The anthracnose pathogen, Colletotrichum gloeosporioides (Penz.) Penz. & Sacc., is a major cause of disease in the avocado industry, causing significant economic losses, and infects all cultivars. In South Africa, Fuerte and Hass varieties are the most widely grown. Identification of genes differentially expressed in avocado during infection with the fungus represents an important step towards understanding the plant’s defence responses and would assist in designing appropriate intervention strategies. In this study, 454 sequencing and analysis of the transcriptome of infected Fuerte avocado fruits were performed using the Roche 454 GS FLX Titanium platform. cDNA libraries enriched for differentially expressed genes were constructed from unharvested and harvested avocado fruit tissues collected after 1, 4 and 24 h post-infection and after 3, 4, 5 and 7 day post-infection, then sequenced.The expression profiles of the genes expressed were measured by a hierarchical clustering algorithm.Subsequently, quantitative real-time PCR was employed to measure the expression of some candidate resistance genes to anthracnose disease and to validate the sequencing results. The single sequencing run produced 215 781 reads from the transcriptome. A total of 70.6 MB of sequence data was generated and subjected to BLAST searches of which about 1500 genes encoding proteins predicted to function in signal transduction, transcriptional control, metabolism, defence, stress response, transportation processes and some genes with unknown functions were identified. The expression profiles studies showed that many expressed genes were either up or down regulated after infection in avocado fruits when compared to the uninfected sample. Salicylic acid and ethylene were identified to be involved in the signalling networks activated in avocado fruit during C. gloeosporioides infection. This study showed that avocado is able to respond to C. gloeosporioides infection by exhibiting a sophisticated molecular system for pathogen recognition and by activating structural and biochemical defence mechanisms.

L'anthracnose constitue pour plusieurs productions végétales une maladie importante qui engendre des pertes post récoltes considérables. Colletotrichum musae est le responsable de cette maladie chez la banane dessert. L'utilisation d'antimicrobiens naturels comme le système lactoperoxydase (LPS) représente une voie naturelle de lutte intéressante contre l'anthracnose. Dans cette étude, nous avons amélioré le LPS en ajoutant de l'iode dans le système existant ou en substituant le thiocyanate par de l'iode. La substitution du thiocyanate à l'iode a permis d'avoir un effet fongicide du LPS. De plus, d'autres substances comme la lactoferrine, le Bioxeda® et l'huile de Neem ont été étudiées pour leur effet antifongique. Les deux dernières substances ont donné une inhibition supérieure respectivement à 90% et à 40%., tandis qu'il n'y a eu aucun effet de la lactoferrine

<p>Since the beginning of mycology, studies of species concept in fungi have been mainly based on morphology, partially due to the history of mycology as part of botany. Current advances in biochemical and molecular research have provided mycologists with powerful tools that can be used for delineation of fungal taxa. Recently, an integrated approach to fungal taxonomy involving both morphological and molecular traits has found a wide application for identification of species, especially in anamorphic (mitosporic) fungi.</p><p>In this thesis, I have tried to use this approach for identification of species units in two rather unrelated groups of organisms. One of the case studies concerned <i>Colletotrichum acutatum</i> – a worldwide economically important plant pathogenic anamorphic fungus, which is exhibiting a high level of variation in both morphological and molecular features. This fungus has been intensively studied during the past decades, and several attempts have been made to find reliable markers to separate it from other closely related species of <i>Colletotrichum</i>. The second case studied in this thesis was <i>Mycelia Sterilia</i> – an artificial group of fungi, which are deficient in production of spores of any kind, therefore lacking the main morphological feature used for assigning them to any certain fungal taxon below class level. Due to this peculiarity, <i>Mycelia Sterilia</i> have usually been neglected, and currently there is no working species concept applicable to these fungi. </p><p>In this work, I have tried to clarify the relationships among <i>C. acutatum</i> and several other anamorphic (<i>C. gloeosporioides</i> and <i>C. fructigenum</i>) and teleomorphic (<i>Glomerella acutata</i>, <i>G. cingulata</i> and <i>G. miyabeana</i>) taxa that are closely related to each other. For this purpose, examination of morphological traits was employed in combination with comparison of DNA sequencing data from three loci and subsequent phylogenetic analysis. As a result, re-description of <i>C. acutatum</i> and separation of (at least) two new species was proposed.</p><p>For studies of <i>Mycelia Sterilia</i>, a large collection of sterile strains was screened in search for biologically interesting organisms. One novel pathogen has been found, and two plant growth promoting strains with antifungal properties were selected. Attempt for tentative identification of those fungi was made based on their morphological, physiological and molecular features. Sequencing of several genes and spacers of the ribosomal DNA array revealed that the plant pathogenic strain is closely related to the teleomorphic basidiomycete genus <i>Campanella</i>, and plant growth-promoting isolates were identified as belonging to the anamorphic ascomycete genus <i>Phoma</i>. However, assigning the sterile strains to any existing species was not possible.</p><p>The main conclusion of the thesis is that species in anamorphic fungi should be defined based on a combination of morphological and molecular methods, both equally important, involving as many aspects of fungal biology as is possible at our current state of knowledge. </p>

