Rozprawy doktorskie na temat „Soil borne diseases- Fusarium”

Banana is one of the world’s most popular fruit crops and Sukali Ndizi is the most popular dessert banana in the East African region. Like other banana cultivars, Sukali Ndizi is threatened by several constraints, of which the Fusarium wilt disease is the most destructive. Fusarium wilt is caused by a soil-borne fungus, Fusarium oxysporum f.sp. cubense (Foc). No effective control strategy currently exists for this disease and although disease resistance exists in some banana cultivars, introducing resistance into commercial cultivars by conventional breeding is difficult because of low fertility. Considering that conventional breeding generates hybrids with additional undesirable traits, transformation is the most suitable way of introducing resistance in the banana genome. The success of this strategy depends on the availability of genes for genetic transformation. Recently, a novel strategy involving the expression of anti-apoptosis genes in plants was shown to result in resistance against several necrotrophic fungi, including Foc race 1 in banana cultivar Lady Finger. This thesis explores the potential of a plant-codon optimised nematode anti-apoptosis gene (Mced9) to provide resistance against Foc race 1 in dessert banana cultivar Sukali Ndizi. Agrobacterium-mediated transformation was used to transform embryogenic cell suspension of Sukali Ndizi with plant expression vector pYC11, harbouring maize ubiquitin promoter driven Mced9 gene and nptII as a plant selection marker. A total of 42 independently transformed lines were regenerated and characterized. The transgenic lines were multiplied, infected and evaluated for resistance to Foc race 1 in a small pot bioassay. The pathogenicity of the Ugandan Foc race 1 isolate used for infection was pre-determined and the spore concentration was standardised for consistent infection and symptom development. This process involved challenging tissue culture plants of Sukali Ndizi, a Foc race 1 susceptible cultivar and Nakinyika, an East African Highland cultivar known to be resistant to Foc race 1, with Fusarium inoculum and observing external and internal disease symptom development. Rhizome discolouration symptoms were the best indicators of Fusarium wilt with yellowing being an early sign of disease. Three transgenic lines were found to show significantly less disease severities compared to the wild-type control plants after 13 weeks of infection, indicating that Mced9 has the potential to provide tolerance to Fusarium wilt in Sukali Ndizi.

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.

International trade in plants, especially with potting substrates, is recognised as the main pathway of plant pathogen dissemination on a global scale. In the last 20 years, the wide use of internet commerce has become common in the nursery sector and, due to the nature of online sales, may be aggravating this risk. Oomycetes in the genera Phytophthora, Pythium and Phytopythium, cause a range of important plant diseases, responsible for serious economic and biological losses. This research focused on the detection of Oomycetes in imported potted ornamental plants in the UK and The Netherlands, including internet sales and asymptomatic plants. Isolation techniques and molecular protocols were developed to quantify pathogen load in ornamental plants, using TaqMan PCR and Next Generation Sequencing (NGS) to assess Oomycete diversity using a multi-locus approach. Survival of Phytophthora cinnamomi and Fusarium verticillioides was estimated in two commercial potting mixes used in ornamental plant production. Oomycetes were detected in all samples analysed with the NGS approach, with 38 Phytophthora spp. and 48 Pythium/Phytopythium spp. identified. Phytophthora ramorum, P. alni subsp. alni and P. cryptogea were common. TaqMan PCR quantification showed high numbers of Oomycetes in all samples, especially in substrates, followed by roots and baiting waters. During sampling by isolation, Pythium kashmirense was recovered from Viburnum plicatum, the first record of this species in the UK. The survival experiment showed that Fusarium verticillioides remained viable after 17 months, whereas Phytophthora cinnamomi was viable up to 7 months after inoculation. This work clearly demonstrated the widespread presence of Oomycete pathogens in the plants for planting pathway. Moreover, the protocols developed and findings of this work contribute greatly to the understanding of the potential for pathogens to spread in the international horticultural trade and may help to improve plant biosecurity protocols in the UK and Europe.

Studies were undertaken to determine the dispersal mechanisms of Verticicladiella procera Kendrick, the causal agent of Procera Root Disease (PRD). Propagule germinability in artificially infested soil decrease rapidly under natural and controlled conditions. Colonization of seedlings in artificially infested soil was rare and symptoms were not displayed by colonized seedlings. Natural populations of V. procera were closely associated with colonized root tissue. Colonization of field planted seedlings was related to proximity to root collars of diseased trees and insect activity on the seedlings. Insects (Coleoptera) contaminated with V. procera were found in plantations both with and without PRD. The percent of weevils and bark beetles contaminated with V. procera was 64 and 0.76 respectively. Verticicladiella procera was transmitted to white pine bolts in the field and under controlled conditions following visitation by contaminated insects. Verticicladiella procera was associated with larval galleries and frass in trap bolts and was observed fruiting in insect galleries in root systems of diseased trees. This evidence suggests that transmission by insects, especially weevils, is the more important mechanism for dispersal and that soil-borne propagules have a minor role in pathogen spread.
M.S.

The nutritional and environmental requirements for mycelial growth, oospore production and germination of Pythium oligandrum were examined. Optimum temperatures for growth of several isolates were in the range of 20 - 30 0 , with little growth occurring below 100 or above 350 Oospore germination occurred over the range of 10-35°. Both growth and oospore germination occurred over the range of pH 4.5 - 9.0 and were optimum between pH 6.0 - 7.5. Growth was reduced markedly below -1.0 to -1.5 MPa osmotic potential and ceased at approximately -2.5 to -3.5 MPa; similar results were obtained for oospore germination. Growth and oospore germination were affected more by low matric than by low osmotic potentials. Oospore production required an exogenous supply of sterols; it was also increased by the presence of calcium and affected by the C:N ratio. Semi-solid, static and aerated culture systems were developed for bulk production of P. oligandrum oospores. A liquid cane molasses medium was particularly convenient and efficient. A range of formulations were prepared using oospores produced mainly in this medium. Formulations were evaluated against pathogens causing damping-off in cress and the level of biocontrol in artificially infested sand was not as good as that obtained in naturally infested soil. Alginate pellets and a perlite preparation survived well in laboratory storage at 5-25° for at least 24 wk. Seeds of cress and sugar beet were coated with oospores using commercial seed-pelleting and film-coating procedures. Both types of seed treatment reduced damping-off of cress caused by P. ultimum in artificially infested sand and potting compost, and by Rhizoctonia solani in artificially infested sand. In general, pelleting of P. oligandrum on cress gave better control than film-coating treatments. P. oligandrum also reduced damping-off of sugar beet in soil naturally infested with Aphanomyces cochlioides and Pythium spp.. Control was equivalent to that achieved with hymexazol fungicide seed-coating treatments and was related to the inoculum potential of A. cochlioides in the soil.

