The effectiveness of the biocontrol product Actinovate® at enhancing tomato plant growth and yield, and reducing the presence of fungal pathogens was studied in greenhouse and field conditions. In the greenhouse, no differences were found among seed germination or plant survival rates, seedling heights, dry root weights, and dry shoot weights of tomato seedlings grown from seeds drenched with Actinovate® or Rootshield®. The effects of one initial Actinovate® seed drench at sowing, repeated applications through the drip irrigation throughout the season, or repeated applications through the drip irrigation plus foliar applications throughout the season at reducing plant infection by fungal plant pathogens, and increasing yield and quality for tomato plants (Solanum lycopersicum) were investigated in Los Alamos, CA, on a sandy loam soil. No significant differences in plant height were found among the four treatments. Marketable fruit weight was greater in the drip plus foliar treatment than in the Actinovate® seed drench treatment. The foliar plus drip treatment resulted in the greatest amount of powdery mildew present, although the disease pressure was low. No significant differences were found among the four treatments in the presence of Verticillium wilt or Sclerotinia.

No description available.

No description available.

Significant mortality of the invasive tree of heaven (TOH), Ailanthus altissima (Mill.) Swingle, was first observed in Pennsylvania in 2002 to be caused by an apparently host-specific strain of Verticillium albo-atrum Reinke & Berthold, a soil-borne, vascular wilt fungus. A limited survey conducted in Virginia revealed two sites where TOH stands were infected with V. albo-atrum. A virulence test confirmed that fungal isolates from both states were found to be highly pathogenic on TOH, killing all inoculated seedlings in 9 weeks. After overwintering, 11% (n = 37) of TOH root sections tested positive for V. albo-atrum, although the origin of the colonies was not identified. The pathogenicity of this pathogen suggests that it could be used together with host-specific insects for the biological control of TOH. A host-specific herbivorous weevil from China, Eucryptorrhynchus brandti Harold (Coleoptera: Curculionidae) that has been extensively studied as another potential biological control agent for TOH is currently pending quarantine release. Quarantine experiments were conducted to test different forms of transmission with E. brandti and V. albo-atrum simultaneously. In one experiment, 75% (n = 32) of adult E. brandti transmitted V. albo-atrum to TOH seedlings after walking on an actively growing culture and feeding on infected plant material. In another study after feeding on infected TOH stems for 24, 48 and 72 h, respectively, 16.7% (n = 120), 15.0% and 12.5% of adult E. brandti ingested and passed viable V. albo-atrum propagules into feces. Surviving weevils (83%, n = 20) overwintering in infested potting mix carried viable V. albo-atrum propagules externally. In addition, all weevil progeny that emerged from infected TOH billets appeared to be as healthy as weevils reared from non-infected billets and wild parents from China. Results from these laboratory studies indicate E. brandti has the ability to spread V. albo-atrum from tree to tree in a laboratory setting. / Master of Science in Life Sciences

