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Thesis (M.S.) -- University of Tennessee, Knoxville, 2005. / Title from title page screen (viewed on June 30, 2005). Thesis advisor: John Sorochan. Document formatted into pages (xiii, 124 p. : ill. (some col.), col. map). Vita. Includes bibliographical references (p. 59-68).

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Anthracnose, incited by Colletotrichum cereale, is a destructive disease of physiologically stressed creeping bentgrass putting greens in Mississippi and Alabama. Anthracnose severity and frequency of occurrence have increased over the past 15 years, and fungicide resistance may have had a role in the increase. In vitro bioassays were performed to evaluate thiophanate methyl and azoxystrobin against C. cereale isolates exposed to the fungicides and baseline isolates that had not been exposed to either fungicide. All isolates collected from creeping bentgrass were uninhibited by both fungicides at discriminatory doses. Partial nucleotide sequences of the â-tubulin 2 (thiophanate methyl) or cytochrome b (azoxystrobin) gene was compared to confirm fungicide resistance. Thiophanate methyl resistance was conferred by either a point mutation from glutamic acid to alanine at position 198, or phenylalanine to tyrosine at position 200. Azoxystrobin resistance was conferred by an amino acid point mutation from glycine to alanine at position 143.

Botrytis cinerea is the principal member of the species complex that causes bunch rot of grapes and gray mold disease on other hosts including fruits and ornamental crops. It has developed resistance to many fungicides, and isolates from eastern US strawberry fields have regularly been identified with resistance to several modes of action. During the 2011-2015 growing seasons, 487 isolates were collected from Virginia wine grapes, strawberries, and ornamental crops and evaluated for sensitivity to eight different fungicides by a germ tube elongation method; for a subset of isolates, a 24-well plate mycelial growth assay was also used, and baseline sensitivity to polyoxin-D was evaluated. Resistance to benzimidazoles and quinone outside inhibitors, and low-level resistance to iprodione were common. Boscalid resistance was common in wine grapes and ornamentals. Resistance to the hydroxyanilide fenhexamid during germ tube elongation was found in only 5% of wine grape isolates, but in 33% of isolates from strawberries and ornamentals. All of the fenhexamid-resistant isolates were identified as B. cinerea carrying various mutations in the erg27 gene. An additional subset of isolates was identified with moderate resistance to fenhexamid during mycelial growth, but not germination and germ tube growth. These were identified as B. cinerea HydR2 isolates, which possess an unknown mechanism of resistance towards fenhexamid in mycelial growth. Moderate resistance to cyprodinil was common, but in grape inoculation tests, moderately resistant isolates were controlled by a field rate of cyprodinil. Diminished sensitivity to fludioxonil and fluopyram was rare. Polyoxin-D controlled most isolates in mycelial growth tests at 100 µg/ml. / Master of Science in Life Sciences

Thesis (MScAgric)--University of Stellenbosch, 2000. / ENGLISH ABSTRACT: Petri grapevme decline, also known as black goo, slow die-back and Phaeoacremonium grapevine decline, causes significant losses of young vines worldwide. Species of Phaeoacremonium, Phaeomoniella chlamydospora and related genera are associated with this grapevine disease. This study investigates the Phaeoacremonium-complex and Phaeomoniella chlamydospora, focussing on the species isolated from grapevines. Fungicide sensitivity of Pa. chlamydospora and the possibility of employing molecular techniques for the detection of Pa. chlamydospora in grapevines were also investigated. In an overview of the literature on Petri grapevine decline the disease history and the relatedness of Petri grapevine decline to esca is discussed. Petri grapvine decline occurs in propagation material or young