A number of experiments were carried out to study the potential of denitrifying bacteria and reduced nitrogen compounds for control of soil-borne damping-off pathogens. Measurement of the rhizosphere pH of growing soybean roots was carried out in soil adjusted to different pH states and packed into sheet microcosms. The results showed that the rhizosphere pH of soybean was lower than the bulk soil. Nitrate reductase activity and nitrite production was then characterised for the rhizosphere of intact 14 day-old soybean roots that were incubated in nitrate substrates adjusted to different pH values under water-logged conditions. The results showed that the rate and the quantity of nitrite production increased with increasing nitrate concentration and pH in the solution. A growth room experiment was carried out to determine root colonization by denitrifying bacteria in relation to disease caused by soil-borne pathogens, which are favoured by high soil moisture (approximately -5 KPa) and low oxygen levels. Nitrite producing bacteria were isolated from soybean roots grown in Grampian (Insch) soils which had not been cropped with soybean and Thai (Phitsanulok) soils which previously had been cropped with soybean. In the first pot experiment, the nitrite producing bacteria were isolated from different root sections of 12 and 19 day-old soybean plants after 8 weeks of continuous cropping of soils with soybean (a new crop was planted every week), and using different isolation media in order to determine the genus/species composition of the denitrifying bacteria on the rhizoplane. The results showed that continuous cropping of Thai soil and Insch soil with soybean increased pre-emergence damping-off disease and decreased fresh weight yields in seedlings that did emerge. ANOVA showed significant differences between root sections for most bacterial groups monitored {Bacillus spp., Pseudomonas and Enterobacteriaceae), with regression analysis generally showing densities increasing with root age or toward the shoot base. All nitrite producing bacterial isolates were screened for antifungal activity against Macrophomina phaseolina on agar plates and between 10 and 25% of nitrite producing bacteria were found to show in vitro antagonism. In a second pot experiment, the nitrite producing bacteria were isolated from root tissue below the crown (5 cm in length) every 2 weeks of continuous cropping of soils with soybean (a new crop was planted every 2 weeks). Plate-counting was carried out to determine the population of nitrite producing bacteria while a liquid culture MPN method was used for determination of NO, N2O and N2 producing bacteria. Linear regression analysis of the incidence of pre-emergence damping-off and soybean yields in seedling that did emerge showed a highly significant negative correlation between these parameters for both soils. ANOVA showed that there was a significant difference between soil type, with the Thai soil showing higher population densities of antagonistic bacteria on soybean roots. All nitrite producing bacterial isolates were screened for antifungal activity, but the plant pathogenic fungus, Pythium ultimum, was used in this experiment. The results showed that between 10 and 40% of nitrite producing bacteria showed in vitro antagonism. However, regression analysis showed that there was no significant increase or decrease in the nitrite producing antagonistic bacterial population with continuous soybean cropping. All 900 isolates of nitrite producing bacteria isolated from the soybean rhizoplane were screened for antagonistic activity towards Pythium ultimum based on a pot trial assay in the greenhouse. As expected, very low numbers of nitrite producing bacteria showed activity against P. ultimum and only one isolate gave a significant reduction in disease incidence in pot trials. The interactive effects of nitrite producing antagonist and an Arbuscular Mycorrhizal fungus (Glomus mosseae) and Bradryrhizobium japonicum, on control of the fungal pathogens, P. ultimum or M. phaseolina were investigated in the greenhouse. The results showed that improved plant growth was obtained with certain combined inocula involving nitrite producing bacterial antagonists, Glomus mosseae and Bradryrhizobium japonicum.

Thesis (PhDAgric (Plant Pathology))--Stellenbosch University, 2010.
ENGLISH ABSTRACT: In the Western Cape onion industry in South Africa, Fusarium oxysporum Schlechtend.:Fr. f.sp. cepae (H.N. Hans.) W.C. Snyder & H.N. Hans. (Focep) has been identified as the leading cause of harvest and storage losses. This pathogen is of world-wide importance and causes Fusarium basal rot of onions (Allium cepa), affecting all onion growth stages. No information is available on the evolution, genetic diversity, molecular detection and inoculum sources of the South African Focep population. Similar to what is the case for South Africa, limited information is available on Focep in other regions of the world. World-wide, four vegetative compatibility groups (VCGs) and two single-member VCGs (SMVs) have been identified among two Japanese and 19 Colorado (USA) isolates. This polyphyletic origin of Focep suggested by VCG analyses was confirmed through molecular analyses of isolates from a few countries. Only the mating type (MAT)1-1 idiomorph has been reported for Focep isolates from Welsh onion (Allium fistulosum). The development of sustainable management strategies of Focep is dependent on knowledge of (i) the genetic diversity and evolution of Focep, (ii) whether high throughput molecular methods can be developed for identifying the most virulent and widespread Focep genotypes and (iii) the role of seedlings and seeds as primary inoculum sources, and the Focep genotypes associated with these growth stages. Therefore, the three main aims of the current study were to investigate the aforementioned three aspects. In the first aim of the study, the genetic diversity and evolution of Focep was investigated using a collection of 79 F. oxysporum isolates from South Africa (27 Focep and 33 non-pathogenic isolates) and Colorado (19 Focep isolates). VCG analyses revealed the presence of six VCGs, four among the Colorado Focep isolates (VCGs 0421, 0422, 0423 and 0424) and two among the South African bulb-associated isolates (VCGs 0425 and 0426). VCG 0421 and VCG 0425 were the two main VCGs in Colorado and South Africa, respectively. Four SMVs and one heterokaryon selfincompatible (HSI) isolate were also identified. The polyphyletic nature of Focep in South Africa and Colorado was shown through a combined translation elongation factor 1α (EF-1α) and mitochondrial small-subunit (mtSSU) phylogeny. The phylogeny divided the Focep isolates into two main clades, of which one contained the two main VCGs (0421 and 0425), SMVs and non-pathogenic isolates. The second, ancestral clade contained the HSI isolate, VCGs 0422, 0423 and 0424, and non-pathogenic isolates. Unlike the clade containing the two main VCGs, which were highly virulent toward onion bulbs, the ancestral clade contained isolates that were mostly moderately virulent. The incongruence of the EF-1α and mtSSU datasets with an intergenic spacer (IGS) region data set, and the presence of both MAT idiomorphs within the same isolate for some isolates, suggested possible exchange of genetic material between isolates. The second aim of the study was to develop molecular methods for identifying the two main Focep VCGs (0425 and 0421), using DNA fingerprinting methods and sequence-characterized amplified region (SCAR) markers. These techniques were first developed