Lipoxygenase (LOX)-derived oxylipins are known to play critical roles in defense against herbivores and pathogens. The objective of this study was to determine the biochemical, molecular and physiological roles of a specific maize lipoxygenase gene, ZmLOX10, with special emphasis on LOX10-derived oxylipins in plant-insect and plant-pathogen interactions. To achieve this goal, independent mutant alleles were generated and genetically advanced to create near-isogenic mutant and wild-type lines suitable for functional analysis. Here we provide genetic evidence that LOX10 is the sole LOX isoform in maize required for the biosynthesis of green leafy volatiles (GLV) in leaves and show that LOX10- mediated GLVs play a significant role in direct and indirect defense responses to insects through their regulation of jasmonic acid and volatile organic compound production. Contrary to the defensive role of LOX10 in plant-insect interactions, tests for susceptibility to fungal pathogens suggest that LOX10-mediated GLVs may contribute to the development of disease symptoms to the economically important maize pathogens, Aspergillus flavus and Colletotrichum graminicola. Specifically, LOX10-derived GLVs may facilitate aflatoxin accumulation in response to A. flavus infection and may play a positive role in anthracnose leaf blight and stalk rot caused by C. graminicola. Collectively, our results suggest that metabolites derived from GLV-regulated pathways have a significant impact on molecular plant-herbivore and plant-pathogen interactions.

Since the beginning of mycology, studies of species concept in fungi have been mainly based on morphology, partially due to the history of mycology as part of botany. Current advances in biochemical and molecular research have provided mycologists with powerful tools that can be used for delineation of fungal taxa. Recently, an integrated approach to fungal taxonomy involving both morphological and molecular traits has found a wide application for identification of species, especially in anamorphic (mitosporic) fungi. In this thesis, I have tried to use this approach for identification of species units in two rather unrelated groups of organisms. One of the case studies concerned Colletotrichum acutatum – a worldwide economically important plant pathogenic anamorphic fungus, which is exhibiting a high level of variation in both morphological and molecular features. This fungus has been intensively studied during the past decades, and several attempts have been made to find reliable markers to separate it from other closely related species of Colletotrichum. The second case studied in this thesis was Mycelia Sterilia – an artificial group of fungi, which are deficient in production of spores of any kind, therefore lacking the main morphological feature used for assigning them to any certain fungal taxon below class level. Due to this peculiarity, Mycelia Sterilia have usually been neglected, and currently there is no working species concept applicable to these fungi. In this work, I have tried to clarify the relationships among C. acutatum and several other anamorphic (C. gloeosporioides and C. fructigenum) and teleomorphic (Glomerella acutata, G. cingulata and G. miyabeana) taxa that are closely related to each other. For this purpose, examination of morphological traits was employed in combination with comparison of DNA sequencing data from three loci and subsequent phylogenetic analysis. As a result, re-description of C. acutatum and separation of (at least) two new species was proposed. For studies of Mycelia Sterilia, a large collection of sterile strains was screened in search for biologically interesting organisms. One novel pathogen has been found, and two plant growth promoting strains with antifungal properties were selected. Attempt for tentative identification of those fungi was made based on their morphological, physiological and molecular features. Sequencing of several genes and spacers of the ribosomal DNA array revealed that the plant pathogenic strain is closely related to the teleomorphic basidiomycete genus Campanella, and plant growth-promoting isolates were identified as belonging to the anamorphic ascomycete genus Phoma. However, assigning the sterile strains to any existing species was not possible. The main conclusion of the thesis is that species in anamorphic fungi should be defined based on a combination of morphological and molecular methods, both equally important, involving as many aspects of fungal biology as is possible at our current state of knowledge.

