Dissertations / Theses on the topic 'Control plant pathogens'

Crabgrass is a major problem in turf in Canada and infestations can be as high as 30% of a residential lawn. Due to the bans and restrictions on the use of chemical herbicides in several provinces, cities and municipalities across Canada, there are currently no effective solutions for controlling crabgrass. Two species of crabgrass, large (Digitaria sanguinalis) and smooth (Digitaria ischaemum), are commonly found in cropland and turf. Several species of phytopathogenic fungi have been studied in China and USA as possible biocontrol agents of Digitaria spp. From among those tested the most promising for the use in Canada are species in the genus Curvularia (C. intermediate, C. lunata, C. eragrostidis). In the present study, twenty-three fungal cultures associated with Digitaria spp. were isolated from leaves with visual symptoms of diseases. They were identified to a genera or species level. Growth and spore production were evaluated for each isolate and slowly growing and poorly sporulating isolates were eliminated from further experiments. Twenty remaining isolates were tested for pathogenicity on large and smooth crabgrass. Isolates belonging to C. eragrostidis species were the most effective. These isolates did not appreciably harm the majority of turf grasses and cereal crops, but caused major damage on forage grass timothy. Due to the absence of difference in the host range and superiority in spore production, isolate Dip0307 (C. eragrostidis) was chosen for further evaluation. Optimal temperature and dew duration conditions and minimal requirements for successful weed control with isolate Dip0307 were determined and compared with those for QZ-2000, the Chinese strain of C. eragrostidis. It was concluded that C. eragrostidis isolate Dip0307 is a strong candidate for development as a bioherbicide against large and smooth crabgrass in Canada.
La digitaire est un problème majeur dans le gazon au Canada et les infestations peuvent être aussi élevées que 30% dans les pelouses résidentielles. En raison de l'interdiction et des restrictions sur l'utilisation des herbicides chimiques dans plusieurs provinces, villes et municipalités du Canada, il n'existe pas actuellement de solution efficace pour contrôler la digitaire. Deux espèces de digitaire sont couramment trouvées dans les terres cultivées et le gazon soit la digitaire sanguine (Digitaria sanguinalis) et la digitaire astringente (Digitaria ischaemum). Plusieurs espèces de champignons phytopathogènes ont été étudiés en Chine et aux États-Unis comme agents de lutte biologique possibles de Digitaria spp. Parmi les espèces testées, l'espèce la plus prometteuse pour l'utilisation au Canada se trouve dans le genre Curvularia (C. intermédiaire, C. lunata, C. eragrostidis). Dans la présente étude, 23 cultures fongiques associées à Digitaria spp. ont été isolées à partir de feuilles présentant des symptômes visuels de maladies. Elles ont été identifiées à un niveau de genre ou d'espèce. La croissance et la production de spores ont été évaluées pour chaque isolat et les isolats qui démontraient une faible croissance et sporulation ont été éliminés des expériences subséquentes. Les 20 isolats restants ont été testés pour leur pathogénie sur la digitaire sanguine et astringente. Les isolats appartenant à l'espèce C. eragrostidis ont été les plus efficaces. Dans un même temps, ils ne nuisaient pas de façon significative à la majorité des graminées à gazon et des cultures céréalières, mais ont causé des dégâts significatifs sur les graminées fourragères testées. En raison de l'absence de différence dans sa gamme d'hôtes et de sa supériorité pour la production de spores, l'isolat Dip0307 (C. eragrostidis) a été choisi pour une recherche plus approfondie. Il a été soumis à différentes températures et durées d'exposition à la rosée et il a été comparé avec la souche chinoise QZ-2000 de C. eragrostidis. Cette étude nous a permis de conclure que l'isolat Dip0307 de C. eragrostidis est un bon candidat pour le développement d'un bioherbicide contre la digitaire sanguine et astringente au Canada.

