M.Sc. / A phytotoxic protein-lipopolysaccharide complex (PLPC) was isolated from 7 day old culture filtrates of Verticillium dahliae. The complex was purified to electrophoretic homogeneily by means of acetone precipitation, gel, chromatography and preparative agarose electrophoresis with a yield of 4.5 mg PLPC per litre culture filtrate ...

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Verticillium wilt, caused by Verticillium dahliae Kleb, is one of the most economically important diseases of woody hosts such as ash (Fraxinus spp.), sugar maple (Acer saccharum), and redbud (Cercis canadensis). The causal agent has a broad host range, including not only woody hosts but also important vegetable and field crops, and it is distributed worldwide. Diagnosis of V. dahliae in infected woody hosts is often based on the occurrence of vascular discoloration and time-consuming isolation. However, not all woody hosts exhibit vascular discoloration symptoms, and not all vascular discoloration symptoms are due to infection by V. dahliae. In this study, real-time PCR-based assays were evaluated and employed for rapid and accurate detection of V. dahliae in different woody hosts. DNA was extracted in large quantities from presumptively infected woody hosts by collecting drill-press shavings from sample tissue, bead-beating, and extracting using a CTAB method. Six published primer sets were evaluated against genomic DNA of V. dahliae as well as selected negative controls, and two sets (VertBt-F/VertBt-R and VDS1/VDS2) showed promise for further evaluation using DNA extracts from field samples. The VertBt primers amplified a species-specific 115-bp fragment of the expected size, while the VDS primers amplified the expected specific 540-bp fragment. However, the VertBt primer set exhibited higher sensitivity in detection of V. dahliae even in asymptomatic trees. The PCR-based methods developed here could be used as rapid tools for pathogen detecting and monitoring, thus informing plant pathogen management decisions.

2 p. / Alfalfa varieties differ in fall dormancy, defined as growth during the fall. Nondormant alfalfa varieties are usually planted in mild winter areas for their ability to grow in the fall. However, fall growth of nondormant alfalfa may be undesirable in areas subject to repeated frosts or freezes. Nondormant, very nondormant, and extremely nondormant alfalfa varieties (fall dormancy class 8, 9, and 10) are adapted to elevations below 4000 feet in Arizona. Other dormancy classes not included in this publication are moderately nondormant varieties (fall dormancy class 7) which may be grown from 3000 to 5000 feet, and semi-dormant and dormant varieties (fall dormancy 6 and below) which are adapted to colder winter areas above 4000 feet.

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

Since the first report of Verticillium wilt of potato in 1950, the disease has been considered to be of minor importance in South Africa. Between 1995 and 2000, however, Verticillium spp. were isolated from 146 samples of symptomatic potato plant material received from 13 of the 14 potato production areas in the country. Of 93 Verticillium isolates that were obtained, 60% were identified as V. dahliae and 8 % V. nigrescens. V. dahliae was present in nine of the regions and V. nigrescens in seven. Unidentified Verticillium species were isolated from six of the regions. Both V. dahliae and V. nigrescens were pathogenic to potato in vivo, with V. dahliae the more virulent of the two species. Ten South African potato cultivars, eight of which have recently been released, were evaluated over two seasons in a greenhouse for resistance to V. dahliae. The cultivars Aviva, BP1, Bravo, Buffelspoort, Caren, Hoevelder and Ropedi were classified as susceptible to Verticillium wilt, whereas Calibra, Dawn and Devlin were rated as very susceptible. No resistance or tolerance was evident. The efficacy of broccoli volatiles on in vitro mycelial growth of Verticillium dahliae, and the effect of incorporation of fresh and dry broccoli residues on the survival of microsclerotia of V. dahliae and infection of potato, were determined in the laboratory and greenhouse. Volatiles emanating from freshly harvested macerated broccoli leaves were inhibitory to mycelial growth of V. dahliae on medium. Fresh and dry residues incorporated into soil artificially infested with V. dahliae, significantly reduced the viability of microsclerotia of the pathogen and the rate of infection of potato plants. Dry residues were more effective than fresh residues in reducing the viability of sclerotia, but suppression of infection was independent of the state of the residues. / Dissertation (MSc (Plant Pathology))--University of Pretoria, 2005. / Microbiology and Plant Pathology / unrestricted

