Polygalacturonase-inhibiting proteins (PGIPs) are plant proteins believed to playa role in the defence against pathogenic fungi. In this study. it was hypothesized that apple PGIPI could be used to confer enhanced resistance against Verticillium-wilt. a major disease of potato caused by the fungus Verticillillm dahliae. Transgenic lines containing the apple pgip1 gene under control of the enhanced CaMV 35S (e35S) promoter had been generated previously. Stable integration of the transgene into the potato genome was shown by the polymerase chain reaction (PCR) and Southern blot with a DIG¬labelled apple pgip1 fragment as probe. Polygalacturonase (PG)-inhibiting assays (the agarose diffusion assay and reducing sugar assays) were employed to investigate the inhibiting activity of apple PGIP I extracts, prepared from the transgenic potato lines. on the PGs secreted by V. dahliae grown on pectin medium. Inhibition was successful for all but one of the transgenic lines. Active PGIPI was expressed in the leaves of in vitro- and glasshouse grown plants, as well as in roots of in vitro-grown plants. Due to the success of the in vitro inhibition results. it was anticipated that the apple pgip1 transgene would protect the transgenic lines against Verticillium-wilt in a subsequent glasshouse trial. The transgenic lines and untransformed BP I potato control were planted in soil inoculated with V. dahliae microsclerotia and control soil. Assessments of the visual symptoms of yellowing and wilt were made on a scale of 1-5. Colonisation of stem sections was determined by plating onto potato dextrose agar plates. Disease index values were calculated from the symptom and colonisation data. Analysis of variance indicated six lines to be significantly different from the rest when grown in the inoculated soil, but five of them also showed significantly slower senescence symptoms when grown in the control soil. It is proposed that the physiological effect of an extended juvenile phase resulted in the apparent increased disease resistance. This could be caused by transformation or tissue culture¬-induced somaclonal variation of the potato plants. The hypothesis that transformation of the apple pgip1 gene into potato would confer enhanced resistance against Verticillium-wilt was not supported by the data that was obtained. Expression of antifungal genes by pathogen-inducible promoters is a valuable strategy in the development of disease resistant crops of importance. A construct containing the apple pgipl gene under control of the pathogen-inducible gst1 promoter from Arabidopsis thaliana (L.) Heynh was generated. Agrobacterium tumefaciens GV31OI(pMP90RK) was transfonned with the plant transformation vector pCAMBIA2300 containing the gst1 and e35S promoter-pgip1 inserts. A. thaliana was transformed using the floral-dip method, and putative transgenic progeny were selected by kanamycin selection of the seeds. PCR verified the insertion of the transgene into the genomes of T2 and T3 lines. Gene expression from the two promoters was compared by performing PGIP extractions and the agarose diffusion assay. The gst1 promoter was active even without induction by methyl-salicylate. Both constructs led to the expression of active apple PGIP1 against V. dahliae PG in the heterologous plant A. thaliana. / Dissertation (MSc (Plant Biotechnology))--University of Pretoria, 2006. / Plant Science / unrestricted

No description available.

Microbial-root associations are important to help plants cope with abiotic and biotic stressors. Managing these interactions offers an opportunity for improving the efficiency and sustainability of agricultural production. By characterizing the bacterial and archaeal community (via 16S rRNA sequencing) associated with the bulk and rhizosphere soil of sixteen strawberry cultivars in two controlled field studies, we explored the relationships between the soil microbiome and plant resistance to two soilborne fungal pathogens of strawberry (Verticillium dahliae and Macrophomina phaseolina). Overall, the plants had a distinctive rhizosphere microbiome relative to the bulk soil, with higher abundances of known beneficial bacteria such as Pseudomonads and Rhizobium. Plant genotype, biomass, leaf nutrient content and mortality were influenced differently by the rhizosphere microbiome in each of the two trials. In the V. dahliae trial, the rhizosphere microbiome was associated with plant biomass and leaf nutrient content and only indirectly to the disease resistance. In the M. phaseolina trial, the rhizosphere microbiome was associated to plant biomass, but not nutrient content; furthermore, resistant cultivars had larger abundances of Pseudomonas and Arthrobacter in their rhizosphere relative to susceptible cultivars. The mechanisms involved in these beneficial plant-microbial interactions and their plasticity in different environments should be studied further for the design of low-input disease management strategies.