Colleotrichum graminicola is the causal agent of anthracnose stalk rot, and is one of the most common and aggressive pathogens of maize. The goal of my Ph.D. project was to contribute to a better understanding of the biology of the interaction between C. graminicola and its host. C. graminicola produces two type of asexual spores: one is produced on the surface of infected tissues and is thought to be involved in the spread of the disease in the field. The second type of spore, oval in shape, is produced inside the infected plant tissues, and was believed to be involved in the movement of the pathogen inside the plant tissues via the vascular system. I tested this hypothesis with both cytological and molecular approaches. I used strains of C. graminicola expressing green fluorescent proteins (GFP) to inoculate wounded plants, and followed the development of the pathogen over time. This study revealed that C. graminicola is not a vascular pathogen. C. graminicola primarily moved through the rind and vascular fibers. Oval spores were produced in colonized parenchyma cells and remained dormant, and did not appear to be involved in the movement of the pathogen, at least during the early stages of the disease development. I also studied pathogen ingress in the absence of a wound. I inoculated unwounded plants with the GFP expressing strains. C. graminicola efficiently colonized the epidermis and, given enough time, penetrated and colonized the deeper parenchyma tissues, after first moving through the fibers. To further test the role of sporulation in colonization of maize tissues, I used targeted mutagenesis to disrupt a major gene known to regulate sporulation and vegetative growth in several other fungi. The gene Cgg1, orthologue of the A. nidulans fadA, was disrupted using the split marker method. The Cgg1 mutants were less pathogenic than the wildtype to wounded plants. This was associated with an apparent increase in production of spores and primary infection hyphae. This suggests that Cgg1 signaling pathway plays a role in maximizing colonization of host tissues, and that this involves negative regulation of sporulation and primary hyphae production in planta.

Oleic acid (18:1) is one of the important monounsaturated fatty acids, which is synthesized upon desaturation of stearic acid and this reaction is catalyzed by the SSI2 encoded stearoyl-acyl-carrier-protein-desaturase. A mutation in SSI2 leads to constitutive activation of salicylic acid (SA)-mediated defense responses. Consequently, these plants accumulate high levels of SA and show enhanced resistance to bacterial and oomycete pathogens. Replenishing 18:1 levels in ssi2 plants, via a second site mutation in GLY1 encoded glycerol-3-phosphate (G3P) dehydrogenase, suppresses all the ssi2-triggered phenotypes. Study of mechanism(s) underlying gly1-mediated suppression of ssi2 phenotypes showed that 18:1 levels are regulated via acylation with G3P and a balance between G3P and 18:1 is critical for the regulation of defense signaling pathways. To establish a role for 18:1 and G3P during host defense, interaction between Colletotrichum higginsianum and Arabidopsis was studied. Resistance to C. higginsianum correlated with host G3P levels. The gly1 plants showed increased susceptibility while act1 plants, defective in utilization of G3P, showed enhanced resistance. Plant overexpessing GLY1 showed enhanced resistance in both wild type as well as camalexin deficient backgrounds. Together, these results suggested that G3P conferred resistance acted downstream or independent of camalexin. Exogenous application of glycerol lowered 18:1 levels and produced ssi2-like phenotypes in wild-type plants. Furthermore, glycerol application or the ssi2 mutation produced similar phenotypes in fatty acid desaturation mutants and mutants defective in SA/resistance gene signaling. Expression studies showed that ssi2 phenotypes were likely due to increased expression of resistance genes. Epistatic analysis suggested that certain components of SA pathway had redundant function and were required for 18:1-regulated signaling.

