides for the management of plant diseases. This book chapter seeks to outline the bioactive compounds and therapeutic benefits of ginger in plant disease management, and the mechanisms of action are also discussed

Evaluation of efficacy of false yam (Icacina oliviformis) as surface protectant against rot pathogens of white yam (Dioscora rotundata Poir)

Rots in yam tubers are the dominant cause of postharvest losses. Therefore, studies were con­ducted to identify the rot-causing organisms in yam and to evaluate the efficacy of false yam (Icacina oliviformis) plant extracts as surface protectants in the storage of yam. The experiment was laid out in a completely randomized design (CRD) design with five treatments (fruit, root and leaf extract of false yam, Mancozeb and tap water) replicated three times. Two concen­trations (50% and 100%) of each extract were also tested. Species were identified based on the structural features, the characteristics and properties of the spore and mycelium. A spore suspension of yam rot fungi was sprayed on healthy yam tubers that had been pretreated with the extracts. Results showed that fungi isolated from rotted yams were Aspergillus niger, A. fla­vus and Penicillium sclerotigenum. Leaf extracts (both 50% and 100%) had the highest growth inhibitions on all the three fungi isolated in vitro. Tubers treated with root and leaf extracts of false yam had a decreased tuber rot lengths of 1.80 mm2 and 2.17 mm2, respectively. The leaves and roots of false yam can be used as a surface protectant of yam in place of Mancozeb. Keywords: Icacina oliviformis; Dioscora rotundata; Mancozeb; Pathogenicity; Aspergillus spp

Variations in the level of resistance to root-knot nematodes (Meloidogyne spp.) infestation among ten cowpeas (Vigna unguiculata L. Walp.) genotypes

The cultivation of cowpea (Vigna unguiculata L. Walp.) cultivars that are tolerant to root-knot nematode attack is among the environmentally safe approach to managing the root-knot nematode menace in cultivated crops. In this study, the tolerance of 10 cowpea genotypes to root-knot nematodes infestation was evaluated in a pot experiment conducted in a Screenhouse, at the University for Development Studies, Nyankpala Campus. The experiment was laid out in a completely randomized design with three replications. The number of second stage juveniles (J2) per 250 cm3 of soil sample were counted while the severity of root-knot nematode damage (root galls) was assessed. The reproduction index (RI) was used to classify the varieties as resistant or susceptible. There was a significant difference (P &lt; 0.05) in the number of second stage juveniles of root-knot nematode (RKN), galling index and RI among the genotypes tested. The study revealed that cowpea genotypes SARI 1-4-90, Padi tuya, Songotra, IT99K-1122, Sanzi and Apagbaala were moderately resistant whereas cowpea genotypes IT86D-610, Zaayura, SARI 5-5-5 and IT07K-299-6 were slightly resistant. Thus, the six moderately resistant cowpea genotypes were suggested to be used as a source of resistance to RKN in future breeding works

Incidence of fungi and aflatoxin contamination of maize in Tolon-Kumbungu district of Ghana