Strawberry (Fragaria x ananassa, Duch.) is an important crop in California, with more than 35,000 acres planted in 2018 resulting in a farm gate value of $3.1 billion. In 2020, California strawberry production accounted for more than 85% of national strawberry production and faces serious threats to production due to various soil-borne diseases. One such disease, Verticillium wilt, is caused by the fungal pathogen Verticillium dahliae and is commonly found in temperate zones around the world where strawberries are grown. Due to the phase-out of efficacious fumigants like methyl bromide, alternative disease management methods have become necessary to alleviate threats to production. Alternative fumigation practices such as crop termination have recently been investigated, and the integration of crop termination with bed fumigation and host resistance can play an integral role in control of Verticillium wilt. A field trial was established at California Polytechnic State University, San Luis Obispo to examine the efficacy of integrative management solutions for control of Verticillium wilt of strawberry in a naturally infested field. The efficacy of sequential fumigation applications of crop termination and bed fumigation was examined. Further, the integration of a resistant cultivar was also implemented in hopes of further decreasing plant mortality and increasing yield. Different fumigant products such as metam potassium, metam sodium, and chloropicrin were used to assess their performance in different fumigation applications. Metam potassium and metam sodium were used for crop termination. When used for crop termination, both products delivered significant reduction in soil inoculum density and adequate crop injury. Metam potassium, metam sodium, and chloropicrin were used for bed fumigation. All products reduced soil inoculum density. Lower plant mortality and higher yield resulted from sequential applications of crop termination and bed fumigation, with average plant mortality for non-treated control plots and sequentially fumigated plots being 67.2% and 24.1%, respectively. There were no significant increases in yield for plots bed fumigated and sequentially crop terminated and bed fumigated, but significant increases in yield for all plots treated versus the non-treated plots were found. The integration of a moderately resistant cultivar Valiant after the fumigation series showed lower mortality and higher yield versus a susceptible cultivar Seascape. A two-year study was also conducted in order to evaluate host resistance to Verticillium wilt in 74 cultivars and elite breeding lines from five strawberry breeding programs. Genotypes were established in a field naturally infested with V. dahliae on the campus farm at California Polytechnic State University, San Luis Obispo. All five breeding programs had a wide range of susceptibility to Verticillium wilt, ranging from 1.5% to 100% mortality for both years of the trial. Twenty-three cultivars and elite breeding lines were common to both years of the trial; of these, five cultivars showed vastly different results between the two years. For example, ‘Monterey’ showed 78.8% mortality in 2021 and 11.5% mortality in 2022. This demonstrates the importance of evaluating host resistance over multiple years under different environmental conditions and field locations.