using the F. oxysporum isolates from the first aim, and were then used to investigate the prevalence of VCG 0425 among 88 uncharacterized F. oxysporum isolates from onion bulbs in South Africa. Two random amplified polymorphic DNA primers provided two diagnostic amplicons for VCG 0425, but attempts to develop SCAR markers from these amplicons were unsuccessful. In contrast, an interretrotransposon amplified polymorphism (IRAP) fingerprinting method enabled the developed of a multiplex IR-SCAR polymerase chain reaction method that detected the VCG 0421, 0425 and SMV 4 isolates as a group. Fingerprinting and SCAR marker testing of the 88 uncharacterized F. oxysporum isolates from South Africa (65 Focep and 23 non-pathogenic) confirmed that VCG 0425 is the main VCG in South Africa associated with mature onion bulbs, since 63 of the Focep isolates had the molecular characteristics of VCG 0425. The third aim of the study was to determine whether seed and seedling transplants are inoculum sources of Focep, and whether the same genotype (VCG 0425) that dominated on mature bulbs could be detected from these sources. Focep isolates were obtained from seven of the 13 investigated onion seed lots, as well as from onion seedling transplants that were collected from all five onion nurseries in the Western Cape. Focep seedling infection more than doubled from the 6-week growth stage to the 14-week growth stage. Seed infections by Focep were low, but the seedborne nature of Focep was confirmed by showing that a green fluorescent protein labelled Focep transformant could be transmitted from infected soil to onion seed via the onion bulbs and seedstalks. It is thus clear that commercial seed and seedlings are inoculum sources of Focep. However, the Focep genotypes on seed and seedlings are different from those in mature bulbs and were not dominated by VCG 0425. Furthermore, most (≤ 60%) of the seed and seedling isolates were moderately virulent, as compared to the mostly highly virulent isolates from mature bulbs.
AFRIKAANSE OPSOMMING: In die Wes-Kaapse uiebedryf in Suid-Afrika is Fusarium oxysporum Schlechtend.:Fr. f.sp. cepae (H.N. Hans.) W.C. Snyder & H.N. Hans. (Focep) geïdentifiseer as die vernaamste oorsaak van oes- en opbergingsverliese. Hierdie patogeen is van wêreldwye belang; dit veroorsaak Fusarium-bolvrot van uie (Allium cepa) en affekteer alle plantgroeistadia. In Suid-Afrika is daar geen inligting beskikbaar oor die evolusie, genetiese diversiteit, molekulêre opsporing en inokulumbronne van die Focep-populasie nie. Soortgelyk aan wat die geval in Suid-Afrika is, is daar beperkte inligting beskikbaar oor Focep in ander wêrelddele. Wêreldwyd is daar vier vegetatiewe versoenbaarheidsgroepe (VVGe) en twee enkellid VVGe (ELVe) geïdentifiseer onder twee Japannese en 19 Colorado (VSA) isolate. Hierdie veelvuldige oorsprong van Focep wat deur VVG-analise voorgestel was, is deur die molekulêre analises van isolate uit ’n paar ander lande bevestig. Slegs die paringstipe (PT)1-1 idiomorf is vir Focep-isolate uit Walliese-tipe uie (ook bekend as ‘lenteuie’ in Suid Africa) (Allium fistulosum) berig. Die ontwikkeling van volhoubare bestuurstrategieë vir Focep steun op kennis van (i) die genetiese diversiteit en evolusie van Focep, (ii) of hoë-deurset molekulêre metodes ontwikkel kan word vir die identifisering van die mees virulente en wydverspreide Focep-genotipes en (iii) die rol van saailinge en saad as primêre inokulumbronne, en die Focep-genotipes wat met hierdie groeistadia geassosieer word. Daarom was die hoof doelstellings van hierdie studie om die bogenoemde drie aspekte te bestudeer. Om die eerste doel van die studie te bereik is die genetiese diversiteit en evolusie van Focep bestudeer deur gebruik te maak van ‘n versameling van 79 F. oxysporum-isolate uit Suid-Afrika (27 Focep en 33 nie-patogeniese isolate) en uit Colorado (19 Focep-isolate). VVG-analises het die teenwoordigheid van ses VVGe aangetoon – vier onder die Colorado Focep-isolate (VVGe 0421, 0422, 0423 en 0424) en twee onder die Suid-Afrikaanse bol-geassosieerde isolate (VVGe 0425 en 0426). VVG 0421 en VVG 0425 was die twee hoof VVGe in onderskeidelik Colorado en Suid-Afrika. Vier ELVe en een meerkernige self-onversoenbare (MSO) isolaat is ook geïdentifiseer. Die veelvuldige oorsprong van Focep in Suid-Afrika en Colorado is ook aangetoon deur ‘n gekombineerde translasie verlengings faktor 1α (VF-1α) en mitokondriale klein-subeenheid (mtKSE) filogenie. Dié filogenie het die Focepisolate in twee groepe verdeel, waarvan die een groep die twee hoof VVGe (0421 en 0425), ELVe en nie-patogeniese isolate bevat het. Die tweede, basal groepering het die MSO-isolaat, VVGe 0422, 0423 en 0424, en nie-patogeniese isolate bevat. In teenstelling met die eersgenoemde groepering wat hoogs virulente isolate van uiebolle bevat het, het die basale groepering isolate bevat wat meestal matig virulent was. Die inkongruensie van die VF-1α en mtKSE-datastelle met ‘n intergeen-gespasieerde (IGS) area datastel – asook die teenwoordigheid van beide PT-idiomorwe binne dieselfde isolaat by sommige isolate – het op ’n moontlike uitruiling van genetiese materiaal tussen isolate gedui. Die tweede doel van die studie was om molekulêre metodes te ontwikkel vir die identifisering van die twee hoof Focep VVGe (0425 en 0421) deur gebruik te maak van DNA-vingerafdrukke en nukleotied-gekarakteriseerde geamplifiseerde area (NKAA) merkers. Hierdie tegnieke is ontwikkel deur van die F. oxysporum-isolate van die eerste doelstelling gebruik te maak en is daarna gebruik om die frekwensie van VVG 0425 onder 88 ongekarakteriseerde F. oxysporum-isolate van uiebolle in Suid-Afrika te ondersoek. Twee gerandomiseerde geamplifiseerde polimorfiese DNS (RAPD) merkers het twee diagnostiese nukleotiedbasis-areas vir VVG 0425 gelewer, maar pogings om NKAA-merkers uit hierdie geamplifiseerde nukleotiedbasis-areas te onwikkel was onsuksesvol. In teenstelling hiermee het ‘n inter-retrotransposon geamplifiseerde polimorfisme (IRAP) vingerafdrukmetode die ontwikkeling van ‘n multipleks IR-NKAA polimerase kettingreaksiemetode moontlik gemaak wat die VVG 0421-, VVG 0425- en ELV 4-isolate as ’n groep aangedui het. Vingerafdruktoetsing en NKAA-merkertoetsing van die 88 ongekaraktariseerde F. oxysporum isolate van Suid-Afrika (65 Focep en 23 nie-patogenies) het bevestig dat VVG 0425 die hoof VVG in Suid-Afrika is wat met volwasse bolle geassosieer word, aangesien 63 van die Focep-isolate die molekulêre eienskappe van VVG 0425 gehad het. Die derde doel van die studie was om vas te stel of saad en saailinge inokulumbronne van Focep is, en of dieselfde genotipe (VVG 0425) wat op volwasse bolle dominant is, waargeneem kon word op hierdie bronne. Focep-isolate is verkry van sewe van die 13 uiesaadlotte asook van uiesaailinge wat in al vyf uiesaailingkwekerye in die Wes-Kaap versamel is. Focep-saailinginfeksie was meer as dubbel in die 14-week groeistadium as wat dit in die 6-week stadium was. Saadinfeksies deur Focep was laag, maar die saadgedraagde aard van Focep is bevestig deur aan te toon dat ’n Focep-transformant wat met ‘n groen fluoreserende proteïen geëtiketeer is, van geïnfekteerde grond na uiesaad oorgedra kon word via die uiebolle en -saadstele. Dit is dus duidelik dat kommersiële saad en saailinge as inokulumbronne van Focep dien. Die Focep-genotipes op saad en saailinge verskil egter van dié in volwasse bolle en is nie deur VVG 0425 gedomineer nie. Verder was die meeste (≤ 60%) saad- en saailingisolate matig virulent, in teenstelling met die meestal hoogs virulente isolate uit volwasse bolle.