Thesis (MScAgric (Plant Pathology))--Stellenbosch University, 2008.
In recent years, several studies have conclusively shown that numerous pathogens, including several species in the Botryosphaeriaceae, Phomopsis, Phaeoacremonium, as well as Phaeomoniella chlamydospora and Eutypa lata, contribute to premature decline and dieback of grapevines. These pathogens have the ability to infect grapevines through pruning wounds, which leads to a wide range of symptoms developing that includes stunted growth, cankers and several types of wood necrosis. Pruning wounds stay susceptible for 2 to 16 weeks after pruning and sustained levels of pruning wound protection is therefore required. The aims of this study were to (i) evaluate the ability of several biological agents to protect pruning wounds, (ii) characterise unknown Trichoderma strains and identify their modes of action and (iii) determine the optimal time of season for biological agent application. Several biological agents were initially evaluated in a laboratory for their antagonism against trunk disease pathogens. The best performing control agents were tested in a field trial conducted on Merlot and Chenin blanc vines in the Stellenbosch region. Spurs were pruned to three buds and the fresh pruning wounds were treated with benomyl as a control treatment, Trichoderma-based commercial products, Vinevax® and Eco77®, Bacillus subtilis, and Trichoderma isolates, USPP-T1 and -T2. Seven days after treatment the pruning wounds were spray inoculated with spore suspensions of four Botryosphaeriaceae spp. (Neofusicoccum australe, N. parvum, Diplodia seriata and Lasiodiplodia theobromae), Eutypa lata, Phaeomoniella chlamydospora and Phomopsis viticola. After a period of 8 months the treatments were evaluated by isolations onto potato dextrose agar. Trichodermabased products and isolates in most cases showed equal or better efficacy than benomyl, especially USPP-T1 and -T2. Moreover, these isolates demonstrated a very good ability to colonise the wound tissue. The two uncharacterised Trichoderma isolates (USPP-T1 and USPP-T2), which were shown to be highly antagonistic toward the grapevine trunk disease pathogens, were identified by means of DNA comparison, and their ability to inhibit the mycelium growth of the trunk disease pathogens by means of volatile and non-volatile metabolite production studied. The two gene areas that were used include the internal transcribed spacers (ITS 1 and 2) and the 5.8S ribosomal RNA gene and the translation elongation factor 1 (EF). The ITS and EF sequences were aligned to published Trichoderma sequences and the percentage similarity determined and the two Trichoderma isolates were identified as Trichoderma atroviride. The volatile production of T. atroviride isolates was determined by placing an inverted Petri dish with Trichoderma on top of a dish with a pathogen isolate and then sealed with parafilm. Trichoderma isolates were grown for 2 days on PDA where after they were inverted over PDA plates containing mycelial plugs. The inhibition ranged from 23.6% for L. theobromae to 72.4% for P. viticola. Inhibition by non-volatile products was less than for the volatile inhibition. Inhibition ranged from 7.5% for N. parvum to 20.6% for L. theobromae. In the non-volatile inhibition USPP-T1 caused significantly more mycelial inhibition than USPP-T2. The timing of pruning wound treatment and subsequent penetration and colonisation of the wound site was also determined. One-year-old canes of the Shiraz and Chenin blanc cultivars were grown in a hydroponic system, pruned and spray treated with a spore suspension of Trichoderma atroviride (USPP-T1) as well as a fluorescent pigment. On intervals 1, 3, 5 and 7 days after treatment, the distal nodes were removed and dissected longitudinally. From the one half, isolations were made at various distances from the pruning surface, while the other half was observed under ultra-violet light to determine the depth of fluorescent pigment penetration. Shortly after spray-inoculation of a fresh pruning wound, Trichoderma was isolated only from the wound surface and shallow depths into the wound (2 to 5 mm). One week after inoculation, Trichoderma was isolated at 10 mm depths, and after 2 weeks, at 15 mm depths. Fluorescent pigment particles were observed to a mean depth of 6 mm, which suggests that initial isolation of Trichoderma at these depths was resultant of the physical deposition of conidia deeper into the pruning wound tissue, whereas the isolation of Trichoderma from deeper depths might be attributed to colonisation of grapevine tissue. In a vineyard trial, fluorescent pigment was spray-applied to pruning wounds of Shiraz and Chenin blanc grapevines during July and September at intervals 0, 1, 3, 7 and 14 days after pruning. One week after treatment, the distal nodes were removed and dissected longitudinally. Each half was observed under UV light and the pigment penetration measured. For Chenin blanc and Shiraz, July pruning wounds showed significant deeper penetration of the pigment than pruning wounds treated in September. Moreover, pruning wounds made in September showed pigment particles in longitudinal sections up to 1 day after pruning, whereas wounds made in July showed pigment particles up to 3 days in the xylem vessels. These findings suggest that the best time for application of a biological control agent should be within the first 24 hours after pruning.

