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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

La verticilliose est une maladie vasculaire des plantes dont les symptômes typiques sont un flétrissement des parties aériennes, des feuilles chlorosées puis séchées, et dans les cas de maladie grave la mort de la plante. Au niveau des racines on observe une coloration brune du tissu conducteur. Cette maladie est causée par un champignon du sol du genre Verticillium. Les espèces majeures V. dahliae et V. albo-atrum sont responsables de pertes importantes de rendement sur de nombreuses cultures. Le champignon entre dans la racine par des blessures ou par des fissures au niveau de sites d’émergence de racines latérales, puis il avance vers le cylindre central et envahit les vaisseaux du xylème. Sa croissance reste pendant longtemps limitée aux vaisseaux qu’il colonise en avançant vers les parties aériennes de la plante. Aux stades tardifs, le champignon sort du cylindre central et colonise les autres tissus. En Europe, V. albo-atrum constitue l’une des principales causes de maladies chez la luzerne pérenne et est à l’origine de pertes économiques très importantes. La capacité de V. albo-atrum de survivre dans le sol ainsi que sa localisation protégée dans le cylindre centrale des plantes infectées en font un pathogène difficile à combattre, la lutte génétique par sélection de variétés tolérantes apparaissant comme une approche prometteuse. Cependant, la capacité des microorganismes pathogènes de s’adapter rapidement à des nouvelles plantes hôtes est une menace bien connue de la durabilité des variétés résistantes. Au laboratoire, des travaux ultérieurs ont établi que la plante modèle des légumineuses Medicago truncatula, une espèce sauvage proche de la luzerne cultivée, peut être utilisée pour étudier les mécanismes de résistance et sensibilité vis-à-vis de V. alboatrum. Une lignée résistant et une lignée sensible ont été identifiées et l’étude de la descendance d’un croisement entre ces deux lignées a permis d’identifier un locus majeur (Quantitative trait locus, QTL) contrôlant la résistance à une souche de V. albo-atrum isolée de la Luzerne (Ben et al., 2013 ; Negahi en 6e co-auteur). Ce travail a également montré qu’il existait une grande biodiversité au sein de l’espèce M. truncatula par rapport à la réponse à cette souche de V. albo-atrum. / Verticillium wilt, caused by Verticillium albo-atrum (Vaa) and Verticillium dahliae (Vd), is responsible for yield losses in many economically important crops. The capacity of pathogenic fungi to adapt to new hosts is a well-known threat to the durability of resistant crop varieties. Medicago truncatula is a good model for studying resistance and susceptibility to Verticillium wilt in legume plants. Phenotyping a population of inbred lines from a cross between resistant parent line A17 and susceptible parent F83005.5 contributed to the identification of a first QTL controlling resistance to an alfalfa strain of Vaa in M. truncatula. Then, 25 M. truncatula genotypes from a core collection and six Vaa and Vd strains were used to study the potential of non-host Verticillium strains isolated from different plant species to infect this legume plant, and the plant’s susceptibility to the pathogens. The experiment was arranged as factorial based on a randomized complete block design with three replications. The wilt symptoms caused by Vaa and Vd were scored on a disease index scale from 0 to 4, during 30 days after inoculation of ten day-old plantlets. Disease severity was quantified by the parameters Maximum Symptom Scores (MSS) and Areas Under the Disease Progress Curves (AUDPC). Highly significant differences were observed among plant genotypes and fungal strains, and their interaction was also significant. The correlation between MSS and AUDPC was 0.86 and highly significant. The most severe symptoms were caused by the alfalfa strain Vaa-V31-2 and the least severe by Vd-JR2, as shown by mean values obtained on the 25 genotypes. M. truncatula genotype TN8.3 was identified as the most susceptible genotype by mean values obtained with the 6 fungal strains, whereas F11013-3, F83005.9 and DZA45.6 were highly resistant to all strains studied. The results were used to choose parents for studying the genetics of resistance in M. truncatula to a nonalfalfa Verticillium strain. So, in the second part of this work, genotype A17 which was susceptible and genotype F83005.5 which was resistant to the potato strain Vaa-LPP0323 and recombinant inbred lines (RILs) from a cross between these genotypes were selected in order to study the genetic control of resistance to this strain of the pathogen. Our experimental design was a randomized complete blocks with 116 RILs and three replications. High genetic variability and transgressive segregation for resistance to Vaa-LPP0323 were observed among RILs. A total of four QTLs controlling resistance to Vaa-LPP0323 were detected for the parameters MSS and AUDPC. The phenotypic variance explained by each QTL (R2) was moderate, ranging from 3 to 21%. A negative sign of additive gene effects showed that favourable alleles for resistance come from the resistant parent.

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Memoria para optar al título profesional de: Ingeniero Agrónomo / El presente estudio se realizó con el objetivo de comprobar la patogenicidad de Verticillium albo-atrum, hongo asociado al decaimiento y muerte de plantas de kiwi dorado (Actinidia chinensis) cultivar Hort16A. Con este propósito plantas de Hort16A de un año de edad injertadas sobre Hayward (Actinidia deliciosa) se inocularon con una suspensión conidial de 106 conidias·ml -1. Al cabo de sesenta días, solamente las plantas inoculadas con V. alboatrum manifestaron síntomas de clorosis, marchitez y defoliación. Desde plantas con síntomas se recuperó en medio de cultivo Agar Agua y Agar Papa Dextrosa a V. alboatrum, identificado morfológicamente y mediante la amplificación del espacio intergénico entre los genes 18S y 28S que codifican para el RNA ribosomal del micelio del hongo. Desde tejido vegetal de plantas de kiwi dorado con síntomas se comprobó la presencia de V. albo-atrum utilizando la técnica de PCR anidado. Se realizó un estudio comparativo de la morfología y temperatura óptima de desarrollo entre V. albo-atrum y V. dahliae. Destacándose como la principal diferencia morfológica la presencia de hifas melanizadas en V. albo-atrum y la formación de microesclerocios en V. dahliae. La temperatura óptima de crecimiento para V. albo-atrum fue de 20°C y de 25°C para V. dahliae. / The objective of this study was to verify the pathogenicity of Verticillium albo-atrum, fungi associated with decline and death of gold kiwifruit (Actinidia chinensis) cultivar Hort16A. For this purpose Hort16A plants of one year old grafted on Hayward (Actinidia deliciosa) were inoculated with a suspension of 106 conidias·ml -1. After sixty days, only plants inoculated with V. albo-atrum showed symptoms of chlorosis, wilting and defoliation. The isolated fungus from symptomatic plant was recovered in water agar and potato dextrose agar medium and identified morphologically and by amplification of intergenic space between rRNA 18S and 28S coding genes of V. albo-atrum mycelium. From symptomatic plants tissue was detected the presence of V. albo-atrum using nested PCR. It was performed a comparative study of morphology and optimum temperature growth between V. albo-atrum and V. dahliae, being the main morphological difference the presence of melanized hiphae on V. albo-atrum and of microesclerotia on V. dahliae. The optimum growth temperature of V. albo-atrum and V. dahliae were 20°C and 25°C respectively.