Maize ( Zea mays L.) is an important staple food crop and a source of income to farmers, as well as foreign exchange earner in most countries in sub-Saharan Africa. Its production is hampered by fungal diseases, which also cause contamination with mycotoxins, especially aflatoxin and its associated health hazards. This study sought to isolate and identify aflatoxigenic fungi, as well as detect the presence of Aflatoxin B1 (AfB1) in maize samples obtained from farmers in the Tolon-Kumbungu district in the northern region of Ghana. Twenty farming communities were randomly selected for the study in consultation with the district office of the Ministry of Food and Agriculture (MoFA). Samples were collected from 200 randomly selected maize farmers by the composite sampling technique, for isolation of aflatoxigenic fungi by the agar plate method and the detection of aflatoxin. Aflatoxin was detected in maize samples with the Black light, rapid screening and immunoassay methods. Aspergillus flavus had the highest percentage of occurrence (63.7%); followed by A. niger (16.5%), Rhizopus stolonifer (9.3%), Penicillium spp. (6.9%) and Fusarium oxysporum (3.7%). Farm samples had more aflatoxin than those from stores and markets. Samples of maize from farms in Gbirimani community had the highest aflatoxin contamination of +60 ppb. Concentrations of Afb1 at or above +20 ppb were recorded in all the communities, except in Tinguli. Apart from Voggu, all market samples were free from aflatoxin contamination.Le ma\uefs ( Zea mays L.) est une importante culture vivri\ue8re de base et une source de revenus pour les agriculteurs, ainsi que des sources de devises dans la plupart des pays Afrique sub-saharienne. Sa production est entrav\ue9e par des maladies fongiques, qui provoquent \ue9galement une contamination par des mycotoxines, en particulier l\u2019aflatoxine et les risques pour la sant\ue9 qui y sont associ\ue9s. Cette \ue9tude visait \ue0 isoler et \ue0 identifier les champignons aflatoxig\ue8nes, ainsi qu\u2019\ue0 d\ue9tecter la pr\ue9sence d\u2019aflatoxine B1 (AfB1) dans des \ue9chantillons de ma\uefs provenant d\u2019agriculteurs du district de Tolon-Kumbungu dans la r\ue9gion nord du Ghana. Vingt communaut\ue9s agricoles ont \ue9t\ue9 s\ue9lectionn\ue9es au hasard pour l\u2019\ue9tude en consultation avec le bureau de district du minist\ue8re de l\u2019Alimentation et de l\u2019Agriculture (MoFA). Des \ue9chantillons ont \ue9t\ue9 pr\ue9lev\ue9s aupr\ue8s de 200 producteurs de ma\uefs s\ue9lectionn\ue9s au hasard par la technique d\u2019\ue9chantillonnage composite, pour l\u2019isolement des champignons aflatoxig\ue8nes par la m\ue9thode de la plaque de g\ue9lose et la d\ue9tection de l\u2019aflatoxine. L\u2019aflatoxine a \ue9t\ue9 d\ue9tect\ue9e dans des \ue9chantillons de ma\uefs avec la lumi\ue8re noire, des m\ue9thodes de d\ue9pistage rapide et d\u2019immunodosage. Aspergillus flavus avait le pourcentage d\u2019occurrence le plus \ue9lev\ue9 (63,7%); suivi par A. niger (16,5%), Rhizopus stolonifer (9,3%), Penicillium spp. (6,9%) et Fusarium oxysporum (3,7%). Les \ue9chantillons de ferme contenaient plus d\u2019aflatoxine que ceux des magasins et des march\ue9s. Les \ue9chantillons de ma\uefs provenant d\u2019exploitations agricoles de la communaut\ue9 de Gbirimani pr\ue9sentaient la contamination d\u2019aflatoxine la plus \ue9lev\ue9e, soit +60 ppb. Des concentrations d\u2019Afb1 \ue9gales ou sup\ue9rieures \ue0 +20 ppb ont \ue9t\ue9 enregistr\ue9es dans toutes les communaut\ue9s, sauf \ue0 Tinguli. \uc0 l\u2019exception de Voggu, tous les \ue9chantillons du march\ue9 \ue9taient exempts de contamination par les aflatoxines

Symbiotic N2 Fixation and Grain Yield of Endangered Kersting's Groundnut Landraces in Response to Soil and Plant Associated Bradyrhizobium Inoculation to Promote Ecological Resource-Use Efficiency

Kersting's groundnut (Macrotyloma geocarpum Harms) is a neglected, endangered food and medicinal legume in Africa. Efforts to harness the benefits of the legume-rhizobia symbiosis have focused on few major legumes to the neglect of underutilized ones such as Kersting's groundnut. This study assessed plant growth, N-fixed and grain yield of five Kersting's groundnut landraces in response to inoculation with Bradyrhizobium strain CB756 at two locations in the Northern Region of Ghana. The transferability of cowpea-derived Simple Sequence Repeat (SSR) markers to Kersting's groundnut was also assessed. The symbiotic results revealed significant variation in nodulation, shoot biomass, δ15N, percent N derived from fixation, amount of N-fixed and soil N uptake. The cross-taxa SSR primers revealed monomorphic bands with sizes within the expected range in all the Kersting's groundnut landraces. The results of the aligned nucleotide sequences revealed marked genetic variability among the landraces. Kersting's groundnut was found to be a low N2-fixer, with 28–45% of its N derived from fixation at Nyankpala and 15–29% at Savelugu. Nitrogen contribution was 28–50 kg N-fixed·ha−1 at Nyankpala, and 12–32 kg N-fixed·ha−1 at Savelugu. Uninoculated plants of the Kersting's groundnut landraces Puffeun, Dowie, Sigiri and Boli, respectively, contributed 22, 16, 13, and 15 kg N-fixed·ha−1 from symbiosis at Savelugu as opposed to 89, 82, 69, and 89 kg N·ha−1 from soil. Landrace Puffeun was highly compatible with the introduced strain CB756 if based on δ15N and %Ndfa values, while Dowie, Funsi and Boli showed greater compatibility with native rhizobia in Ghanaian soils. The unimproved Kersting's groundnut in association with soil microsymbionts could produce grain yield of 1,137–1,556 kg ha−1 at Nyankpala, and 921–1,192 kg ha−1 at Savelugu. These findings suggest the need for further work to improve the efficiency of the Kersting's groundnut-rhizobia symbiosis for increased grain yield and resource-use efficiency in cropping systems