Thesis (MScAgric)--University of Stellenbosch, 2006.
ENGLISH ABSTRACT: Phaeomoniella chlamydospora, Eutypa lata, Phomopsis, Phaeoacremonium, and Botryosphaeria spp. are important trunk disease pathogens that cause premature decline and dieback of grapevine. Previous research has focused primarily on wine grapes and the incidence and symptomatology of these pathogens on table grapes were largely unknown. A survey was therefore conducted to determine the status and distribution of these pathogens and associated symptoms in climatically diverse table grape growing regions. Fifteen farms were identified in the winter rainfall (De Doorns, Paarl and Trawal) and summer rainfall (Upington and Groblersdal) areas. Samples were taken in July and August 2004 from Dan-ben-Hannah vineyards that were 8 years and older. Distal ends of arms were removed from 20 randomly selected plants in each vineyard. These sections were dissected and isolations were made from each of the various symptom types observed: brown or black vascular streaking, brown internal necrosis, wedge-shaped necrosis, watery necrosis, esca-like brown and yellow soft wood rot, as well as asymptomatic wood. Fungal isolates were identified using molecular and morphological techniques. Pa. chlamydospora was most frequently isolated (46.0%), followed by Phaeoacremonium aleophilum (10.0%), Phomopsis viticola (3.0%), Botryosphaeria obtusa (3.0%), B. rhodina (2.2%), B. parva (2.0%), Fusicoccum vitifusiforme (0.6%), B. australis, B. dothidea and an undescribed Diplodia sp. (0.2% each), while E. lata was not found. Most of these pathogens were isolated from a variety of symptom types, indicating that disease diagnosis can not be based on symptomatology alone. Pa. chlamydospora was isolated from all areas sampled, although most frequently from the winter rainfall region. Pm. aleophilum was found predominantly in Paarl, while P. viticola only occurred in this area. Although B. obtusa was not isolated from samples taken in De Doorns and Groblersdal, it was the most commonly isolated Botryosphaeria sp., being isolated from Upington, Paarl and Trawal. B. rhodina occurred only in Groblersdal and B. parva in Paarl, Trawal and Groblersdal, while B. australis was isolated from Paarl only. The rest of the isolates (33%) consisted of sterile cultures, Exochalara, Cephalosporium, Wangiella, Scytalidium, Penicillium spp. and two unidentified basidiomycetes, which were isolated from five samples with yellow esca-like symptoms from the Paarl area. These findings clearly illustrate that grapevine trunk diseases are caused by a complex of fungal pathogens, which has serious implications for disease diagnosis and management. Protection of wounds against infection by any of these trunk disease pathogens is the most efficient and cost-effective means to prevent grapevine trunk diseases. However, previous research on the effectiveness of chemical pruning wound protectants has mostly focused on the control of Eutypa dieback only. Fungicide sensitivity studies have been conducted for Pa. chlamydospora, P. viticola and Eutypa lata, but no such studies have been conducted for the pathogenic Botryosphaeria species from grapevine in South Africa. Ten fungicides were therefore tested in vitro for their efficacy on mycelial inhibition of the four most common and/or pathogenic Botryosphaeria species in South Africa, B. australis, B. obtusa, B. parva and B. rhodina. Iprodione, pyrimethanil, copper ammonium acetate, kresoxim-methyl and boscalid were ineffective in inhibiting the mycelial growth at the highest concentration tested (5 μg/ml; 20 μg/ml for copper ammonium acetate). Benomyl, tebuconazole, prochloraz manganese chloride and flusilazole were the most effective fungicides with EC50 values for the different species ranging from 0.36-0.55, 0.07-0.17, 0.07-1.15 and 0.04-0.36 μg/ml, respectively. These fungicides, except prochloraz manganese chloride, are registered on grapes in South Africa and were also reported to be effective against Pa. chlamydospora, P. viticola and E. lata. Results from bioassays on 1-year-old Chenin Blanc grapevine shoots indicated that benomyl, tebuconazole and prochloraz manganese chloride were most effective in limiting lesion length in pruning wounds that were inoculated with the Botryosphaeria spp after fungicide treatment. The bioassay findings were, however, inconclusive due to low and varied re-isolation data of the inoculated lesions. Benomyl, tebuconazole, prochloraz manganese chloride and flusilazole can nonetheless be identified as fungicides to be evaluated as pruning wound protectants in additional bioassays and vineyard trials against Botryosphaeria spp. as well as the other grapevine trunk disease pathogens.
AFRIKAANSE OPSOMMING: Phaeomoniella chlamydospora, Eutypa lata, Phomopsis, Phaeoacremonium, en Botryosphaeria spesies is die mees belangrikste stamsiekte patogene wat agteruitgang en vroeë terugsterwing van wingerd veroorsaak. Voorafgaande navorsing het hoofsaaklik gefokus op wyndruiwe en die voorkoms en simptomatologie van hierdie patogene op tafeldruiwe is dus grootliks onbekend. ‘n Opname is gevolglik gedoen in verskillende klimaaatsareas waar tafeldruiwe verbou word om die voorkoms en verspreiding, asook die simptome geassosieer met hierdie patogene, te bepaal. Vyftien plase is geïdentifiseer in die winter- (De Doorns, Paarl en Trawal) en somer-reënval (Upington en Groblersdal) streke. Wingerde (8 jaar en ouer) met die kultivar Dan-ben-Hannah is gekies vir opname en monsters is gedurende Julie en Augustus 2004 geneem. Die distale deel van ‘n arm is verwyder vanaf 20 lukraak gekose plante in elke wingerd. Hierdie dele is ontleed en isolasies is gemaak vanuit elke simptoomtipe wat beskryf is, naamlik bruin en swart vaskulêre verkleuring, bruin interne nekrose, wig-vormige nekrose, waterige nekrose, esca-geassosieerde bruin en geel sagte houtverrotting en asimptomatiese hout. Identifikasie van die swamagtige isolate is gedoen op grond van morfologiese eienskappe en molekulêre tegnieke. Pa. chlamydospora is die meeste geïsoleer (46.0%), gevolg deur Phaeoacremonium aleophilum (10.0%), Phomopsis viticola (3.0%), Botryosphaeria obtusa (3.0%), B. rhodina (2.2%), B. parva (2.0%), Fusicoccum vitifusiforme (0.6%), B. australis, B. dothidea en ‘n onbeskryfde Diplodia sp. (0.2% elk), terwyl E. lata nie geïsoleer is nie. Hierdie patogene is elk geïsoleer vanuit ‘n verskeidenheid simptoomtipes, wat daarop dui dat siektediagnose nie alleenlik op simptomatologie gebaseer kan word nie. Pa. chlamydospora is geïsoleer vanuit al die gebiede, alhoewel die patogeen opmerklik meer voorgekom het in die winter-reënval area. Pm. aleophilum het hoofsaaklik voorgekom in Paarl, terwyl P. viticola slegs in hierdie area voorgekom het. Alhoewel B. obtusa nie voorgekom het in die De Doorns en Groblersdal areas nie, was dit die mees algemeen geïsoleerde Botryosphaeria sp. en het in Upington, Paarl en Trawal voorgekom. B. rhodina het slegs in Groblersdal voorgekom, B. parva in Paarl, Groblersdal en Trawal en B. australis het slegs in Paarl voorgekom. Die res van die isolate (33%) het bestaan uit steriele kulture, Exochalara, Cephalosporium, Wangiella, Scytalidium, en Penicillium spesies asook twee onbekende basidiomycete isolate, geïsoleer vanuit vyf monsters met geel eska-geassosieerde simptome vanuit die Paarl area. Hierdie resultate illustreer dus die feit dat wingerdstamsiektes deur ‘n kompleks van swampatogene veroorsaak word, wat belangrike implikasies het vir die bestuur en diagnose van hierdie siektes. Wondbeskerming teen infeksie van enige van hierdie stamsiekte patogene is die mees doeltreffende en koste-effektiewe manier om wingerdstamsiektes te voorkom. Vorige navorsing aangaande die effektiwiteit van chemiese wondbeskermingsmiddels het egter slegs gefokus op die beheer van Eutypa terugsterwing. In vitro swamdoder sensitiwiteitstoetse is gedoen vir Pa. chlamydospora, P. viticola en Eutypa lata, maar geen studies is al gedoen ten opsigte van die patogeniese Botryosphaeria spesies op wingerd in Suid-Afrika nie. Tien swamdoders is dus getoets vir inhibisie van in vitro miseliumgroei van die vier mees algemene en/of patogeniese Botryosphaeria spesies wat in Suid-Afrika voorkom, naamlik B. australis, B. obtusa, B. parva en B. rhodina. Iprodione, pyrimethanil, koper ammonium asetaat, kresoxim-metiel en boscalid was oneffektief by die hoogste konsentrasies getoets (5 μg/ml; 20 μg/ml vir koper ammonium asetaat). Benomyl, tebuconasool, prochloraz mangaan chloried en flusilasool was die mees effektiewe swamdoders met EC50 waardes tussen 0.36-0.55, 0.07-0.17, 0.07-1.15 en 0.04-0.36 μg/ml, onderskeidelik vir die verskillende spesies. Hierdie fungisiedes, behalwe prochloraz mangaan chloried, is geregistreer op druiwe in Suid-Afrika en is ook effektief gevind teenoor Pa. chlamydospora, P. viticola en E. lata. Resultate van biotoetse op 1-jaar-oue Chenin Blanc wingerd lote het getoon dat benomyl, tebuconasool en prochloraz mangaan chloried die effektiefste was om die lengte van letsels in snoeiwonde, geinokuleer met Botryosphaeria spesies na die aanwending van swamdoder behandelings, te verminder. Die bevindinge was egter onbeslis as gevolg van die lae en variërende her-isolerings data. Benomyl, tebuconasool, prochloraz mangaan chloried en flusilasool kan egter geïdentifiseer word as swamdoders wat verder geevalueer kan word as snoeiwond beskermingsmiddels teen Botryosphaeria spesies asook ander wingerd stamsiekte patogene in verdere biotoetse en wingerdproewe.