A series of experiments were conducted to determine the potential of a new method of steam treatment for the control of soil-borne diseases, insects and weeds in sustainable crop production.  The new method involved rapidly heating a bed of prepared soil on a porous base by passing an upward flow of steam through it.  The aim was to determine whether the new method would be suitable for use in a field machine, making large-scale field steaming practically and economically viable. In the first experiments, the feasibility of the new method was tested.  It was shown that it was an effective and rapid way in which a soil bed could be steamed.  However, for some soils at, or near, permanent wilting point, the entrainment of aggregates in the steam flow was shown to be a problem.  The second series of experiments showed that the key factor determining the flow rate at which aggregate entrainment occurred was the mean aggregate diameter of the soil being treated.  The third series of experiments examined the rate at which heated soil would cool when placed in the field.  It was shown that where there was contact with unheated field soil, cooling was very rapid.  The final series of experiments investigated the effects of the new steaming method on the soil.  A three minute steaming time was used to account for the short time it had been shown some of the heated soil would remain at steam temperature when replaced in the field.  The effects of the new method, including the effectiveness of disinfection, were shown to be similar to those of a conventional steam treatment. It was concluded that the new steaming method was an effective way to steam treat soil and should be suitable for use in a field machine.

Fusarium graminearum est un champignon pathogène des plantes, responsable de la fusariose de l'épi (plus connue sous le nom de Fusarium Head Blight : FHB) sur céréales, notamment sur le blé et le maïs. En interaction avec la plante, le champignon produit des mycotoxines, parmi lesquellse le déoxynivalénol (DON), dont la finalité pour le champignon producteur est méconnue mais qui sont toxiques pour les humains et les animaux. Ainsi la qualité des grains contribue fortement aux pertes de rendement observées et les résidus contaminés restent au champ. Une première revue bibliographique (Leplat et al 2012) a mis en évidence l'importance des résidus de culture (habitat écologique) pour la survie saprophyte du champignon, pour sa reproduction sexuée et pour l'établissement de l'inoculum primaire susceptible d'infecter la prochaine culture. Une seconde revue bibliographique a souligné les lacunes en ce qui concerne le rôle que les mycotoxines pourraient jouer dans la survie de F. graminearum dans un cet habitat. L'objectif principal de cette thèse était donc de vérifier si la présence de mycotoxines dans les résidus de récolte donne un avantage compétitif à F. graminearum vis-à-vis des composantes biotiques du sol et des résidus et notamment les champignons, les bactéries, les protozoaires, les nématodes et les vers de terre. L'impact du DON sur ces différentes communautés a été évalué dans des résidus de maïs et de blé, au champ et en microcosmes, en condition de labour et de travail superficiel du sol. Le développement de la maladie et ses conséquences sur le rendement ont été observés dans l'expérience de terrain à l'Unité Expérimentale de l'INRA de Dijon.Au cours de cette étude, la survie et les dynamiques de développement de la souche modèle d'étude F. graminearum MIAE00376 et des communautés fongiques et bactériennes ont été mesurées en utilisant la réaction de polymérisation en chaîne en temps réel (Q-PCR) ainsi que par comptage sur boîtes. Dans le même temps, l'évolution des structures des communautés microbiennes a été déterminée par analyse du polymorphisme de longueur des fragments de restriction terminaux (T-RFLP). Les nématodes et les vers de terre ont été quantifiés par extraction et observations à l'œil ou a la loupe binoculaire. Le DON introduit dans le sol et les résidus a été extrait et quantifié au cours du temps par chromatographie liquide haute performance (CLHP). Des dynamiques de population de la souche MIAE00376 associée à différents microorganismes isolés de paille en décomposition et sélectionnés pour leur résistance au DON, à des bactéries fixatrice d'azote et à des Fusarium sp. appartenant au complexe fongique du FHB ont été mesurées en microcosmes de paille en présence ou non de DONLes résultats suggèrent que le DON dans les résidus de culture a une incidence sur les composantes biotiques du sol, mais l'impact dépend des communautés et de la localisation des résidus (en surface ou incorporés dans le sol). La biomasse moléculaire montre que les densités bactériennes et fongiques ont été significativement affectées par la présence de DON. La présence de DON a joué un rôle significatif sur la structure des communautés bactériennes et protozoaires, plus faible sur les communautés fongiques et nul sur les nématodes voire positif sur les vers de terre.Il est conclu que le DON est rapidement inaccessible en profondeur et un peu moins rapidement en surface (immobilisation ou dégradation), qu'il ne confère pas d'avantage compétitif au champignon producteur et que la gestion de l'habitat privilégié que constituent les résidus de culture pour F. graminearum peut être envisagée par le travail du sol en favorisant la décomposition rapide des résidus, par le labour ou l'utilisation d'organismes décomposeurs indigènes ou introduits
Fusarium graminearum is a plant pathogenic fungus, causing devastating disease “Fusarium head blight” (FHB) in cereals including wheat and maize. It also contaminates the grains with mycotoxins including deoxynivalenol (DON) which are toxic to human and animals. This disease has resulted in the serious losses in grain yield and quality. We established through a first bibliographic review that during off season fungus survives saprophytically on the crop residues (ecological habitat) and serves as primary inoculum for the next season crop. However, we noticed also that the literature was poor about the role mycotoxins could play in the establishment of F. graminearum in such a habitat. The main aim of this thesis was therefore to test whether the presence of mycotoxins in the crop residues gives an advantage to F. graminearum to survive and develop a primary inoculum in the presence of the whole soil biota including fungi, bacteria, protozoa, nematodes and earthworms. We studied the impact of DON on the soil communities in the field as well as in microcosms, in wheat as well as in maize residues under tillage and no-tillage conditions. The disease development and the yield were noted in the field experiment. Some DON resistant active fungal decomposers and nitrogen fixing bacteria were picked and the dynamics of F. graminearum was observed by accelerating decomposition of crop residue in their presence, in the presence or absence of DON.During this study, the dynamic and survival of F. graminearum and total fungal and bacterial communities were examined by using quantitative real time polymerase chain reaction (qPCR) as well as by plate counting. At the same time, the structures of microbial communities were determined by using terminal restriction fragment length polymorphism analysis (T-RFLP). The DON resistance of isolated fungal decomposers and nitrogen fixers was tested by using minimal inhibitory concentration test (MIC). Nematodes and earthworms were quantified through binocular observations. The fate of DON was determined by quantifying the mycotoxin by high performance liquid chromatography (HPLC).The results suggested that DON in crop residues showed an impact on the biotic components of the soil but the impact depended on the communities and on the location of the residues (on surface or incorporated in the soil). The molecular biomass shows that the fungal and bacterial densities were significantly affected by the presence of DON. The presence of DON played significant role on the structure of bacterial and protozoan community while the nematodes and fungal communities remained unaffected. MIC results showed that the susceptibility of some competitive fungal strains towards DON was dependent on the dose of mycotoxin. The earthworms (Lumbricus terrestris) were not affected by the presence of mycotoxin. The degradation of DON in the residues was dependent on the time, the location of residues and the soil biota. The quantification of F. graminearum suggested that the presence of DON gave no advantage in the survival and development of primary inoculum during the decomposition of crop residues in the soil. We conclude that fungal decomposers can be selected on their enzymatic potential towards organic matter more than on the DON resistance to increase the degradation of the straw left at the surface and limit the subsequent development of F. graminearum

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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior
The melon cultivation in semi-arid northeast in consecutive cycles has generated problems caused by fungi inhabitants of the soil, such as Fusarium solani and M. cannonballus. The use of resistant cultivars is an interesting measure for the management of the disease. For this reason, it is important identify sources of resistance and study their inheritance. The objectives of this work were: a) to evaluate the reaction of melon accessions to F. solani f. sp. Cucurbitae; b) to evaluate the reaction of accessions and study the inheritance of resistance of accession AC-33 to M. cannonballus. In the first experiment, we evaluated twenty-one accessions in a completely randomized design in greenhouse. Two isolates were inoculated fifteen days after sowing by the toothpick. The assessment was done Thirty days after inoculation with a scale scored from zero to four. The accessions AC-01, AC-09, AC-42, AC-45, AC-50 and the cultivar 'Doublon' are the most promising materials for use in breeding programs for resistance to F. solani or as rootstocks. In the second experiment, sixteen accessions and line OF-02 were evaluated in a completely randomized design. We used the isolated MC-16 to infestation of a mixture (1:1:1) with soil, peat, and sand previously sterilized with the addition of a concentration of 20 u.f.c./g soil. The evaluation was performed at 45 days using a rating scale (1-5). In the third experiment, we investigated the inheritance of resistance of accession AC-33 crossed with line OF-02 (susceptible). We observed variability in the germplasm investigated for reaction to the fungus. The AC-33 is highly resistant to access M. cannonballus and its resistance is controlled by a major gene with additive and dominant effects and polygenes with additive effects
O cultivo do meloeiro no semiárido nordestino em ciclos consecutivos tem gerado problemas causados por fungos habitantes do solo, como Fusarium solani e Monosporascus cannonballus. O uso de cultivares resistentes é uma medida interessante para o manejo da doença. Em razão disso, é importante que fontes de resistência sejam identificadas e se conheça a herança da resistência. Os objetivos do presente trabalho foram: a) avaliar a reação de acessos de meloeiro a F. solani f. sp. cucurbitae e b) avaliar a reação de acessos e estudar a herança da resistência do acesso AC-33 a M. cannonballus. No primeiro experimento, foram avaliados 21 acessos em um delineamento inteiramente casualizado em casa-de-vegetação. Foram inoculados dois isolados, com o método do palito, aos 15 dias após a semeadura. A avaliação dos acessos foi realizada 30 dias após a inoculação, com uma escala de 0 a 4. Os acessos AC-01, AC-09, AC-42, AC-45 e AC-50 e a cultivar ‘Doublon’ são os materiais mais promissores para uso em programas de melhoramento genético visando à resistência a F. solani ou como porta-enxertos. No segundo experimento, foram avaliados 16 acessos e a linhagem OF-02 em um delineamento inteiramente casualizado. Utilizou-se o isolado MC-16 para infestação de mistura em volume de 1:1:1 de terra, turfa e areia, previamente esterilizada com a adição de uma concentração 20 u.f.c./g de solo. A avaliação foi realizada aos 45 dias utilizando uma escala de notas (1 a 5). No terceiro experimento, investigou-se a herança da resistência do acesso AC-33 (resistente) cruzado com a linhagem OF-02 (suscetível). Observou-se a existência de variabilidade no germoplasma investigado para reação ao fungo. O acesso AC-33 é altamente resistente a M. cannonballus e sua resistência é controlada por um gene maior de efeito aditivo e dominante e poligenes de efeitos aditivos
2017-01-10