vines. Infected material can appear asymptomatic and therefore the possibilities of molecular techniques for identification were also investigated in the literature. In South Africa Pa. chlamydospora is the dominant organism causing Petri grapevine decline and therefore different fungicides were evaluated to control this fungus. Six isolates of Pa. chlamydospora, from Stellenbosch, Wellington, Somerset West and Malmesbury of Western Cape province, South Africa, were screened against twelve fungicides testing their effect on mycelial inhibition in vitro. These fungicides included benomyl, chlorothalonil, fenarimol, fosetyl-Al, iprodione, kresoxim-methyl, mancozeb, metalaxyl, prochloraz manganese chloride, quintozene, tebuconazole and thiram. Results provided the base-line sensitivity of South African isolates of Pa. chlamydospora. Benomyl, fenarimol, kresoxim-methyl, prochloraz manganese chloride and tebuconazole were the most effective (with EC50 values ranging from 0.01 to 0.05 ug/ml) for inhibiting mycelial growth of Pa. chlamydospora in vitro. This in vitro test gave a good indication of which fungicides could be selected for further studies in glasshouses and nurseries. The molecular phylogeny of Phaeoacremonium and Phaeomoniella isolates from grapevines of South Africa, or isolates obtained from the Centraalbureau voor Schimmelcultures (CBS) in the Netherland, were investigated. Sequence data were created from the rONA region and partial B-tubulin gene of 33 of these isolates using the PCR technique. This sequence data were analysed with PAUP* version 4.Ob2a. An analysis of the sequence data confirmed the genus Phaeomoniella to be distinct from Phaeoacremonium (Pm.) based on DNA phylogeny. Although morphologically similar, the species status of Pm. aleophi/um and Pm. angustius was confirmed with DNA phylogeny and cultural characteristics. Pm. aleophilum has an optimum growth rate at 30°C and the ability to grow at 35°C, where as Pm. angustius has an optimum growth rate at 25°C and cannot grow at 35°C_ Pm. viticola was shown to be synonymous with Pm. angustius, and a new species, Pm. mortoniae, was newly described from grapevine occurring in California. Futhermore, Pm. aleophilum was newly reported from South Africa and grapevine isolates thought to be Pm. inflatipes were all re-identified as Pm. aleophilum. These findings therefore also shed some doubt on the possible role of Pm. inflatipes in Petri grapevine decline. It was confirmed that Pa. chlamydospora, Pm. aleophilum and Pm. angustius are the species involved in Petri grapevine decline. Pm. mortoniae was isolated from grapevines, but its pathogenicity should still be confirmed and the role of Pm. injlatipes in Petri grapevine decline remains unclear. Pa. chlamydospora has been routinely isolated from symptomless propagation and nursery material. Because the disease can take years to develop, it is crucial that healthy propagation material is used at planting. Pa. chlamydospora is a slowgrowing fungus, and positive identification from symptomless grapevine tissue can take up to 4 wks. The possibility of employing molecular techniques for the detection of Pa. chlamydospora in apparently healthy grapevines was investigated. Speciesspecific primers (PCLI and PCL2) based on the regions ITSI and ITS2 were designed for Pa. chlamydospora. These primers were highly sensitive and amplification was achieved from genomic DNA of Pa. chlamydospora from as low as 16 pg. Phaeoacremonium spp., related genera and common fungal taxa from grapevines were tested with these primers, but positive amplification was achieved for Pa. chlamydospora only. The presence of Pa. chlamydospora in symptomless grapevine tissue culture plants was confirmed by PCR within 24 hours. These primers therefore allow rapid and accurate identification of Pa. c~lamydospora. Testing on a larger scale with nursery material should be conducted to determine the feasibility of using these species-specific primers in the grapevine industry. / AFRIKAANSE OPSOMMING: Petri-terugsterwing van jong wingerde, ook algemeen