Foodborne pathogens are a threat to public health worldwide. Because many consumers prefer natural compounds to synthetic additives, research on safe plant-derived compounds with antimicrobial activity against foodborne pathogens is vital. The aim of this investigation was to evaluate the antimicrobial activities of plant essential oils (oregano, cinnamon, lemongrass), their active components (carvacrol, trans-cinnamaldehyde, citral) and plant-extracts such as green tea, apple skin extract, black and decaffeinated black tea, grapes seed and pomace extracts against foodborne bacteria. Salmonella enterica serotype Typhimurium DT104, and serotype Newport, were selected conducting an antibiotic screening on 23 Salmonella isolates using seven antibiotics to determine antibiotic resistance. Listeria monocytogenes (strain 101M; beef and pork sausage isolate; resistant to antimicrobials in past investigations) was included to represent gram-positive bacteria. Escherichia coli O157:H7 virulent isolates (932- apple juice isolate; ATCC 35150- human isolate; F4637- sprouts isolate; used as a cocktail) were selected after conducting a Multiplex PCR over nine E. coli O157:H7 isolates to detect shiga-toxin 1 and 2 genes. All antimicrobials were evaluated in vitro in phosphate buffered saline. In general, all pathogens were more susceptible to essential oils and their active components, than powder extracts. The most active antimicrobials from each category were directly applied on foods. The activity of oregano oil (0.5%) and green tea (3%) was evaluated against S. Typhimurium on chicken and S. Newport on tomatoes and sprouts, and the results showed that oregano oil was more effective. In addition, baby spinach leaf samples inoculated with green fluorescent protein labeled S. Newport were examined under confocal scanning laser microscope before and after antimicrobial treatments. Antimicrobial experiments against L. monocytogenes on sprouts, ham and bologna, carvacrol at 0.5% and grape seed extract at 3% were used and carvacrol showed better activity. Antimicrobial activity against E. coli O157:H7 was tested on romaine lettuce, spinach and ground beef using oregano oil at 0.5% and green tea at 3%. Both compounds were effective showing no recovery of E. coli O157:H7 from lettuce and spinach; however, was not reduced in ground beef. Antimicrobial plant compounds have the potential for reducing foodborne pathogenic bacteria on/in various foods.

A fungicide trial was established at The University of Arizona Marana Agricultural Center in April 2000 to evaluate rotation and timing of application for several fungicides used for control of powdery mildew on cantaloupe. Treatments included seven registered fungicides: azoxystrobin, micronized sulfur, neem extract, potassium bicarbonate, benomyl, thiophanate methyl and trifloxystrobin. Different rotations and timing of application of these fungicides were applied either before or immediately after initial signs of powdery mildew infection and up to three times thereafter depending on rotation scheme. By the second application, disease severity was mild but increased rapidly, and it was severe by the time of the last application. Powdery mildew was controlled to some degree on the upper leaf surface by all treatments. However, efficacy was more variable on the lower leaf surface and was reduced when applications were made only at dates 1 and 2. Results show the increased efficacy of fungicides with systemic or trans-laminar activity and the possibilities of rotations with contact fungicides for resistance management.

Powdery mildew on lettuce is caused by the fungus Erysiphe cichoracearum. This disease is favored by moderate to warm temperatures and dry weather conditions. Several potential new fungicides were evaluated for control of powdery mildew on lettuce in 2001. Powdery mildew appeared in our plots by Jan 16 and reached high levels by plant maturity on Mar 13. Nontreated lettuce plants were heavily infected with powdery mildew at plant maturity, whereas the level of disease was low to virtually nonexistent in plots treated with BAS 500, Flint, Rally, Rally alternated with Microthiol, Microthiol and Quinoxyfen. The future availability of one or more of these chemistries under development could help in efforts to control powdery mildew of lettuce and to establish and maintain a fungicide resistance management program for plant disease control products of importance for this crop.

A fungicide trial was established in a commercial style greenhouse at The University of Arizona Campus Agricultural Center in November 2000 to evaluate efficacy of several fungicides for control of powdery mildew on bell pepper. Treatments included five registered fungicides: Microthiol Special (micronized sulfur), Trilogy (neem extract), Flint (trifloxystrobin), Serenade (Bacillus subtilis QST713) and AQ10 (Ampelomyces quisqualis) applied as single treatments every 10-14 days to each of four replicates. In samples to determine the percentage of leaf area affected by powdery mildew lesions and the number of leaves infected within different treatments, Microthiol Special and Serenade were significantly different from non-treated controls, while Flint and AQ10 had fewer lesions and number of leaves infected but were not significantly different from the control. Although Trilogy was not different from the control, this treatment had more lesions and number of leaves infected than all treatments.

Powdery mildew on lettuce is caused by the fungus Golovinomyces cichoracearum (Erysiphe cichoracearum). This disease is favored by moderate to warm temperatures and dry weather conditions. Several potential new fungicides were evaluated for control of powdery mildew on lettuce in 2003. Powdery mildew appeared in our plots by Jan 9 and reached high levels by plant maturity on Feb 19. Compared to non-treated plants, all treatments significantly reduced the final severity of powdery mildew on lettuce statistically. However, only a limited number of compounds, such as Rally, Microthiol Disperss, Quinoxyfen, Flint, Zoxamide, Maneb, Pristine and Cabrio, provided the degree of disease control that would be of value to growers. The trial was intended to be a downy and powdery mildew trial; therefore, some of the treatments within this study were specifically included for downy mildew. No downy mildew developed; however, the downy mildew test products did offer some protection against powdery mildew.