Multiple species of Colletotrichum can cause bitter rot disease of apple, but the identities and relative representation of the species causing the disease in Kentucky are unknown. A total of 475 Colletotrichum isolates were collected from diseased apples in 25 counties and characterized both morphologically and by using various molecular approaches. Four morphotypes corresponded to reported descriptions of bitter rot species. Morphotype 1, distinguished by the production of a pink color on potato dextrose agar (PDA), orange conidial masses, and fusiform spores, was consistent with C. acutatum. Morphotype 2, which produced gray or white mycelial colonies with orange conidial masses and fusiform spores, was also similar to C. acutatum. Morphotype 3 had abundant gray mycelium and rounded spores and was identical to C. gloeosporioides. Morphotype 4 produced ascospores and resembled Glomerella cingulata. Species-specific polymerase chain reaction (PCR) indicated that both Morphotype 1 and Morphotype 2 belonged to the C. acutatum species complex, whereas Morphotype 3 and Morphotype 4 corresponded to the C. gloeosporioides complex. Multigene sequence analyses revealed that sample isolates belonged to several newly erected species within these species complexes. Morphotype 1 was identified as C. fioriniae, which resides within the C. acutatum species complex. Morphotype 2 was identified as C. nymphaeae, which is also a species within the C. acutatum species complex. Some isolates of Morphotype 3 were identified as C. siamense and some as C. theobromicola; both species are grouped within the C. gloeosporioides species complex. Morphotype 4 was identified as C. fructicola, which is also placed within the C. gloeosporioides species complex. C. fioriniae was the most common species causing bitter rot in Kentucky, comprising more than 70% of the isolates. Molecular fingerprinting using random amplified polymorphic DNA (RAPD) suggested that isolates within C. fioriniae belonged to a relatively homogeneous population, while isolates within C. siamense, C. theobromicola and C. fructicola were more diverse. Infectivity tests on detached fruit showed that C. gloeosporioides species-complex isolates were more aggressive than isolates in the C. acutatum species complex. However, isolates within the C. acutatum species complex produced more spores on lesions compared to isolates within the C. gloeosporioides species complex. Aggressiveness varied among individual species within a species complex. C. siamense was the most aggressive species identified in this study. Within the C. acutatum species complex, C. fioriniae was more aggressive than C. nymphaeae, causing larger, deeper lesions. Apple cultivar did not have significant effect on lesion development. However, Colletotrichum species produced more spores on Red Stayman Winesap than on Golden Delicious. Fungicide sensitivity tests revealed that the C. acutatum species complex was more tolerant to thiophanate-methyl, myclobutanil, trifloxystrobin, and captan compared to the C. gloeosporioides species complex. The study also revealed that mycelial growth of C. siamense was more sensitive to tested fungicides compared to C. fructicola and C. theobromicola. These research findings emphasize the importance of accurate identification of Colletotrichum species within each species complex, since they exhibit differences in pathogenicity and fungicide sensitivity.

Greenhouse and laboratory experiments were conducted on the fungal bioherbicides Colletotrichum graminicola and Gloeocercospora sorghi on shattercane (Sorghum bicolor), to evaluate any potential interactions among and between these two plant pathogens for management of this weed. Intraspecific interactions were evaluated with back to back applications of C. gramincola or G. sorghi, and results indicate additive or no effects. Results indicate that interspecific interactions between C. graminicola and G. sorghi lower expected disease incidence when C. gramincola is applied first, while the opposite effect occurs when G. sorghi is applied first. Tank mixtures were also evaluated, and results indicate that no synergistic relationships occur when both species are applied simultaneously. When using spores in conjunction with the chemical herbicide RoundUp (a.i. glyphosate), applying spores with or before glyphosate results in decreased biomass loss, while applying glyphosate prior to spore application results in increased weed biomass loss. Data from intra- and interspecific interactions evaluated in vitro support these findings. / Department of Biology