D.Phil. / The fungus, Verticillium dahliae, is the causative agent of Verticillium wilt, which results in significant cotton (Gossypium hirsutum) crop losses worldwide. This study contributes to the elucidation of cotton defence responses against V. dahliae. The identification, cloning and characterization of three genes that were differentially expressed in response to elicitation with a cell wall-derived (CWD) V. dahliae elicitor are described. It was hypothesized that the molecular architectures of the three characterized genes are supportive of a role in cotton defence against V. dahliae. As one of these genes was present as two homoeologous copies, this study also reports on the molecular characterization of both homoeologs, thus providing further insight into the processes of genomic evolution between homoeologous loci in allotetraploid cotton. The three genes were initially represented as expressed sequence tags (ESTs), obtained from a previous differential display reverse transcription polymerase chain reaction (DDRT-PCR) study by Zwiegelaar (2003), as part of an MSc project. These ESTs, designated C1B10, C4B5 and C4B4, were differentially induced upon elicitation with a CWD V. dahliae elicitor (Zwiegelaar, 2003). In the present study, the genes represented by the three ESTs were identified and characterized by genome walking and 5‘/3‘ rapid amplification of cDNA ends (RACE). Additionally, PCR and reverse-transcription PCR (RT-PCR) were utilized, where necessary, to obtain internal sequences, not covered by the genome walking and RACE reactions. Through the use of these molecular techniques, the full transcript and genomic sequences of each of the three genes was obtained, including their promoters. The promoter of each gene was analyzed for cis-elements driving gene transcription, through bioinformatic analysis. Furthermore, the copy number of each gene was determined through Southern blot analysis. The genes were translated to reveal their encoded protein sequences. The amino acid sequences were submitted to a basic local alignment (BLAST) search of the NCBI database to identify, and align them with, homologous proteins from other plant species (and those from G. hirsutum, if any). An in silico analysis of the encoded protein of each gene was also performed. This examination included domain architecture, post-translational modification, subcellular location and tertiary structure predictions. This study also involved the isolation of the elicitor from the cell walls of V. dahliae fungal cultures. The potency of the freshly-isolated elicitor was investigated with a triphenyltetrazolium chloride (TTC) viability assay on cotton cell suspensions. Its potential to induce PR-proteins was also explored but these results were inconclusive. In addition, expression studies were performed with real-time PCR (q-PCR), to confirm the up- or down-regulation of each gene upon elicitation of cotton cell suspensions with the CWD V. dahliae elicitor, and to investigate the time frame/kinetics of induction. The gene corresponding to the C1B10 EST was designated GhLIPN as this study revealed that it encodes a lipin protein. Lipins are novel proteins with phosphatidate phosphatase 1 (PAP1) activity, exclusive to eukaryotes. They play a fundamental role in the lipid metabolism of organisms ranging in complexity from yeast to animals and plants. In plants, this role includes lipid membrane remodelling during phosphate (Pi) deficiency. During the study of the GhLIPN gene, it was discovered that it occurred as two distinct homoeologous copies from the A- and D-co-resident genomes of allopolyploid G. hirsutum. The GhLIPN homoeologs were named GhLIPN I and N for Insert present and No insert, respectively, based on the presence or absence of a 13 base pair (bp) insertion/deletion (indel) site in intron 6.