Powdery mildew on lettuce is caused by the fungus Erysiphe cichoracearum. This disease is favored by moderate to warm temperatures and dry weather conditions. Several potential new fungicides were evaluated for control of powdery mildew on lettuce in 2002. Powdery mildew appeared in our plots by February 15 and reached moderate levels of severity by plant maturity on March 6 to 8th. Nontreated lettuce plants were moderately infected with powdery mildew at plant maturity, whereas the level of disease was low to virtually nonexistent in plots treated with Microthiol Disperss, Rally, Quinoxyfen, Flint, and Rally alternated with Kaligreen. Furthermore, other tested products provided moderate suppression of powdery mildew. The future availability of one or more of the tested chemistries not currently registered for lettuce could help in efforts to control powdery mildew on this crop and to establish and maintain a fungicide resistance management program for plant disease control products.

Powdery mildew can occur on melons annually in Arizona. Podosphaera xanthii (Sphaerotheca fuliginea) is the plant pathogenic fungus that causes powdery mildew of cucurbits, such as cantaloupe, honeydew, watermelon, cucumber and squash. When environmental conditions are favorable, disease incidence and severity can reach economically significant levels. Development of powdery mildew on melons is favored by moderate temperatures and relative humidity, succulent plant growth and reduced light intensity brought about by a dense plant canopy. Potential new fungicides were evaluated and compared to existing chemicals for control of powdery mildew of cantaloupe in a field trial conducted during the spring of 2002 at the Yuma Agricultural Center. Among treatments, the degree of powdery mildew control ranged from minimal to essentially complete. One notable observation was the relative decrease in performance of Flint compared to earlier field trials. An isolate of the fungus from this trial was tested at Cornell University and found to be less sensitive to Flint compared to other isolates of the pathogen not previously exposed to this fungicide. This potential development of resistance by the pathogen to Flint will be examined in further studies. A moderately high level of disease had developed by crop maturity (Jun 25) on non-treated plants. The better performing treatments included Cabrio, Flint+pHortress, Foliar Supreme, Microthiol Disperss, Pristine, Procure, Quadris+Latron B-1956, Quadris+LatronB- 1956+Actigard, Quinoxyfen, Rally, Topsin M+Trilogy, and UCC-A1639. The potential availability of chemistries with new modes of action could help improve overall control of powdery mildew as well as facilitate the implementation of effective fungicide resistance management strategies.

Powdery mildew can occur on melons annually in Arizona. Sphaerotheca fuliginea is the plant pathogenic fungus that causes powdery mildew of cucurbits, such as cantaloupe, honeydew, watermelon, cucumber and squash. When environmental conditions are favorable, disease incidence and severity can reach economically significant levels. Development of powdery mildew on melons is favored by moderate temperatures and relative humidity, succulent plant growth and reduced light intensity brought about by a dense plant canopy. Potential new fungicides were evaluated and compared to existing chemicals for control of powdery mildew of cantaloupe in a field trial conducted during the spring of 2000 at the Yuma Agricultural Center A moderately high level of disease had developed by crop maturity (June 22) on nontreated plants. All treatments significantly reduced the level of powdery mildew on both sides of leaves, compared to nontreated plants. The best treatments among those tested with respect to disease control on the underside of leaves, where disease is more difficult to control than on the tops of leaves, included Actigard, Armicarb+Quadris, BAS 500, Benlate+Microthiol, Flint, Flint+Trilogy, Microthiol, Quadris+Actigard, Quadris+Benlate, Quinoxyfen, Nova, Nova+KHHUBF, Topsin, Topsin+Trilogy, Benlate, Benlate+Trilogy, Folicur, Quadris and Topsin+Microthiol. The potential availability of new chemistries for management of powdery mildew of cantaloupe and other cucurbits could help improve overall control of powdery mildew as well as facilitate the development of fungicide resistance management strategies, which strive to minimize the risk of resistance development by the pathogen to these compounds.

Powdery mildew on lettuce is caused by the fungus Erysiphe cichoracearum. This disease is favored by moderate to warm temperatures and dry weather conditions. Several potential new fungicides were evaluated for control of powdery mildew on lettuce in 2000. Powdery mildew appeared in our plots by Feb 9 and reached high levels by plant maturity on Mar 2. Nontreated lettuce plants were heavily infected with powdery mildew at plant maturity, whereas the disease ranged from low to virtually nonexistent levels in plots treated with BAS 500, Quadris+Actigard, Flint, Flint+Actigard, Flint alternated (alt.) with Trilogy, Rally, Microthiol, EksPunge alt. with Microthiol, KHHUBF-99-001, Quinoxyfen, Flint alt.with Serenade, Rally alt. with Serenade, and Serenade alt. with Microthiol. These compounds have various modes of action, and some could be available for “organic” production. The future availability of one or more of these chemistries under development could help in efforts to control powdery mildew of lettuce and to establish and maintain a fungicide resistance management program for plant disease control products of importance for this crop.

Powdery mildew on melons is an annual disease problem in Arizona. Sphaerotheca fuliginea is the plant pathogenic fungus that causes powdery mildew of cucurbits, which include cantaloupe, honeydew, watermelon, cucumber and squash. When environmental conditions are favorable, disease incidence and severity can reach economically significant levels. Factors that favor development of powdery mildew on melons include moderate temperatures and relative humidity, succulent plant growth, and reduced light intensity brought about by a dense plant canopy. Potential new fungicides were evaluated and compared to existing chemicals for control of powdery mildew of cantaloupe in a field trial conducted during the spring of 1999 at the Yuma Agricultural Center. A high level of disease had developed by crop maturity (June 29). On nontreated plants 43% of the upper leaf surface was covered by powdery mildew, whereas the level on the underside of leaves was 78%.. All of the 34 different treatments significantly reduced the level of powdery mildew on both sides of leaves, compared to nontreated plants. The best treatments among those tested with respect to disease control on the underside of leaves, where disease control is more difficult than on the tops of leaves, included Topsin+Trilogy, Benlate, Benlate+Trilogy, Quadris, A815, Topsin+Microthiol, and Topsin. The potential availability of new chemistries for management of powdery mildew of cantaloupe and other cucurbits could help improve overall control of powdery mildew as well as the implementation of fungicide resistance management strategies, which strive to minimize the risk of resistance development by the pathogen to these compounds.