S'avaluaren 58 soques de Pseudomonas fluorescens i Pantoea agglomerans per la seva eficàcia en el biocontrol de la malaltia causada per l'oomicet Phytophthora cactorum en maduixera i pel nematode formador de gal·les Meloidogyne javanica en el portaempelt GF-677.
Es desenvolupà un mètode ex vivo d'inoculació de fulla amb l'objectiu de seleccionar soques bacterianes com a agents de control biològic de P. cactorum en maduixera. Tres soques de P. fluorescens es seleccionaren com a soques eficaces en el biocontrol del patogen en fulles i en la reducció de la malaltia en plantes de maduixera. La combinació de soques semblà millorar la consistència del biocontrol en comparació amb les soques aplicades individualment.
Tres soques de P. fluorescens es seleccionaren per la seva eficàcia en la reducció de la infecció de M. javanica en portaempelts GF-677. La combinació d'aquestes soques no incrementà l'eficàcia del biocontrol, però semblà reduir la seva variabilitat.
58 Pseudomonas fluorescens and Pantoea agglomerans strains were evaluated for their biocontrol efficacy against the oomycete Phytophthora cactorum in strawberry and the root-knot nematode Meloidogyne javanica in GF-677 rootstocks.
An ex vivo detached leaf inoculation method was developed to select bacterial strains as biological control agents of P. cactorum in strawberry. Three P. fluorescens strains were selected as effective in biocontrol of the pathogens on leaves and in disease reduction in strawberry plants. Combination of strains improved biocontrol consistency compared to strains applied individually.
Three P. fluorescens strains were selected for their efficacy in M. javanica infection reduction in GF-677 rootstocks. Combination of these strains did not increase biocontrol efficacy, but reduced its variability.

False codling moth (FCM), Thaumatotibia leucotreta is an extremely important pest of citrus in South Africa and with the shift away from the use of chemicals, alternate control options are needed. One avenue of control which has only recently been investigated against the soil-borne life stages of FCM is the use of entomopathogenic fungi (EPF). In 2009, 12 entomopathogenic fungal isolates collected from South African citrus orchards showed good control potential during laboratory conducted bioassays. The aim of this study was to further analyse the potential of these isolates through concentration-dose and exposure-time response bioassays. After initial re-screening, concentration-dose response and exposure-time response sandconidial bioassays, three isolates were identified as exhibiting the greatest control potential against FCM in soil, Metarhizium anisopliae var. anisopliae (G 11 3 L6 and FCM Ar 23 B3) and Beauveria bassiana (G Ar 17 B3). Percentage mycosis was found to be directly related to fungal concentration as well as the amount of time FCM 5th instar larvae were exposed to the fungal conidia. LC50 values for the three isolates were not greater than 1.92 x 10⁶ conidia.ml⁻ₑ and at the LC₅₀, FCM 5th instar larvae would need to be exposed to the fungus for a maximum of 13 days to ensure a high mortality level. These isolates along with two commercially available EPF products were subjected to field persistence trials whereby net bags filled with a mixture of autoclaved sand and formulated fungal product were buried in an Eastern Cape citrus orchard. The viability of each isolate was measured on a monthly basis for a period of six months. All isolates were capable of persisting in the soil for six months with the collected isolates persisting far better than the commercially used isolates. Two of the isolates, G 11 3 L6 and G Ar 17 B3, were subjected to small scale laboratory application trials. Two formulations were investigated at two concentrations. For each isolate, each formulation and each concentration, FCM 5th instar larvae were applied and allowed to burrow into the soil to pupate before fungal application or after fungal application. Contact between fungi and FCM host is essential as, in contrast to pre-larval treatments, percentage mortality in post-larval treatments was low for both formulations and both isolates. For isolate G Ar 17 B3, a conidial suspension applied as a spray at a concentration of 1 x 10⁷ conidia.ml⁻ₑ obtained the highest percentage mortality (80 %). For isolate G 11 3 L6 however, both formulations performed equally well at a high, 1 x10⁷ conidia.ml⁻ₑ concentration (conidial suspension: 60 %; granular: 65 %) The results obtained thus far are promising for the control of FCM in citrus, but if these EPFs are to successfully integrate into current FCM control practices more research, some of which is discussed, is essential

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The cowpea (Vigna unguiculata L.) is one of the main crops in the Northeast of Brazil especially for the small farmers. The Fusarium wilt and Rhizoctonia canker caused by Fusarium oxysporum f.sp. tracheiphilum and Rhizoctonia solani, respectively are the cowpea diseases showing more frequency and intensity in the Northeast of Brazil. This work aimed to evaluate the natural suppressiveness of 66 soils of this region to the Fusarium wilt and Rhizoctonia canker, and to analyze the physical, chemical and biological characteristics of this soils associated with disease suppressiveness or conducivity. The evaluated soils were grouped from highly suppressive to highly conducive in relation to Fusarium wilt and Rhizoctonia canker severities. The main variables involved in Fusarium wilt suppressiveness were high levels of phosphorus and potassium, basal respiration (CO2 evolution) and indexes of microbial diversity and equitability. For Rhizoctonia canker important correlations were determined with levels of phosphorus, potassium and sodium, basal respiration and enzymatic activity of fluorescein diacetate. There was no correlation between physical factors and suppressiveness to Fusarium wilt, but it was possible to correlate the levels of sand, clay and silt with suppressiveness and/or conducivity of Rhizoctonia canker. Three soils previously classified as highly supressive to Rhizoctonia canker were evaluated in relation to eight strains and three inoculum densities of R. solani. There was significant difference among soils and strains in relation to levels of disease severity. In the three soils the severity levels induced by the strain CMM-1053 were similar to those observed in former studies. Most of the strains showed different behavior in relation to soils, except for CMM-1064 and CMM-1066. There was significant difference among disease severity levels and different inoculum densities. The three soils presented good stability in relation to the different R. solani strains, but the inoculum density may be a limiting factor in the implementation of the natural soil suppressiveness or the supressivity induction in conducive soils.
O caupi (Vigna unguiculata L.) é uma das culturas mais importantes da região Nordeste do Brasil, principalmente na economia de pequenos produtores rurais. A murcha-de-fusário e a rizoctoniose, causadas pelos fungos Fusarium oxysporum f.sp. tracheiphilum e Rhizoctonia solani, respectivamente, são as doenças mais freqüentes e de maior intensidade em caupi no Nordeste brasileiro. Esta tese teve como objetivos avaliar a supressividade natural de 66 solos do Nordeste brasileiro à murcha-de-fusário e rizotoniose do caupi, analisar as características físicas, químicas e biológicas dos solos associadas com a supressividade ou conducividade às doenças, bem como avaliar a estabilidade da supressividade de três solos à rizoctonose do caupi, considerando diferentes isolados e densidades de inóculo de R. solani. Em relação à severidade da murcha-de-fusário e da rizoctoniose do caupi, os solos avaliados foram agrupados desde fortemente supressivos a altamente conducivos. As principais variáveis envolvidas na supressividade da murcha-de-fusário foram elevados teores de fósforo e potássio, respiração basal (CO2 evoluído) e os índices de diversidade e eqüitabilidade microbiana. Para a rizoctoniose, foram determinadas correlações importantes com os níveis de fósforo, potássio e sódio, respiração basal e atividade enzimática de diacetato de fluoresceína. Não foram correlacionados fatores físicos com a supressividade à murcha-de-fusário, porém foi possível correlacionar os teores de areia, argila e silte com asupressividade e/ou conducividade da rizoctoniose. Três solos classificados como fortemente supressivos à rizoctoniose foram avaliados em relação a oito isolados e três densidades de inoculo de R. solani. Houve diferença significativa entre os solos e os isolados quanto aos níveis de severidade da doença. Nos três solos os níveis de severidade induzidos pelo isolado CMM-1053 foram similares aos verificados nos estudos prévios. A maioria dos isolados apresentou comportamentos diferente em função dos solos, com exceção dos isolados CMM-1064 e CMM-1066. Foi verificada diferença significativa entre os níveis de severidade da doença e as diferentes densidades de inoculo. Os três solos evidenciaram estabilidade em relação aos diferentes isolados de R. solani, porém a densidade de inóculo pode ser um fator limitante na implementação da supressividade natural dos solos ou da indução da supressividade em solos conducivos.