The invasive tree-of-heaven, Ailanthus altissima (Miller) Swingle, is widespread and damaging throughout North America. Verticillium wilt disease is emerging as a potentially exciting biological control option for this difficult to control tree. In Virginia, Verticillium nonalfalfae has been confirmed causing significant mortality to A. altissima, while V. dahliae is suspected to be present and causing lower levels of disease. Little else is known regarding these two fungal species in this state. The purpose of this research was to gain a better understanding of how Verticillium wilt impacts A. altissima and its potential as a biological control agent. We first confirmed V. dahliae's presence in Virginia and its pathogenicity to A. altissima using Koch's postulates. We then completed a regional field-inoculation experiment to show that V. nonalfalfae effectively kills and spreads to adjacent A. altissima, regardless of V. dahliae presence or other climate and stand variables. Additionally, we showed that V. dahliae causes lower levels of disease than V. nonalfalfae, and does not spread rapidly. Next, we surveyed all Virginia A. altissima stands known to be naturally infected with V. nonalfalfae to determine whether V. nonalfalfae persists long-term, that it considerably reduces A. altissima numbers, and that its local prevalence may be higher than initially suspected. However, we were unable to infect A. altissima seedlings using soil collected at these infested sites, suggesting that V. nonalfalfae's survival within field soil may be limited. Lastly, using paired A. altissima invaded-uninvaded sites, we found that A. altissima presence is associated with a decreased proportion of native plants and species in the woody and herbaceous understory, but not the germinable seedbank. Furthermore, we found that this impact on the woody understory appears to increase over time, supporting early management actions and helping us predict post-management restoration needs. We conclude that V. nonalfalfae has a high potential of successfully limiting A. altissima throughout Virginia, supporting its registration as a biopesticide. / Doctor of Philosophy / Commonly called the tree-of-heaven, the nonnative invasive forest-tree Ailanthus altissima, is extensive, damaging, and spreading throughout North America. After finding large areas of declining tree-of-heaven being killed by two different fungal species (Verticillium nonalfalfae and V. dahliae), research has been focused on how to use these fungi to help us manage the tree-of-heaven. In Virginia, V. nonalfalfae has been confirmed killing large numbers of tree-of-heaven, while V. dahliae is suspected to be present in areas with lower levels of decline. The purpose of our research was to gain a better understanding of how these pathogens impact tree-of-heaven and their potential as biological control agents in Virginia. We first confirmed that V. dahliae is present in Virginia and can cause disease on tree-of-heaven. We then inoculated tree-of-heaven stands throughout the state to confirm that V. nonalfalfae effectively kills and spreads to adjacent tree-of-heaven regardless of V. dahliae presence or other climate or site variables. In contrast, we found that V. dahliae only causes low levels of disease and does not spread effectively. Next, we surveyed all known naturally infected V. nonalfalfae sites in Virginia and demonstrated that V. nonalfalfae persists long term within these stands, considerably reducing but not eradicating the tree-of-heaven, and that V. nonalfalfae's local prevalence may be higher than initially suspected. However, when tree-of-heaven seedlings were planted into soil collected from these infested sites, no disease developed, suggesting that V. nonalfalfae's survival within the soil may be limited. Lastly, by looking at tree-of-heaven stands, we found that the tree-of-heaven's presence is associated with a lower percentage of native plants and species in the understory, but not in the seeds present in the soil. In addition, we found that this impact on the woody plants in the understory appears to become more severe over time, supporting managing the tree-of-heaven as soon as possible. We conclude that V. nonalfalfae used as a biological control has a good potential of successfully limiting the tree-of-heaven in Virginia and support its registration as a biopesticide.