Made available in DSpace on 2016-12-08T16:44:49Z (GMT). No. of bitstreams: 1 PGPV14MA158.pdf: 622907 bytes, checksum: 4bef911008ae5cffa26706180f710db4 (MD5) Previous issue date: 2014-07-31 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The addition and incorporation of organic matter to the soil, besides favoring crops by improving soil physical, can increase nutrients and add specific biochemicals capable of renewing the native microflora and microfauna. These compounds may, depending on the organic material to act as a suppressant effect and biocontrol. The aim of this study was to evaluate the effect of swine manure compost (DSC) in an orchard of apple, pear and grape vines on the population dynamics of Verticillium dahliae, Fusarium solani, Fusarium oxysporum, Fusarium verticillioides and Trichoderma sp. Soil samples for quantification of fungal colonies and nutrient analysis were collected at a depth of 0-10 cm soil of the orchard with apple, pear and grape vines. The population of pathogenic soil fungi and Trichoderma sp. were obtained by dilution and plating of 10 g of soil samples from soil orchard who received two doses of DSC (50 to 100%) compost and two (50 and 100%), using two culture media (BDA potato-dextrose-agar) and Sabouraud-ágar-chloramphenicol. The application of different doses of DSC and chemical fertilizer began in December 2012, repeated at intervals of 60 days until the 2014 harvest analysis of macronutrients (nitrogen, phosphorus, potassium, calcium and magnesium) and micronutrients (iron, copper, zinc and Manganese) DSC and chemical fertilizer were run using Mehlich-1, spectrophotometry, acid-base titration and Kjeldahl method, all described by Tedesco et al. (1995). Results in the concentration of nutrients was related to the population of Verticillium dahliae, Fusarium solani, Fusarium oxysporum, Fusarium verticillioides and Trichoderma sp. The experimental design was completely randomized, factorial 2 x 5, repeated in time (months). The data were analyzed using the MIXED procedure of SAS (SAS Inst. Inc., Cary, NC, v.9.2) and mean comparisons using Tukey least significant difference p ≤ 0.05. In the apple orchard, Fusarium oxysporum and Fusarium solani showed higher populations in Q100 treatments (0-110 x 103 CFU / g of soil) and Q50 (0-70 x 103 CFU/g of soil) respectively. There were differences in the population periods. Phosphorus, Potassium and Sodium showed significant differences among the treatments tested. In the orchard of pear trees the largest population of Fusarium solani was the S100 treatment (0-50 x 103 CFU/ g of soil). Treatments Q50 and Q100 had higher populations of Verticillium dahliae, Fusarium oxysporum and Fusarium verticillioides in different periods. Concentrations of Nitrogen and Potassium differ between treatments tested. In vineyards the largest populations of Fusarium solani and Fusarium oxysporum were found in December-2012 periods (0-70 x 103 CFU / g of soil) and August 2013 (0-60 x 103 CFU / g of soil) respectively. Concentrations of potassium, phosphorus and sodium were higher in treatment S50 and S100. The orchard of apple, pear and grape vines have different response to chemical and organic fertilization. The intensity of response to fertilization has little influence population dynamics of plant pathogens in soil and Trichoderma / A adição e incorporação de matéria orgânica ao solo, além de favorecer as culturas pela melhoria física do solo, podem potencializar nutrientes e adicionar compostos bioquímicos específicos capazes de renovar a microfauna e microflora nativas. Estes compostos podem, dependendo do material orgânico, agir como efeito supressor e como biocontrole. O objetivo deste trabalho foi avaliar o efeito da aplicação de dejeto suíno compostado (DSC) em pomar de macieiras, pereiras e videiras, sobre a dinâmica populacional de Verticillium dahliae, Fusarium solani, Fusarium oxysporum, Fusarium verticillioides e Trichoderma sp. Amostras de solo para quantificação de colônias fúngicas e análise de nutrientes foram retiradas na profundidade 0-10 cm de solo do pomar de macieiras, pereiras e videiras. A população de fungos fitopatogênicos de solo e Trichoderma sp. foram obtidas pela diluição e plaqueamento de 10 g de amostras de solo provenientes do solo do pomar que receberam duas doses de DSC (50 e 100%) e duas de adubo químico (50 e 100%), utilizando dois meios de cultura, BDA (batata-dextrose-agar) e Sabouraud ágar-cloranfenicol. A aplicação das diferentes doses de DSC e adubo químico tiveram início em dezembro-2012, repetidas em intervalos de 60 dias até a safra 2014. A análise dos macronutrientes (nitrogênio, fósforo, potássio, cálcio e magnésio) e micronutrientes (ferro,cobre,zinco e Manganês) do DSC e da adubação química foram realizados pelos métodos de Mehlich -1, espectrofotometria, titulação ácido-base e método Kjeldahl, todas descritas por Tedesco et al. (1995). Resultados da concentração de nutrientes foi relacionado à população de Verticillium dahliae, Fusarium solani, Fusarium oxysporum, Fusarium verticillioides e Trichoderma sp. O delineamento experimental foi inteiramente casualisado, em arranjo fatorial 2 x 5, repetidos no tempo (meses). Os dados foram analisados pelo procedimento MIXED do SAS (SAS Inst. Inc., Cary, NC, v.9.2) e as comparações de médias usando a diferença mínima significativa de Tukey p ≤ 0,05. No pomar de macieiras, Fusarium oxysporum e Fusarium solani apresentaram maiores populações nos tratamentos Q100 (0-110 x 103 UFC/g de solo) e Q50 (0-70 x 103 UFC/g de solo) respectivamente. Houve diferenças da população nos períodos avaliados. Fósforo, Potássio e Sódio apresentaram diferenças significativas entre os tratamentos testados. No pomar de pereiras a maior população de Fusarium solani foi ao tratamento S100 (0-50 x 103 UFC/g de solo). Os tratamentos Q50 e Q100 apresentaram maiores populações de Verticillium dahliae, Fusarium oxysporum e Fusarium verticillioides em diferentes períodos de avaliação. Concentrações de Potássio e Nitrogênio apresentaram diferenças nos tratamentos testados. Na cultura da videira as maiores populações de Fusarium solani e Fusarium oxysporum foram encontradas nos períodos dezembro-2012 (0-70 x 103 UFC/g de solo) e agosto-2013 (0-60 x 103 UFC/g de solo) respectivamente. Concentrações de Potássio, Fósforo e Sódio foram superiores nos tratamento S50 e S100. O pomar de macieiras, pereiras e videiras apresentam diferentes resposta a adubação química e orgânica. A intensidade de resposta da adubação pouco influencia a flutuação da população de fitopatógenos de solo e Trichoderma