The overall goal of this research was to determine what, if any, role grapevine rhizosphere bacteria play in the differing susceptibilities of New Zealand grown rootstocks to Cylindrocarpon black foot disease. The size and diversity of bacterial populations associated with the rhizospheres of grapevine rootstocks: 101-14, 5C, Schwarzmann and Riparia Gloire were evaluated. Dilution plating showed that total bacterial (P=0.012, P=0.005 for NA and KB, respectively) and fluorescent Pseudomonad (P=0.035) rhizosphere counts differed between rhizosphere and bulk soils but did not correlate with the differing susceptibilities of the rootstock varieties to black foot. No varietal differences were found for spore forming bacteria (P=0.201). SSCP banding patterns showed that species diversity was similar for most rootstocks, but that there were some differences in the composition of bacterial populations, probably attributable to vigour. Some functional characteristics of the bacteria isolated from the rhizospheres of the most and least susceptible rootstock varieties were assessed to investigate their potential to suppress the pathogen. In dual culture, bacteria from Riparia Gloire, 101-14 and the control soil all had little ability to antagonise Cylindrocarpon destructans. However, they differed in their degrees of activity for glucanase (P=0.000), protease (P=0.001) and siderophores (P=0.000). In all tests, bacterial isolates from the rhizosphere of 101-14 had the largest number of active isolates (P≤0.002); however, those from Riparia Gloire had the greatest degree of positive responses for the glucanase and siderophore assays. Bacterial isolates from the control soil produced few glucanases and no siderophores, but had the highest degree of protease activity. Bands excised and sequenced from SSCP gels frequently matched to other ‘uncultured bacteria’ in GenBank, as well as to other bacterial phyla, classes and genera commonly isolated from soil and sediment samples. These included members of the Firmicutes, Proteobacteria (α, δ, γ), Verrucomicrobia, Acidobacteria and Chromatiales. The pathogenicity of C. destructans and Fusarium oxysporum was investigated by inoculating soil containing wounded ungrafted rootstocks of 101-14, 5C, Schwarzmann and Riparia Gloire. Results indicated that F. oxysporum might be a more aggressive pathogen than C. destructans. Inoculation with F. oxysporum or C. destructans increased disease severity, P=0.018 and P=0.056, respectively at 0 cm. Rootstock variety influenced disease severity caused by C. destructans (P<0.001) and F. oxysporum (P=0.090), with rootstocks 101-14 and 5C being most susceptible to C. destructans, and Riparia Gloire and Schwarzmann most susceptible to F. oxysporum. There was also an indication that inoculation with one pathogen increased plant susceptibility to the other, with increased F. oxysporum infection in the C. destructans inoculated treatments of Riparia Gloire and Schwarzmann (P<0.05). The effect of carbohydrate stress (leaf trimming) and inoculation on C. destructans disease severity, incidence, and rootstock rhizosphere bacterial populations was evaluated by inoculating the soil containing one year old plants of Sauvignon Blanc scion wood grafted to rootstocks 101-14 and Schwarzmann. Disease severity and incidence was similar for both Schwarzmann (8.4% and 29.3%, respectively) and 101-14 (14.9% and 31.0%, respectively). When data for the moderate and no stress treatments were combined, because their effects were similar, the disease severity was significantly higher for the highly stressed plants(P=0.043). Stress did not influence disease incidence (P=0.551). Infection occurred in the non-inoculated plants, but disease severity was higher in the plants inoculated with C. destructans than those that were not. Root dry weight of highly stressed plants was lower than in both the moderately stressed (P=0.000) and unstressed plants (P=0.003). An interaction between inoculation and stress (P=0.031) showed that inoculated and highly stressed plants had the lowest root dry weight but there was no effect of rootstocks (P=0.062). There was no significant effect of carbohydrate stress (P=0.259) or inoculation (P=0.885) on shoot dry weight. SSCP banding patterns showed that bacterial diversity was generally similar between rootstocks, but stress and inoculation altered rhizosphere bacterial communities. This study has demonstrated that functionality of grapevine rhizosphere bacteria do differ between grapevine rootstock varieties that have different susceptibilities to black foot disease, but that this role needs to be further investigated if more accurate and practically relevant conclusions are to be drawn.

Thesis (M.Sc. (Agriculture)) --University of Limpopo, 2018
Tomato is second most cultivated crop globally and in South Africa it is planted by both commercial and smallholder farmers. However, the crop is susceptible to a number of diseases including those caused by fungal pathogens. Fusarium wilt caused by Fusarium oxysporum f. sp. lycopersici and seedling damping-off caused by Rhizoctonia solani, are known to cause serious yield loss in tomato production. Their management is mainly based on the application of synthetic fungicides and cultural practices. However, both methods have limitations which result in their inefficiency. Synthetic fungicides also have negative impact on the environment and human health. The ability of fungal pathogens to develop resistance to fungicides has also resulted in their reduced application. These challenges have led to a need to identify novel methods using plant extracts and biological control agents which can be used to manage these diseases. The objectives of this study were therefore to, firstly determine the efficacy of both plant extracts on mycelial growth of F. oxysporum f. sp. lycopersici and R. solani under laboratory conditions and secondly, to evaluate the effectiveness of both plant extracts as well as antagonistic fungi Trichoderma harzianum against Fusarium wilt and damping-off of tomato under greenhouse conditions. Food poisoning assay was used to investigate the efficacy of M. burkeana and M. oleifera extracts in vitro. Six (0, 2, 4, 6, 8, 10 g/ml) treatments were arranged in a completely randomised design and replicated four times. After 7 days of incubation at 25 °C, radial growth colony was measured. For the greenhouse xp im nt, Fusa ium wilt was t st d on cv. ‘HTX14’ as th most susc ptibl cultiva whilst seedling damping-off was t st d on cv. ‘Mon y-make ’. Aqu ous xt acts were prepared by decocting different concentrations of M. burkeana (4, 6, 8 g/ml) xiv and M. oleifera (2, 4 and 6 g/ml) in 100 ml of distilled water at 100 °C for 15 minutes then left to cool before filtering and applying as a treatment. Trichoderma harzianum as a treatment was applied 7 days after inoculating the soil-borne pathogens. In-vitro M. burkeana treatments concentrations had the highest mycelia growth suppression against both F. oxysporum f. sp. lycopersici at 10 g/ml (76 %) whilst suppression on R. solani was at 8 g/ml (71 %) relative to control. Moringa oleifera xt acts’ highest pathogen suppression for both F. oxysporum f. sp. lycopersici and R. solani were respectively 35 % and 60 % relative to control at concentration 6 g/ml. Under greenhouse conditions shoot disease severity had highest suppression at 0.6 g/ml of M. burkeana and 0.4 g/ml of M. oleifera treatment concentrations resulting to 32 and 49 % relative to control. Whereas, treatment 0.8 g/ml of M. burkeana and 0.4 g/ml of M. oleifera suppressed stem and root discoloration by 39 and 54 % respectively. Trichoderma harzianum significantly (P ≤ 0.05) reduced shoot severity and root and stem discolouration contributing the highest suppression of 49 % relative to control. In damping-off treatments, both plant extracts and T. harzianum also significantly duc d (P ≤ 0.05) pre- and post-emergence damping-off incidence with M.burkeana recording the highest suppression at 78 % followed by M. oleifera at 64 %. Trichoderma harzianum reduced incidence of damping-off by 60 % relative to untreated control on both M. burkeana and M. oleifera experiments. The results of this study showed that M. burkeana, M. oleifera extracts and T. harzianum can be highly suppressive to both tested plant diseases. However, further studies should be conducted to determine their mode of action, application method and their effect on other soil microorganisms. Keywords: Damping-off, Fusarium wilt, Plant extracts, T. harzianum, Tomato plant