bekend as "black goo" en Phaeoacremonium-terugsterwing, veroorsaak wêreldwyd groot geldelike verliese in die wingerdbedryf. Spesies van Phaeoacremonium, Phaeomoniella chlamydospora en verwante genera word met hierdie wingerdsiekte geassosieer. In die tesis word In oorsig gegee van die geskiedenis van hierdie siekte, die verwantskap tussen Petriterugsterwing en esca, en moontlike maniere van siektebestuur. Swamme wat by die siektekompleks betrokke is, kan in simptoomlose plantweefsel voorkom en daarom is die moontlikhede van die gebruik van molekulêre tegnieke vir swamidentifikasie in oënskou geneem. In Suid-Afrika is Pa. chlamydospora die dominante swam wat met Petriterugsterwing geassosieerword, gevolglik is verskillende fungisiedes vir die chemiese beheer van Pa. chlamydospora geëvalueer. Ses isolate van Pa. chlamydospora, versamel vanaf verskillende areas in die Wes-Kaap provinsie, is in dié studie gebruik. Benomyl, chlorothalonil, fenarimol, fosetyl-Al, iprodione, kresoxim-methyl, mancozeb, metalaxyl, prochloraz manganese chloride, quintozene, tebuconazole en thiram se effek op miselium inhibisie van Pa. chlamydospora is in vitro geëvalueer. Benomyl, fenarimol, kresoxim-methyl, prochloraz manganese chloride en tebuconazole was die mees effektiewe middels. Die effektiewe konsentrasie waarby 50% van die miselium groei geïnhibeer is (EKso),was tussen 0.01 en 0.05 ug/ml vir die mees effektiewe groep middels. Benomyl, fenarimol, kresoxim-methyl, prochloraz manganese chloride en tebuconazole het in vitro goeie potensiaal getoon, en verder toetse moet in vivo uitgevoer word. 'n Molekulêre studie is van Phaeoacremonium en Phaeomoniella isolate; verkry uit Suid-Afrikaanse wingerde, of vanaf die "Centraalbureau voor Schimmelcultures" (CBS) van Nederland; gedoen. Deur van die PKR tegniek gebruik te maak, is die basispaaropeenvolgingsdata van 33 isolate, van die ITSl, 5.8S, ITS2 rDNA area en die gedeeltelike B-tubullen geen verkry. Gekombineerde molekulêre data het die teorie ondersteun dat Phaeomoniella (Herpotrichiellaceae) gedistansieerd is van Phaeoacremonium (Magnaporthaceae). Pm. aleophilum en Pm. angustius was morfologies moeilik onderskeibaar, maar kon op grond van molekulêre data en kulturele eienskappe onderskei word. Pm. aleophilum se optimum groeitemperatuur was by 30°C en die swam besit die vermoë om by 35°C te groei. Pm. angus/ius se optimum groeitemperatuur was by 25°C, maar het nie by 35°C gegroei nie. 'n Studie van molekulêre en kulturele eienskappe het getoon dat Pm. angus/ius en Pm. viticola sinoniem is. 'n Nuwe spesie, Pm. mortoniae, wat uit wingerde van Kalifornie geïsoleer is, is beskrywe. Verder is Pm. aleophilum die eerste keer in Suid-Afrikaanse wingerde aangetref en Pm. tnflatipes isolate, wat vanuit wingerde geïsoleer is, is almal met molekulêre data gewys om Pm. aleophilum te wees. Hierdie bevindinge trek die rol van Pm. inflatipes in Petri-terugsterwing van wingerde in twyfel. Phaeomoniella chlamydospora IS m voortplantingsmateriaal en kwekerystokkies opgespoor. Omdat dit jare kan duur voordat siektesimptome ontwikkel, is dit belangrik om vroegtydig te weet of jong stokkies met Pa. chlamydospora geïnfekteer is. Pa. chlamydospora groei baie stadig en positiewe identifikasie van simptoomlose infeksies duur tot vier weke. Die toepassing van molekulêre tegnieke vir die vinnige identifikasie van Pa. chlamydospora in wingerde is dus ondersoek. Spesie-spesifieke oligonukleotiedes (PCU en PCL2) is vir Pa. chlamydospora ontwerp. Hierdie oligonukleotiedes is uiters sensitief en genomiese DNA van Pa. chlamydospora is van so laag as 16 pg geamplifiseer. Phaeoacremonium spp., verwante genera en algemene swamme vanuit wingerdmateriaal is met die oligonukleotiedes getoets, maar positiewe amplifikasie was slegs met Pa. chlamydospora moontlik. Die teenwoordigheid van Pa. chlamydospora is binne 24 uur in asimptomatiese wingerd weefselkultuurplantjies bevestig. Hierdie oligonukleotiedes identifiseer Pa. chlamydospora vinnig en akkuraat en toetsing op 'n groter skaal moet vervolgens met kwekerymateriaal onderneem word.