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.

No description available.

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.

Field and lab experiments were conducted to determine the efficacy of sudangrass (Sorghum X drumondii (Nees ex Steud.) Millsp. & Chase) cover crop management techniques and soil solarization on important agricultural weeds and pathogens in organic strawberry production in Central California. Lab experiments assessed the time needed to kill weed seeds at temperatures typically achieved during soil solarization (40°C, 45°C, 50°C, 55°C, and 60°C) in California. Seeds tested included little mallow, redstem filaree, bristly oxtongue, annual sowthistle, common purslane, nettleleaf goosefoot, and redroot pigweed. Efficacy of simulated solarization temperatures differed between different species. Cool-season annuals annual sowthistle and bristly oxtongue were more susceptible to heat treatments than warm-season annuals common purslane, redroot pigweed and nettleleaf goosefoot. Hard seeded weed species little mallow and redstem filaree were the least susceptible to heat treatments. Annual sowthistle, bristly oxtongue and nettleleaf goosefoot were affected at all temperatures. Redroot pigweed and little mallow were not affected by temperatures below 40°C. Common purslane was not affected by temperature below 45°C and redstem filaree was not affect by any temperatures tested. Hours of exposure and percent mortality of weed seeds were used to create thermal death models for weed seeds. Field experiments were conducted at the Cal Poly Organic Farm in San Luis Obispo, CA testing the effects of soil solarization and sudangrass residues on weeds, Verticillium dahliae populations, plant health, and yields in organic strawberry production. Using a split plot design, sudangrass was grown, mowed and then developed into two treatments: surface mulch or incorporated into the soil. The sudangrass treatments and a control were tested with and without soil solarization (n=4). Maximum soil temperatures in solarized treatments were 53°C at a soil depth of 5 cm and 42°C at a soil depth of 15 cm. Solarization reduced weed biomass between 49.8 and 95.2% during the first 3.5 months after tarp removal (p=0.03), reduced Verticillium dahliae populations by 80.7% (p=0.01), reduced plant mortality by 54.9% (pV. dahliae populations (p=0.33) or yields (p=0.25). However, mulched treatments reduced weed biomass between 45.0 and 61.3% (p=0.03) compared to other sudangrass treatments. Results indicate solarization can be used in central coast organic strawberry production to reduce hand-weeding, disease incidence, and increase yields.

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.