Differences in pea (Pisum sativum L.) and chickpea (Cicer arietinum L.) microbial compatibility and/ or their associated farming practices may influence root fungi of the following crop and affect the yield. The main objective of this research was to explain the difference in durum wheat (Triticum turgidum L.) yield the year after pea and chickpea crops through changes in the functional diversity of wheat root fungi. The effect of fungicides used on chickpea on the root fungi of a following durum wheat crop was studied using plate culture and pyrosequencing. Pyrosequencing detected more Fusarium spp. in the roots of durum wheat after fungicide-treated chickpea than in non-fungicide treated chickpea. Plate culture revealed that the functional groups of fungi responded differently to fungicide use in the field but the effect on total community was non-significant. Highly virulent pathogens were not affected, but antagonists were suppressed. More fungal antagonists were detected after the chickpea CDC Luna than CDC Vanguard. Fungal species responded differently to the use of fungicides in vitro, but the aggregate inhibition effect on antagonists and highly virulent pathogens was similar. The effect of chickpea vs. pea previous crop and different chickpea termination times on root fungi of a following durum wheat crop was studied. The abundance of Fusarium spp. increased after cultivation of both cultivars of chickpea as compared to pea according to pyrosequencing and was negatively correlated with durum yield. Plate culture analysis revealed that fungal antagonists were more prevalent after pea than both cultivars of chickpea and chickpea CDC Vanguard increased the abundance of highly virulent pathogens. The abundance of highly virulent pathogens in durum wheat roots was negatively correlated to durum yield. Early termination of chickpea did not change the community of culturable fungi in the roots of a following durum crop. It is noteworthy that Fusarium redolens was identified for the first time in Saskatchewan and its pathogenicity was confirmed on durum wheat, pea and chickpea. The classical method of root disease diagnostics in cereals is based on the examination of the subcrown internode. I evaluated the method by comparing the fungal communities associated with different subterranean organs of durum wheat. The fungal community of the subcrown internode was different from that of roots and crown, suggesting cautious use of this method.

碩士
亞洲大學
創意商品設計學系
104
Soil-borne diseases is a problem farming must face. To maintain agricultural products under the premise of quality, agricultural facilities start being applied. In cultivation of agricultural facilities to guard against climate change, natural disasters and pests, and effectively manage the environment for crop growth through automation. Modern agriculture mostly uses pesticides and other chemical additives to resist and suppress a variety of pests and diseases. In addition to the efficacy of being easily absorbed by soil particles, if it fail to be managed and used properly, it would not only cause permanent damage to the soil but also indirectly threaten other creatures such as bees living environment. The deduction of bees has an impact on the opportunities for bees to pollinate plants. Therefore, a vicious cycle poses a threat to biology and environment. Actually we may attribute the disasters to man-made factors. The target of this research is to convert a conversational microwave oven into a movable one and to record the speed and wattage microwave machine at the time of microwave soil sterilization that measured by the heat treatment process. After comparing soil depth with temperature through the data, soil insects harmful bacteria with rising temperature of the elimination of levels. We expect to apply microwave technology in the prevention of job processing of agricultural land management. This study of literature case in 1946, Raytheon Company (Raytheon) radar engineer Percy Spencer, the test generating microwaves 'magnetron', the unexpected discovery pocket candy dissolved, presumably wave having a heating function, leading to the invention of the microwave oven. In this study, case studies, examples of microwave heating of the soil, using the energy directly into the technology. Of "medium" for disinfection, for the effects of combustion or decomposition of the constitution concerns the impact, which save space and time and energy purposes, sterile and achieve consistent operation of the production line. In this study, experimental analysis of the scientific research methods of experiment. Starting in 1920, created by the mathematician Ronald Fisher. As an experimental method which operates under the control of the academic research environment variables to test the hypothesis. As deemed appropriateness, the use of methods necessary for the performance of this study. Confirmed by this study that the conclusions are as follows: Conclusions first, In order to reduce the harm effects of pesticides on soil environment, the present study the experiment proved that due to the soil via microwave for 2 to 3 minutes, can lower soil depths of 10 cm reached a temperature of Celsius 50 to 55 degrees, by the present study demonstrated that temperature can kill In addition to pathogens and nematode populations. Use the heat treatments characteristics for reaching and solving the impact of pet disaster. Conclusion second, the wattage of the microwave heat treatment temperature level of influence, the experiment proved that the present study, increasing the relative humidity of the soil, can reach temperature microwave heat treatment to enhance the soil. Conclusion third, after the experiment that, to maintain the soil temperature time up sterilization effect, can be used with spray and soil vapor after forming of silicon production industry microwave.

Banana production is under threat globally from Fusarium wilt, caused by the soil-borne pathogen Fusarium oxysporum f.sp. cubense (Foc) Tropical Race 4. Foc Tropical Race 4 was first discovered in Australia’s primary banana production region of North Queensland in 2015 and has slowly spread since. To date there are no agronomically suitable disease resistant varieties and no known treatments, so quarantine has been the primary management tool, though it is not a permanent solution. The objective of this thesis was to increase understanding of how soil physico-chemical characteristics affect the severity of Fusarium wilt of banana.

Seedling blight of maize has significantly influenced field crop stands and seedling vigour over various localities and seasons. The extent of the problem is influenced by a number of factors which includes soil temperature (generally below 13 °C), waterlogged soils, inadequate fertilization, herbicide damage and fungal pathogens. The fungi generally causing seedling damping off are often involved in a complex and succession over time varying in importance depending on the field circumstances at a given time. These generally include the Pythium spp., Rhizoctonia spp. and various Fusarium spp. These have been recorded in a number of studies conducted by local researchers in the late 1980’s and early 1990’s on sorghum but to a lesser degree on maize. Uncertainty regarding the status of the etiology of maize seedling blights as maize production practices have changed dramatically in the last 10 years with increased plant populations, reduced tillage, increased crop rotation options and new short season maize hybrids. It is therefore essential to determine the present status of seedling blights in South Africa to confirm the necessity of fungicide seed treatments to ensure adequate plant densities and seedling vigour. Cob and tassel smut caused by Sphacelotheca reiliana is a disease of maize that was a problem in the 1970’s. Due to improved fertilisation, fungicide seed treatments and hybrid resistance this disease was reduced to such levels that the disease was only found to occur on research farms where seedlings were inoculated. Since 2007, the disease was reported to reach epidemic proportions on the heavy clay soils in the Standerton area. This disease has since spread over the last seven seasons to a range including northern KwaZulu/Natal, namely as far as Underberg/Swartberg, the Witbank, Ermelo, Middelburg and Delmas area in Mpumalanga and to Harrismith in the eastern Free State maize production area. This may be due to susceptible hybrids coming onto the local market or the inability of traditional fungicide seed treatments to contain infection. New and unregistered seed treatments available will be tested for their ability to control cob and tassel smut in two fields over two seasons. The aims of this dissertation were to determine the extent of the seedling blight problem in commercial fields throughout the maize industry. To determine the efficacy of fungicide seed treatments for the control of maize seedling blights using both field and greenhouse studies, and to determine the efficacy of fungicide seed treatments for the control of cob and tassel smut of maize in field trials. A total of 101 localities were sampled throughout the maize producing region of South Africa with root discolouration varying from 0 to 90 % root discolouration. Seventy different fungal species were isolated from the maize seedlings roots which include species such as Aspergillus, Clonostachus, Fusarium, Trichoderma and Penicillium. The most commonly isolated fungi which included Aspergillus niger, Fusarium solani, Fusarium verticillioides and Fusarium oxysporum were evaluated in glasshouse studies to determine their pathogenicity. Pathogenicity differed between isolates of the same fungal species, which were collected from different geographical regions, in the glasshouse studies. Field trials for seedling blight disease showed significant differences between the localities (P < 0.001) the trials were planted at, and between seed treatments. Significant season (P < 0.001) and locality (P < 0.05) differences were also found for cob and tassel smut trials planted at Potchefstroom, North-West province and Greytown, KwaZulu/Natal Province respectively. Fungicide seed treatments also showed significant differences for cob and tassel smut regarding plants infected (P < 0.001) and yield loss (P < 0.05). Overall seed treatments can be seen as an effective controlling agent for the control of seed- and soil-borne fungi on maize.
MSc (Environmental Sciences), North-West University, Potchefstroom Campus, 2015