Phoma stem canker is a disease of oilseed rape (Brassica napus) caused by closely related plant pathogens Leptosphaeria maculans and L. biglobosa. It is an economically important disease, causing annual yield losses of approximately £770 million worldwide. When colonising oilseed rape, L. maculans and L. biglobosa exist in close proximity on the leaf, competing for resources as they move through the main leaf vein and into the stem. Fungicides are commonly used to decrease severity of phoma stem canker on oilseed rape. However, the efficacy and longevity of active chemicals is under threat from evolution of resistance in pathogen populations and government legalisation. Moreover, it has been suggested that both L. maculans and L. biglobosa differ in their sensitivity to azoles, and important class of fungicides that are used to control the disease through the inhibition of lanosterol 14-α demethylase (erg11, CYP51). This project aims to further understand the role that fungicides have in controlling phoma stem canker by investigating their efficacy against L. maculans and L. biglobosa in crops, in vitro and in planta. In field experiments, established in Cambridgeshire across four cropping seasons, the fungicide mixture penthiopyrad (SDHI) plus picoxystrobin (QoI) was as effective at controlling phoma leaf spotting and phoma stem canker in winter oilseed rape as prothioconazole (DMI), suggesting that both fungicides could be used to reduce phoma stem canker symptoms. The two pathogens differed in their growth rates in vitro, with L. biglobosa growing faster than L. maculans when untreated or treated with lower fungicide concentrations. Fungicide sensitivity assays suggest that L. maculans and L. biglobosa are both sensitive to DMI, SDHI and QoI fungicides and that differences between the species are minor. Prothioconazole and penthiopyrad + picoxystrobin had a similar efficacy on oilseed rape cotyledons colonised with either L. maculans or L. biglobosa. There was no difference between species on prothioconazole treated plants, although there was a difference between L. maculans and L. biglobosa when treated with 20 μg/ml penthiopyrad + picoxystrobin. Heterologous yeast expression of LmCYP51B and LbCYP51B with fungicide sensitivity testing of the yeast transformants suggests that LmCYP51B and LbCYP51B are similarly sensitive to azole fungicides flusilazole, prothioconazole-desthio and tebuconazole. These findings are supported by homology protein modelling, which predicts that LmCYP51B and LbCYP51B are structurally very similar, specifically at the azole-binding site. In conclusion, fungicides are still an effective control method for reducing phoma stem canker symptoms caused by Leptosphaeria species in the UK, and a useful tool to in the sustainable production of oilseed rape.

Strawberry powdery mildew, caused by Podosphaera aphanis, affects leaves, fruit, and runners of strawberry plants. Infected leaves have reduced photosynthetic capability and infected fruit become unmarketable. Both of these factors translate to economic loss for the grower and therefore merit taking measures to control the disease. One objective of this study was to evaluate the resistance developed in populations of strawberry powdery mildew to chemical control measures. A fungicide assay was developed to evaluate the efficacy of six treatments (penthiopyrad, quinoxyfen, myclobutanil, trifloxystrobin, cyflufenamid, fluopyram + trifloxystrobin) for control of the disease. Nineteen isolates of strawberry powdery mildew were collected from Balico, Salinas, Watsonville, San Luis Obispo, Santa Maria, Ventura, and Oxnard CA and tested through the assay. The number of isolates resistant to each treatment was: penthiopyrad (7), quinoxyfen (6), myclobutanil (7), trifloxystrobin (2), cyflufenamid (1), fluopyram + trifloxystrobin (0). This documents resistance in P. aphanis to multiple chemicals used for its control. Documentation of any resistance is novel in California and novel worldwide with resistance to Fungicide Resistance Action Committee (FRAC) codes 7 and 13. Another objective of this study was to evaluate host plant resistance to strawberry powdery mildew. Twelve cultivars were evaluated in a winter greenhouse trial, sixteen cultivars in a summer greenhouse trial, and the ten cultivars shared in both trials were also evaluated in two fields. The cultivars found to be most susceptible to mildew infection were BG 3.324 and Royal Royce. The cultivars found to be the least susceptible to mildew infection were Fronteras, San Andreas, and Sweet Ann. The cultivars evaluated represent more than 55% of the state’s acreage and the host plant resistance information will be a valuable tool to growers looking to culturally control powdery mildew.