Thesis (MSc)--Stellenbosch University, 2014.
ENGLISH ABSTRACT: Apples are an important export commodity for the South African market, and postharvest losses that occur as a result of decay due to infection with pathogenic fungi such as Botrytis cinerea Pers., Penicillium expansum (Link) Thom. and Neofabraea alba (E.J. Guthrie) are of major concern for all parties concerned with fruit production and distribution. Decay control of these fungi is primarily managed through the use of synthetic fungicides; however, pathogen development of resistance to these fungicides and recent worldwide concern over healthier living and a greener environment has called for the discriminate use of synthetic chemicals. This has opened up an avenue for the development of safer and more environmentally friendly alternatives to control postharvest decays. The use of plant extracts and essential oils are favoured as natural sources of antimicrobials whilst still being safe for human consumption and having no negative impact on the environment. Allium sativum (garlic) is one such plant species that is well documented for its value in improving human health and is readily available for consumption not just as a flavour component of food but also to be taken as a daily herbal diet supplement. Given the antimicrobial effectiveness of garlic against human pathogens and ailments, its value as an antifungal agent against postharvest pathogens causing grey mould, blue mould and bull’s eye rot of apples was investigated in vitro and in vivo within this study. Furthermore, an attempt was made to elucidate the chemical components of garlic extracts by gas chromatography-mass spectrometry (GC-MS). All experiments in this study were carried out with garlic extracts prepared from fresh garlic bulbs. For the in vitro experiments, two extract preparations of garlic, one containing ethanol (Extract 1) and one where ethanol had been removed by evaporation (Extract 2), was tested for antifungal action within an amended media experimental design. Both extract preparations were each subjected to two dilution series (0-80% garlic extract) with water and ethanol as diluents. Both extract preparations were successful at retarding pathogen mycelial growth and spore germination; however, concentrations of Extract 2 (ethanol evaporated) and diluted with distilled water provided markedly better inhibition of B. cinerea and P. expansum than the ethanolic dilutions of extract 2. Both extract preparations yielded similar inhibitory results when tested against N. alba. Due to the results achieved in the amended media experiments, the use of a crude garlic extract without ethanol and diluted in water was considered to be the best option for further tests throughout the remainder of the study. In vitro volatile effects of crude garlic extracts at concentrations between 0 and 40% garlic extract were subsequently tested. Garlic volatiles were effective in inhibiting pathogen mycelial growth and spore germination of all three pathogens, at lower concentrations compared to the amended media experiments. In vitro volatile exposure with garlic extracts was more effective at inhibiting N. alba than direct application of the extracts. Curative and protective application of garlic extracts and clove oil for increased fungal inhibition through synergism was tested by direct and volatile exposure to the pathogens in vivo on three economically important apple cultivars; ‘Granny Smith’, ‘Golden Delicious’, and ‘Pink Lady’. Direct exposure of artificially wounded and inoculated fruit to the garlic extract and clove oil revealed that garlic extracts applied curatively but not protectively effectively controlled decay caused by B. cinerea and P. expansum on all apple cultivars. Both curative and protective applications were ineffective in controlling N. alba. In vivo volatile exposure to the garlic extracts and clove oil did not inhibit decay on any of the cultivars and was not effective against any of the three pathogens investigated. A full chemical profile analysis was done by GC-MS analysis of garlic extract samples. The compounds diallyl disulphide, allyl methyl trisulphide, allyl methyl disulphide and dimethyl trisulphide were detected in relatively high amounts. This result suggests that the abundance of sulphur and sulphur related compounds detected may be responsible for the antifungal action noted in the experimental studies. In conclusion, garlic was shown to have antifungal activity against B. cinerea, P. expansum and N. alba. The pathogens used in this study were not compared with each other, but undoubtedly each pathogens reacts differently to exposure to the garlic extracts. It would therefore be advisable to investigate the effects of the extracts on each of the pathogens in a more in-depth study. More investigations into the application of the garlic extracts is required before it may be recommended for use; however, results for the use of garlic extracts against these postharvest pathogens and the postharvest decay they cause are promising.
AFRIKAANSE OPSOMMING: Appels is ‘n belangrike uitvoerproduk vir die Suid-Afrikaanse vrugtebedryf, maar noemenswaardige na-oes verliese word weens bederf deur patogeniese swamme soos Botrytis cinerea Pers., Penicillium expansum (Link) Thom. en Neofabraea alba (E.J. Guthrie) ervaar. Dit raak alle partye betrokke met die produksie en verspreiding van hierdie vrugsoort. Hierdie swamme word hoofsaaklik met behulp van kunsmatige swamdoders beheer, alhoewel weerstand-ontwikkeling en wêreldwye bewusmaking van ‘n gesonder leefstyl en omgewing die gebruik van kunsmatige middels streng aanspreek en die ontwikkeling van veiliger en meer omgewingsvriendelike alternatiewe middels verlang. Plant-ekstrakte en essensiële olies kan dien as sulke middels en is natuurlike bronne van anti-mikrobiese aktiwiteit, is veilig vir menslike verbruik en het ook geen negatiewe invloed op die omgewing nie. Allium sativum (knoffel) is so ‘n plantspesie wat as alternatiewe middel gebruik kan word. Dit is bekend vir sy waarde in die verbetering van menslike gesondheid, is maklik bekombaar en word nie net as ‘n geurmiddel vir voedsel gebruik nie, maar ook as ‘n daaglikse krui-aanvulling. Gegewe die anti-mikrobiese doeltreffendheid van knoffel teenoor menslike patogene en kwale, is die werking (in vitro en in vivo) teen na-oes patogene wat grys skimmel, blou skimmel en teikenvrot in appels veroorsaak, in hierdie studie ondersoek. Bepaling van die chemiese samestelling van die knoffel-ekstrak is ook met behulp van gaschromatografie massa spektrometrie (GK-MS) onderneem.Vars knoffelbolle is vir elke eksperiment in hierdie studie gebruik met die voorbereiding van die knoffel-ekstrak. Vir die in vitro eksperiment is twee knoffel-ekstrakte voorberei, naamlik: ‘n ekstrak wat etanol bevat (Ekstrak 1) en een waarvan die etanol verwyder is met verdamping (Ekstrak 2). Die ekstrakte is getoets vir werking teen fungi in kultuur-medium.. Albei ekstrakte is verdun tot twee konsentrasie reekse (0-80%) met water en etanol as verdunningsmiddels. Albei ekstrakte het suksesvolle werking getoon teenoor die patogene ten opsigte van vertraging van miseliumgroei en spoor-ontkieming, alhoewel konsentrasies van Ekstrak 2, verdun met gesuiwerede water, patogene B. cinerea en P. expansum beter onderdruk het as Ekstrak 2 verdunnings met etanol.. Beide ekstrakte en hul afsonderlike verdunnings met etanol en water het soortgelyke resultate gelewer met onderdrukking van N. alba. Volgens resultate wat verkry is van die kultuur-medium eksperimente, is Ekstrak 2 verdun met gesuiwerde water beskou as die geskikste vir verdere toetse in hierdie studie. Die vlugtige effek van Ekstrak 2 is in vitro getoets by konsentrasies tussen 0 tot 40%. Die vlugtige stowwe van knoffel het al drie patogene se groei en spoor-ontkieming effektief onderdrukby laer konsentrasies as wat gebruik is in die kultuur-medium eksperiment. Dus is in vitro blootstelling van N. alba aan die vlugtige stowwe meer effektief as direkte toediening van die ekstrakte. Die voorkomende en beskermende effek van die knoffel-ekstrak, asook naeltjie-olie, is in vivo ondersoek om te bepaal of die stowwe saam sterker onderdrukking van die patogene kon bewerkstellig. Direkte en vlugtige blootstelling is op drie ekonomies-belangrike appel-kultivars getoets, naamlik: ‘Granny Smith’, ‘Golden Delicious’ en ‘Pink Lady’. Direkte blootstelling met die knoffel-ekstrak en naeltjie-olie aan gewonde en ge-inokuleerde vrugte het aangedui dat B. cinerea- en P. Expansum-bederf net beheer kon word indien knoffel voorkomend toegedien is vir al die ondersoekte appel-variëteite. Voorkomende en beskermende toediening was onsuksesvolle om N. alba te beheer. In vivo blootstelling van die drie patogene aan die knoffel-ekstrak en naeltjie-olie se vlugtige stowwe kon nie enige van die patogene effektief onderdruk nie en was onsuksesvol in bederf-beheer. ‘n Volledige chemiese profiel is saamgestel deur GK-MS ontleding van die knoffelekstrakte. Hoë vlakke van verbindings dialliel disulfied, alliel-metiel-tri-sulfied, alliel-metieldisulfied en dimetiel-trisulfied is bespeur. Die aantal vrye sulfied en sulfied-verwante verbindings in die ekstrak kan moontlik ‘n verduideliking bied vir die anti-swam werking waargeneem gedurende hierdie studie. Ten slotte: knoffel toon ‘n anti-swam werking teenoor B. cinerea, P. expansum en N. alba. Die patogene in hierdie studie is nie met mekaar vergelyk nie, omdat elkeen uniek en uiteenlopend op knoffel reageer het. Alhoewel die huidige studie alreeds belowende resultate gelewer het, moet die ekstrak se effek op elke patogeen onderskeidelik nog in diepte ondersoek word, asook die wyse van die toediening in die na-oes praktyk voordat hierdie middel aanbeveel kan word vir gebruik.