La verticilliose est causée par des agents pathogènes telluriques du genre Verticillium. Elle est, depuis sa découverte dans les années 50, maladie majeure du tournesol en Argentine où des sources de résistances ont été identifiées. En France, c’est une maladie de plus en plus fréquente, observée chaque année sur de nouvelles zones de production. Elle suscite désormais des efforts dans la recherche de moyens de lutte sur ce territoire. Ce travail s’est concentré sur l’étude des interactions Tournesol - agents causals de la verticilliose à deux niveaux d’observation : celui du système de culture (français vs. argentin) et celui de l’individu. L’objectif est d’apporter des connaissances sur l’agent causal et sur le déterminisme moléculaire dans la résistance à la verticilliose du tournesol afin de développer un modèle de criblage de résistances à grande échelle. L’étude de la maladie dans les systèmes de culture a permis de mettre en évidence l’existence d’une différence significative de la réponse du tournesol à Verticillium entre la France et l’Argentine. L’étude moléculaire des pathogènes vasculaires in planta, échantillonnés dans les 2 systèmes de culture, a permis de confirmer l’implication majeure de V. dahliae dans la verticilliose du tournesol. En conditions contrôlées, une étude comparative de la pathogénicité de plusieurs isolats de V. dahliae (de la tomate, du coton, du sol) sur le tournesol a mis en évidence que seul l’isolat 85S, isolé à partir du tournesol, est capable de le coloniser et de provoquer des symptômes. L’étude du génome de l’isolat 85S révèle que cet isolat n’appartient à aucune branche existante de l’arbre phylogénétique ; il forme un groupe per se, associé aux isolats non défoliant du coton mais infectant la tomate. L’hypothèse de la spécificité de la réponse induite dans l’hypocotyle et les feuilles du tournesol par certains isolats de V. dahliae a été confirmée en étudiant la cinétique de l’expression de 9 gènes associés à la défense, 5 semaines après inoculation. Le tournesol met uniquement en place son système de défense en réponse à l’infection par 85S. La réponse semble induite ; la colonisation n’est pas systémique, la biomasse fongique n’a pas été détectée dans l’hypocotyle et les feuilles de l’hybride asymptomatique. L’ensemble de ces travaux a conduit au développement d’un modèle pour le criblage de résistances à Verticillium chez le tournesol. Celui-ci répond aux contraintes liées à la diversité du pathogène dans les méga-environnements, conséquences de pressions de sélection différentielles. / Verticillium wilt is caused by soil-borne fungi of the genus Verticillium. From its discovery in the 50’s, sunflower Verticillium wilt is a major disease in Argentina where sources of resistances have been identified. Since few years, the disease occurs more frequently in France raising concerns on sources of resistances discoveries regarding its spread to other sunflower French production areas. This work focus on the study of Sunflower- causals agent of Verticillium wilt interaction at 2 levels of observation: at the cultural system (French vs Argentinian) and at the plant individual scale. The aim is to provide identification of the causal agents and knowledge on the molecular determinism of sunflower resistances to implement high-throughput plant phenotyping approach. Disease symptom studies within the cultural systems reveal a significant difference in the phenotypic expression of sunflower against Verticillium pressure according to the location in France or in Argentina. Molecular studies of isolates in planta, from naturally infested field in cultural systems reveal the major implication of V. dahliae in the sunflower Verticillium wilt disease. In controlled conditions, comparative studies of V. dahliae isolates pathogenicity (isolated from cotton, tomato and soil) on sunflower reveal that only 85S, isolated from sunflower is able to colonize and provoke symptoms. Genomic studies of 85S isolates reveal that the isolate did not belong to any branch of the current phylogenetic tree; 85S makes a 'per se' group within the cotton non-defoliating but tomato infecting strains. Specificity of induced responses in sunflower hypocotyl and leaves by only some of V. dahliae isolates have been confirmed through a gene expression kinetic analysis of 9 defenses related genes on 5 weeks post inoculation. Sunflower genotype is responding only to the 85S isolate. Resistance seems to be induced; colonization is not systemic as pathogen biomass has been detected but not quantify in symptomless cultivar. Our study finally leads to the implementation of Verticillium resistances screening model on the sunflower with respect to the constraints related to the pathogen diversity from the different environments, according to differential selection pressure.