Anthracnose Disease of Mango: Epidemiology, Impact and Management Options

Mango is one of the frequently cultivated seasonal fruit crops in several tropical and subtropical regions. It is consumed as whole fruits apart from serving as raw materials for most industries that are into mineral production. Mango production is, however, constrained by diseases, pests, and poor post-harvest handling of fruits. Anthracnose disease, caused by Colletotrichum gloeosporioides Penz and Sacc, is one of the most important yields limiting constraint in mango production across the globe. The disease occurs in both the field and post-harvesting. In the field, it affects aboveground parts, such as the stem, branches, leaves, flowers, and fruits. Anthracnose disease reduces the shelve life and marketability of mango fruit. In Ghana, anthracnose disease is responsible for about 30% yield/fruit loss. Most farmers do not control it, although some have resorted to the application of various fungicides not registered for mango anthracnose disease management in Ghana. This chapter will highlight on the importance of the disease on the mango industry in Ghana, control strategies currently employed thereby reducing the over-reliance on chemical control option and propose ways to minimize the effect of the disease in the country

Vertical transmission of fungal endophytes is widespread in forbs

To date, it has been thought that endophytic fungi in forbs infect the leaves of their hosts most commonly by air-borne spores (termed “horizontal transmission”). Here, we show that vertical transmission from mother plant to offspring, via seeds, occurs in six forb species (Centaurea cyanus, C. nigra,Papaver rhoeas,Plantago lanceolata,Rumex acetosa, and Senecio vulgaris), suggesting that this may be a widespread phenomenon. Mature seeds were collected from field-grown plants and endophytes isolated from these, and from subsequent cotyledons and true leaves of seedlings, grown in sterile conditions. Most seeds contain one species of fungus, although the identity of the endophyte differs between plant species. Strong evidence for vertical transmission was found for two endophyte species, Alternaria alternata and Cladosporium sphaerospermum. These fungi were recovered from within seeds, cotyledons, and true leaves, although the plant species they were associated with differed. Vertical transmission appears to be an imperfect process, and germination seems to present a bottleneck for fungal growth. We also found that A. alternata and C. sphaerospermum occur on, and within pollen grains, showing that endophyte transmission can be both within and between plant generations. Fungal growth with the pollen tube is likely to be the way in which endophytes enter the developing seed. The fact that true vertical transmission seems common suggests a more mutualistic association between these fungi and their hosts than has previously been thought, and possession of endophytes by seedling plants could have far-reaching ecological consequences. Seedlings may have different growth rates and be better protected against herbivores and pathogens, dependent on the fungi that were present in the mother plant. This would represent a novel case of trans-generational resistance in plants

Analysis of cryptic, systemic Botrytis infections in symptomless hosts

Botrytis species are generally considered to be aggressive, necrotrophic plant pathogens. By contrast to this general perception, however, Botrytis species could frequently be isolated from the interior of multiple tissues in apparently healthy hosts of many species. Infection frequencies reached 50% of samples or more, but were commonly less, and cryptic infections were rare or absent in some plant species. Prevalence varied substantially from year to year and from tissue to tissue, but some host species routinely had high prevalence. The same genotype was found to occur throughout a host, representing mycelial spread. B. cinerea and B. pseudocinerea are the species that most commonly occur as cryptic infections, but phylogenetically distant isolates of Botrytis were also detected, one of which does not correspond to previously described species. Sporulation and visible damage occurred only when infected tissues were stressed, or became mature or senescent. There was no evidence of cryptic infection having a deleterious effect on growth of the host, and prevalence was probably greater in plants grown in high light conditions. Isolates from cryptic infections were often capable of causing disease (to varying extents) when spore suspensions were inoculated onto their own host as well as on distinct host species, arguing against co-adaptation between cryptic isolates and their hosts. These data collectively suggest that several Botrytis species, including the most notorious pathogenic species, exist frequently in cryptic form to an extent that has thus far largely been neglected, and do not need to cause disease on healthy hosts in order to complete their life-cycles