Spot blotch is one of the most important diseases of barley (Hordeum vulgare) in Libya and worldwide. The overall aim of this study was to investigate the potential of biological control in combination with disease resistance to control spot blotch without the potential hazards of chemical application. Fungi were isolated from barley plants with spot blotch symptoms from different areas in Libya. As well as the commonly known spot blotch pathogen Bipolaris sorokiniana (teleomorph Cochliobolus sativus), Bipolaris spicifera (teleomorph Cochliobolus spicifer), Curvularia inaequalis, and Alternaria alternata were identified by their morphology and ribosomal DNA sequences. Bipolaris sorokiniana was the most serious pathogen under the test conditions; the others infected barley but caused less severe symptoms. Spot blotch resistance of barley seedlings was tested under greenhouse conditions with four Libyan cultivars (ACSAD, Nibola, Rehan, and Wadi Utbah) and two UK cultivars (Gaelic and Pastoral). Nibola was the most resistant. The ability of the organisms in three commercial biocontrol products, Trichoderma harzianum T-22 (Trianum), Streptomyces lydicus WYEC 108 (Actinovate) and Bacillus subtilis QST 713 (Serenade), to control spot blotch individually and in combination was investigated. On agar plates, all three inhibited growth of the pathogens completely on the second day of culture, except that for B. spicifera with S. lydicus there was an inhibition zone and the pathogen grew in the opposite direction. Disease severity was lowest when T. harzianum T-22 was applied individually to the most resistant cultivar, Nibola. Foliar application, soil treatment and seed coating all reduced disease severity. With foliar application, T. harzianum T-22 was more effective when applied at the same time as the pathogen than when applied one week before or four days after. In a field experiment with T. harzianum T-22, foliar application combined with seed treatment suppressed spot blotch more effectively than either method individually.