Strawberries are considered an important crop in California where in 2018 it was in the top 5 valued fruit and vegetable commodities valued at $2.84 billion accounting for 88% of the total U.S. production. Strawberry production can be severely impacted by soilborne pathogens that can affect strawberry roots, crowns and leaves which can result in plant mortality. As much as 50 to 60% mortality can occur in one field. Pathogens responsible for such losses include Colletotrichum acutatum (syn.C. nymphaeae), Macrophomina phaseolina and Verticillium dahliae. With the phaseout of methyl bromide, host resistance and an understanding of host-pathogen interactions can play an important role in control of these diseases. A two-year study was conducted in order to evaluate host resistance of anthracnose in 105 cultivars and elite breeding lines developed by six strawberry breeding programs. Cultivars and elite breeding lines were inoculated using three local isolates in both years. All breeding programs provided genotypes that had a wide range of anthracnose susceptibility ranging from 0 to 100% mortality during both years. In both years an average of 78% of all the plant mortality occurred by 1 January. From the 105 cultivars and elite breeding lines, 30 cultivars were common to both years. Of these 30 cultivars, nine of them differed in their disease susceptibility between experiments by more than 20%. This suggests that several years of field evaluation may be necessary to determine susceptibility to anthracnose. Popular cultivars that represent the spectrum of susceptibility are Monterey (susceptible), Festival (moderately resistant), and Sensation (resistant). A second study was conducted toevaluate pathogen colonization of resistant and susceptible strawberry cultivars, testing interactions among crown and root plant tissue and two sampling timings. These cultivars were challenged with two soilborne pathogens, Macrophomina phaseolinaand Verticillium dahliae,over two years. Existing qPCR protocols for M. phaseolina and V. dahliae were used in order to quantify how much pathogen DNA was detected in crown and root samples. For the 2016-2017 V. dahliae trial there were significant effects for cultivar. Cultivar Benicia had significantly higher pathogen DNA compared to resistant cultivars Marquis, UC-12 and Camino Real. Susceptible cultivar BG 1975 had significantly less pathogen DNA compared to resistant cultivars San Andreas and Petaluma. In the 2017-2018 V. dahliaetrial pathogen DNA amount was not significantly different based on cultivar, plant part colonization, or the sampling period. In the 2017-2018 M. phaseolina trial all three of the fixed factors, cultivars, plant part colonization and sampling period were statistically significant. Cultivar ‘Sweet Ann’ had a significantly higher level of M. phaseolinaDNA in the early vs. the late sampling.

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.

Surveys of the four major soilborne pathogens of strawberry (Fusarium oxysporum f. sp. fragariae, Macrophomina phaseolina, Phytophthora spp., and Verticillium dahliae) to determine their relative prevalence were conducted in Watsonville-Salinas, CA in 2021 and in Santa Maria, CA in 2022. All four major pathogens were detected at relatively similar prevalence in Watsonville-Salinas, between 22% and 31% of sampled fields. In Santa Maria, M. phaseolina was far more prevalent at 52% of sampled fields, the other three falling between 14% and 17%. Additionally replicated greenhouse and field trials were conducted to evaluate the effects of wheat as a single season cover crop on Macrophomina root rot of strawberry and the soil microbiome. Greenhouse trials and the first year of the field trial are described here and demonstrate a lack of substantial disease mitigation or pathogen reduction in the soil following wheat growth compared to no-treatment control. Significant changes were seen in the soil microbiome following wheat growth, including the significant amplification of several bacterial species known to be antagonistic to plant-pathogenic fungi.

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.