La verticilliose de la luzerne cultivée (Medicago sativa L.) est une maladie de flétrissement vasculaire causée par le champignon du sol Verticillium alfalfae. C’est une des maladies les plus dévastatrices et les plus difficiles à contrôler. Les symptômes sont un jaunissement des feuilles suivi de flétrissement et défoliation. Les structures de dormance produites en fin de cycle de maladie constituent une source de contamination pour plusieurs années. Aucun traitement fongicide n’est efficace, la seule méthode de contrôle reste la production de variétés résistantes. En raison de sa nature tétraploïde et de son allogamie, il est difficile de réaliser des études génétiques sur M. sativa. Un pathosystème entre la légumineuse modèle Medicago truncatula et V. alfalfae a été mis au point pour étudier les mécanismes mis en place au cours de l’interaction entre V. alfalfae et son hôte. Les lignées A17 et F83005.5 ont été identifiées comme étant respectivement résistante et sensible à la souche V31-2 de V. alfalfae. La première partie de ce travail de thèse est une étude comparative du processus d’infection de V. alfalfae V31-2 au cours d’une interaction compatible et incompatible. Nous avons étudié la cinétique de colonisation des racines d’A17 et F83005.5 avec la souche V31-2 exprimant le gène marqueur GFP ce qui confère une fluorescence verte au champignon. Les observations en microscopie confocale ont montré que le champignon se développait dans les racines des deux lignées contrastées de façon similaire pendant les premières étapes d’infection. Quelques jours plus tard, il n’était plus détectable dans la lignée résistante, tandis qu’il colonisait les vaisseaux du xylème dans la lignée sensible et avançait vers les parties aériennes. La lignée résistante A17 était donc capable d’inhiber totalement le développement du pathogène dans la partie racinaire. Ce résultat a été confirmé par la quantification de l’ADN du pathogène dans la racine et dans les parties aériennes. Nous avons conclu que la lignée A17 exprime une résistance totale à V. alfalfae. Dans la deuxième partie de cette thèse, nous avons cherché à identifier le rôle des hormones dans les mécanismes de défense de M. truncatula en réalisant des traitements exogènes avec l’acide salicylique (SA), le méthyl jasmonate (MeJA), l’éthylène (ET), l’auxine et l’acide abscissique (ABA). Ces traitements n’avaient aucun effet sur la résistance d’A17, mais toutes les hormones, à l’exception du MeJA, protégeaient la lignée sensible contre les symptômes de la maladie. La quantification de l’ADN du champignon in planta a montré que seule l’ABA inhibait significativement le développement du pathogène. Dans la troisième partie, nous avons cherché à identifier des acteurs moléculaires impliqués dans la résistance et la sensibilité en comparant le transcriptome de la lignée F83005.5 et A17 dans la phase précoce de l’infection. L’analyse des gènes différentiellement exprimés en réponse à l’inoculation montre que les deux lignées induisent des gènes impliqués dans la production de métabolites secondaires, et des gènes des voies de signalisation hormonale. Mais seule la lignée résistante montre une induction de l’expression de gènes de résistance et de gènes impliqués dans les voies de signalisation tels que des gènes de la synthèse de l’ABA et des facteurs de transcription. Ces résultats renforcent l’hypothèse que l’ABA serait un facteur important dans la résistance à V. alfalfae chez M. truncatula. L’analyse des réseaux de gènes coexprimés a montré une désorganisation de la réponse de la lignée F83005.5. En revanche, dans la lignée A17, on observe une réponse organisée et orientée vers la défense. Ce travail décrit pour la première fois les mécanismes de défense de M. truncatula contre V. alfalfae. L’ensemble des résultats montre que la résistance exprimée chez la lignée A17 est différente des mécanismes de résistance contre la verticilliose décrits chez la tomate et le coton. / Verticllium wilt of alfalfa (Medicago sativa L.) is a vascular disease caused by the soil fungus Verticillium alfalfae. It is one of the most devastating diseases and most difficult to control. Symptoms are leaf yellowing followed by wilting and defoliation. Survival structures which are produced at the end of the disease cycle are a source of inoculum for many years. Fungicide treatment is not efficient, and the only way to control this disease is to breed resistant cultivars. Genetic studies are difficult in M. sativa because it is tetraploid and outcrossing. A pathosystem has been set up in our laboratory in order to study the mechanisms involved in the interaction between V. alfalfae and its host. It involves the model legume plant M. truncatula and strain V31-2 of V. alfalfae. The lines A17 and F83005.5 were identified as respectively resistant and susceptible to V31-2. The first part of this thesis is a comparative study of the infection process of V. alfalfae V31-2 in a compatible and incompatible interaction. The time course of root colonization in lines A17 and F83005.5 was studied with a GFP-expressing strain which confers green fluorescence to the fungus. Observations by confocal microscopy showed that the fungus developed in a similar way in roots of both lines during the first stage of the interaction. Some days later the fungus was not detectable anymore in roots of the resistant line, but has colonized the xylem vessels and grew towards the aerial part of the plant in the susceptible line. Quantification of fungal DNA in roots and aerial parts confirmed these results. This showed that the resistant line A17 was able to suppress the pathogen’s development in the root. It can be concluded that line A17 presents total resistance towards V. alfalfae. The second part of the thesis concerns the role of phytohormones for defence mechanisms against V. alfalfae in M. truncatula. Susceptible and resistant plants were treated with salicylic acid (SA), methyl jasmonate (MeJA), ethylene (ET), auxine and abscissic acid (ABA). Resistance of line A17 was not affected by these treatments, but all hormones except MeJA protected the susceptible line against disease symptoms. However, when fungal DNA was quantified in planta in these assays, only ABA inhibited the pathogen’s development significantly. The third part of this thesis aims at identifying molecular factors involved in resistance and susceptibility. To address this topic, the transcriptome of lines A17 and F83005.5 was compared during the early stages of infection, in inoculated or mock-inoculated plants. A bioinformatics analysis of differentially expressed genes showed that both lines responded to inoculation by inducing genes involved in secondary metabolism and hormone signaling pathways. However, only resistant line A17 showed induction of the expression of putative resistance and signaling genes, genes involved in ABA synthesis and transcription factors. This result confirms our hypothesis that ABA might be an important factor in M. truncatula resistance against V. alfalfae. Gene network analysis of co-expressed genes showed a disorganised response in the susceptible line, whereas in the resistant line the response was highly organised and turned to defence. Taken together, this work describes for the first time defence mechanisms against V. alfalfae in M. truncatula. The results show that resistance of line A17 is different from resistance mechanisms Verticillium resistance described in tomato and cotton. Several approaches for future research are presented in order to test our hypotheses concerning genes and molecules putatively involved in this interaction. With regard to applied research, defence and signaling genes identified in this work may be useful for the improvement of alfalfa, after functional validation.

No description available.

No description available.