Thesis (M.A. Agricultural Management (Plant Production)) -- University of Limpopo, 2015
Tomato (Solanum lycopersicum L.) production had been ranked as the most important commodity in terms of job and wealth creation within the auspices of the National Development Plan (NDP) framework in Limpopo Province. However, soil-borne diseases including plant-parasitic nematodes preclude the successful monoculturing of this commodity and therefore inducing instability in job creation. Generally, after growing a tomato crop for one season in commercial tomato-production systems, the land is being fallowed for 3-5 years under natural grasses. Attempts are being initiated to ensure that during the 3-5 years the land be occupied by an economic alternative crop in order to level off job instability as broadly articulated in the NDP framework. The production of sweet stem sorghum (Sorghum bicolor L.) for ethanol production during the 3-5 years fallowing period could potentially be attractive to commercial tomato-producing famers. Preliminary agronomic evaluations demonstrated that sweet stem sorghum var. ndendane-X1 had attributes to fulfil the identified need. However, the degree of nematode resistance of the variety to Meloidogyne incognita race 2 and M. javanica, which are dominant in Limpopo Province, along with the compatibility of var. ndendane-X1 to phytonematicides used in tomato production had not been documented. The objectives of the study were, therefore, to determine whether sweet stem sorghum var. ndendane-X1: (1) had any degree of nematode resistance to M. incognita race 2 under both greenhouse and microplot conditions, (2) had any degree of nematode resistance to M. javanica under greenhouse conditions, and (3) would be compatible with phytonematicides used in suppression of population densities of xiv Meloidogyne species in tomato production under field conditions. In the greenhouse trials, seeds were sown in 20-cm-diameter plastic pots and each seedling inoculated with 0, 600, 1 000, 1 400, 1 800 and 2 200 eggs and second-stage juveniles (J2s) of M. incognita race 2 or M. javanica. Treatments were arranged in a randomised complete block design (RCBD), with 10 replicates (n = 60). In the microplot trial, seeds were sown in 30-cm-diameter plastic pots and buried 75% deep in a 0.30-m intra-row and 0.25-m inter-row spacing. Treatments, namely, 0, 200, 600, 1 000, 1 400, 1 800 and 2 200 J2s of M. incognita race 2 were arranged in RCBD, with 14 replications (n = 98). In a Meloidogyne-infested field trial, seeds were sown at 0.2-m inter-row and 0.3-m intra-row spacing, with treatments 0, 2, 4, 6, 8 and 10 g nemafric-BG phytonematicide/plant, arranged in RCBD, with 13 replications (n = 78). The degree of nematode resistance was measured using host-status and host-sensitivity, which provide information on reproduction of the target nematode and plant damage due to nematode infection, respectively. Nematode reproduction was measured through the reproductive factor (RF), which is a proportion of final nematode population density (Pf) to initial nematode population density (Pi), summarised as RF = Pf/Pi. In all nematode resistance trials, RF was equivalent to zero, which implied that var. ndendane-X1 was a non-host to both M. incognita race 2 and M. javanica. Additionally, in both greenhouse and microplot trials, sweet stem sorghum var. ndendane-X1 did not suffer any significant damage due to infection by Meloidogyne species. Using nematode-plant relation concepts, sweet stem sorghum var. ndendane-X1 was resistant to M. incognita race 2 and M. javanica under greenhouse and microplot conditions. Under field conditions, nemafric-BG phytonematicide reduced eggs and J2s of Meloidogyne species in root and soil samples xv by 76-85% and 24-65%, respectively, without nematode effect on plant growth, suggesting that nemafric-BG could be integrated with nematode resistance in var. ndendane-X1 to manage nematode population densities. In conclusion, pilot projects where sweet stem sorghum var. ndendane-X1 could be used during the 3-5 years fallowing period in a tomato-sweet stem sorghum crop rotation system should be established to assess: (i) the economics of the proposed cropping system, (ii) the effect of the cropping system on soil-borne diseases, including plant-parasitic nematodes, and (iii) the effect of the cropping system on soil health.

This research has examined the effects of soil compaction, and common agricultural management strategies used to overcome soil compaction, on soil bacterial and fungal activity and diversity. Soil microbial communities, bacteria and fungi in particular, play essential roles in the maintenance of soil health, where high soil microbial diversity might strongly contribute to the natural disease suppression. The activity and diversity of soil microbial communities is, however, strongly dependent on other soil characteristics, especially soil physical parameters. Soil compaction indicated by increased bulk density is the most soil physical parameter that directly modifies soil environment where crops and diseases exist in, but it might also indirectly cause more disease by affecting the composition of soil microbial communities in the soil. Many strategies have been used to attempt to overcome compaction in temperate environments, but they have been poorly studied/less successful in tropical and subtropical environments. This thesis, therefore, looks at the impact of compaction, and methods used to reduce compaction, on the soil microbial profile, and its capacity to resist introduced diseases.

Resting spores are important for the spread and survival of Spongospora subterranea, the causative agent of potato powdery scab and root disease. However, resting spores must germinate and release zoospores (the infective agents) to cause infection. Understanding of the germination process and factors is, therefore, important. The knowledge can potentially have implications for resting spore-inoculum management and Spongospora disease control. The biology and ecology of S. subterranea during resting spore germination is not well understood. Knowledge of factors influencing resting spore germination have been limited. This thesis studied the properties of S. subterranea resting spores and investigated some chemical factors stimulating resting spore germination. The role of germination-chemical stimulants was also examined. This study used a combination of tomato-plant and zoospore in vitro bioassays, light microscopy, a targeted hydrophilic interaction ultra-high performance liquid chromatography–mass spectrometry metabolomic approach, and a greenhouse chemical-soil treatment study. Some of S. subterranea resting spores exhibited dormant spore characteristics, which required specific stimuli to germinate. Although a proportion retains constitutive dormancy characteristics, chemical germination-stimulants were found in potato root exudates and in Hoagland’s solution. The low-molecular weight organic amino acids – tyramine and L-glutamine – and sugars – cellobiose and L-rhamnose – compounds stimulated resting spore germination at 0.1 mg/ml solution. The release of these compounds in potato roots were influenced by the plant’s physiology and growth conditions. Hoagland’s solution contains Iron-EDTA, which stimulated resting spore germination. Germination stimulant Fe-EDTA, in Hoagland’s solution, in the presence a susceptible host plant, enhanced root infection. However, Fe-EDTA and Hoagland’s solution added into S. subterranea-infested soil, a month prior to planting, reduced pathogen inoculum DNA levels in the soil. Further field studies underpinning the use of germination stimulant compounds could lead to a novel, safe and sustainable chemical approach for the management of S. subterranea inoculum in the soil and thus will augment other Spongospora disease control measures. This thesis advances our understanding of S. subterranea biology and chemical ecology during resting spore germination. Substantial development is now beginning to be made in the facet of resting spore germination biology and chemical ecology, which are important aspects of Spongospora disease epidemiology and disease control development. There remains much to be learned, but the knowledge presented here will encourage additional studies and research efforts. Powdery scab, Spongospora root infection, plasmodiophorid, chemical ecology, potato metabolomics, HILIC UHPLC-MS, resting spore germination, spore dormancy.