Made available in DSpace on 2015-04-09T12:28:07Z (GMT). No. of bitstreams: 0 Previous issue date: 2014-07-28Bitstream added on 2015-04-09T12:48:21Z : No. of bitstreams: 1 000818168.pdf: 338028 bytes, checksum: e610bbb669509fc3b15eadffe717b866 (MD5) / A Podridão floral dos citros (PFC), ocasionada por Colletotrichum acutatum e C. gloeosporioides, reduz consideravelmente a produção de citros nas principais áreas produtoras e a utilização de fungicidas ainda é a estratégia de controle mais eficaz. No ano de 2012 os benzimidazóis, grupo químico que por muitos anos foi importante no controle de PFC, tiveram seu uso suspenso em pomares de citros destinados à produção de suco para exportação, uma exigência de adequação aos padrões de qualidade dos EUA, principal comprador. Em decorrência disso, fez-se necessária a busca por novos fungicidas, que apresentassem boa eficiência agronômica e que atendessem às taxas de resíduos permitidas. Sendo assim, o objetivo do presente estudo foi avaliar novos grupos químicos de fungicidas, isoladamente ou em combinação, bem como reguladores de crescimento, indutores de resistência e fertilizantes químicos no controle da PFC, em pomares comerciais, nas safras 2012/13 e 2013/14. Os experimentos foram realizados em plantas de laranjeira „Pera‟ localizadas nos municípios paulistas de Itapetininga (IT), Santa Cruz do Rio Pardo (SC) e Casa Branca (CB). O delineamento experimental utilizado foi o de blocos casualizados (DBC), com cinco repetições. Dentre as novas estratégias de manejo foram avaliadas: i) Fungicidas dos grupos das estrobilurinas em combinação com outros grupos químicos; ii) Reguladores de crescimento aplicados isoladamente ou em combinação com reguladores hormonais e fungicidas; iii) Eficiência de misturas de estrobilurinas e triazóis, aplicadas em conjunto com reguladores hormonais e fertilizantes químicos; iv) Eficiência de clorotalonil combinado a estrobilurinas e triazóis, e v) Eficiência de Stimulate combinado a Hold e fungicida. Observou-se que fungicidas do grupo das estrobilurinas em combinação com triazóis ou clorotalonil, bem como a utilização de biorreguladores e indutores ... / The postbloom fruit drop (PFD), caused by Colletotrichum acutatum and C. gloeosporioides, considerably reduces citrus production in the main producing areas and the use of fungicides is still the most effective control strategy. In 2012 benzimidazoles, chemical group which, for many years, was important in controlling PFD, have had their use suspended in citrus orchards for the production of exportation juice, an adaptation requirement to the quality standards of the USA, the main buyer. As a result, it was made necessary to search for new fungicides enhancing good agronomic efficiency and heeding the rate of waste allowed. Thus, the aim of this study was to evaluate new chemical groups of fungicides, alone or in combination, as well as growth regulators, resistance inducers and chemical fertilizers to control PFC in commercial orchards, during 2012/13 and 2013/14 crop seasons. The experiments were performed with sweet orange 'Pera' trees located in the municipalities of Itapetininga (IT), Santa Cruz do Rio Pardo (SC) and Casa Branca (CB). The experimental design was a randomized block design (RBD) with five replications. Among the new management strategies were evaluated: i) fungicides of the strobilurin group in combination with other chemical groups; ii) growth regulators applied alone or in combination with hormonal regulators and fungicides; iii) efficiency of strobilurin and triazole mixtures applied in conjunction with hormonal regulators and chemical fertilizers; iv) efficiency of strobilurins and triazoles combined with chlorothalonil, v) efficiency Stimulate® combined to Hold® and fungicide. It was observed that fungicides of the strobilurin and triazoles groups or in combination with chlorothalonil, as well as the use of bioregulators e resistance inducers are efficient alternative preventive control of the PFC