The aim of this thesis was to gain an understanding of the molecular and ecological basis for the biological control of Pythium by fluorescent pseudomonads. A fluorescent pseudomonad biocontrol agent (BCA), Pseudomonas fluorescens 54/96, identified as a potential candidate for commercial development, was analysed together with transposon induced mutants in a variety of assays for anti-fungal activity (Chapter 2). It was revealed that 54/96 had a fungistatic effect generated by a number of different mechanisms, which included nutrient competition and antibiosis. The synecology of this organism with Pythium was then compared to a similar organism (P. fluorescens SBW25) demonstrating a similar degree of anti-fungal activity (Chapter 3). The similarity of the population dynamics of these two strains prompted an examination of the genetic basis for the anti-fungal activity of the second strain, with the intention of comparing with 54/96 (Chapter 4). Again this revealed a multifactorial mode of action of SBW25 against Pythium. Whilst some mutants with reduced anti-fungal activity were deficient in growth on seed exudate others were unaffected, but the mechanisms appeared to be different to those utilized by 54/96. The comparison of strains was expanded to a larger collection of pseudomonad BCAs which were contrasted by a number of phenotypic and genotypic methods (Chapter 5). Various markers were identified which showed commonality within the different classes of BCA, the most useful of which was cyclopropanated fatty acids. These may prove to be a useful marker when screening for new pseudomonad BCAs. It was concluded that a greater understanding of the molecular, physiological and ecological basis of anti-fungal activity of bacterial will lead to the development of biocontrol strategies with improved efficacy.

Macrophomina phaseolina (Mp) and Fusarium oxysporum f. sp. fragariae (Fof) are emerging soilborne pathogens causing crown rot and Fusarium wilt, respectively, in commercial strawberry production in California. Fungicides representing eight active ingredients from four different mode of action groups (FRAC groups 1, 3, 7 and 12) were evaluated for their efficacy against each pathogen in vitro and each disease in planta. Fungicide active ingredients were evaluated for their ability to inhibit mycelial growth of both pathogens in vitro. Half-strength potato dextrose agar was amended with six different concentrations (0.01, 0.1, 1.0, 5.0, 10, 50 µg a.i./ml) of seven fungicides in FRAC groups 3, 7 and 12. Concentrations that inhibited fungal growth by 75% (EC75) compared to unamended media were determined for two different isolates each of Mp and Fof and were used to determine fungicide rates for subsequent in planta studies. Tebuconazole strongly inhibited the mycelial growth of both pathogens (average EC75 for Mp was 2.4 ppm; average EC75 for Fof was 7.48 ppm), as did metconazole (average EC75 for Mp was2.53 ppm; average EC75 for Fof was 1.28 ppm). Fludioxonil strongly inhibited mycelial growth of Mp, but had no impact on the growth of Fof. Penthiopyrad, fluopyram, flutriafol, and flutolanil were less effective at inhibiting fungal growth of either fungus. Greenhouse in planta studies evaluated twenty-four fungicide treatments (eight fungicides at low, med and high rates) that were drench applied to infested potting media two days prior to planting of pathogen susceptible strawberry cultivars (San Andreas for Mp and Monterey for Fof) and again at day 21. Controls were a non-inoculated and an inoculated water-drench treatment. Buried inoculum was recovered at days 2 and 23 and plated on selective media for colony forming unit (CFU) quantification. Plant disease assessments were made each week for 11 weeks. An analysis of variance (ANOVA) of CFUs revealed no significant differences (p > 0.05) among treatments and when compared to the non-treated control for both Mp and Fof, but showed significant decreases (p < 0.05) in CFUs between weeks 1 and 3 for both Mp and Fof. An ANOVA for disease assessments in the form of area under the disease progress curve (AUDPC) showed significant decreases of disease severity in treatments with penthiopyrad only (low, medium and high rates) (p < 0.05). There were no significant differences (p > 0.05) in AUDPC among treatments and when compared to the non-inoculated and no-fungicide controls for Fof. The data indicates that these fungicides used alone are not effective against these pathogens in planta. A strawberry plant extract (germination stimulant) was assessed for its ability to stimulate germination of Mp microsclerotia in vitro and in planta. The germination stimulant was applied as a drench at six different concentrations (0, 10, 100, 1,000, 10,000 and 30,000 ppm) to soil containing filter disk packets of microsclerotia of Mp at day 0 and 14. Filter disk packets were retrieved three days after the drench and microsclerotia were observed microscopically for germination. Results showed that the number of germinating microsclerotia was significantly higher after the application of the germination stimulant compared to non-drench and 0 ppm controls (p < 0.001). An integrated container trial was also conducted using the germination stimulant at 10,000 ppm applied three days prior to a fungicide drench with tebuconazole or thiophanate-methyl to determine the effect of fungicides on the germinated microscleotia. The use of the germination stimulant with label rates of the fungicides lowered the number of germinated intact microsclerotia significantly (p < 0.001) especially after two drench applications. The use of the germination stimulant with fungicides could be investigated further as one method for controlling soilborne diseases of strawberry.

A number of experiments were carried out to study the potential of denitrifying bacteria and reduced nitrogen compounds for control of soil-borne damping-off pathogens. Measurement of the rhizosphere pH of growing soybean roots was carried out in soil adjusted to different pH states and packed into sheet microcosms. The results showed that the rhizosphere pH of soybean was lower than the bulk soil. Nitrate reductase activity and nitrite production was then characterised for the rhizosphere of intact 14 day-old soybean roots that were incubated in nitrate substrates adjusted to different pH values under water-logged conditions. The results showed that the rate and the quantity of nitrite production increased with increasing nitrate concentration and pH in the solution. A growth room experiment was carried out to determine root colonization by denitrifying bacteria in relation to disease caused by soil-borne pathogens, which are favoured by high soil moisture (approximately -5 KPa) and low oxygen levels. Nitrite producing bacteria were isolated from soybean roots grown in Grampian (Insch) soils which had not been cropped with soybean and Thai (Phitsanulok) soils which previously had been cropped with soybean. In the first pot experiment, the nitrite producing bacteria were isolated from different root sections of 12 and 19 day-old soybean plants after 8 weeks of continuous cropping of soils with soybean (a new crop was planted every week), and using different isolation media in order to determine the genus/species composition of the denitrifying bacteria on the rhizoplane. The results showed that continuous cropping of Thai soil and Insch soil with soybean increased pre-emergence damping-off disease and decreased fresh weight yields in seedlings that did emerge. ANOVA showed significant differences between root sections for most bacterial groups monitored {Bacillus spp., Pseudomonas and Enterobacteriaceae), with regression analysis generally showing densities increasing with root age or toward the shoot base. All nitrite producing bacterial isolates were screened for antifungal activity against Macrophomina phaseolina on agar plates and between 10 and 25% of nitrite producing bacteria were found to show in vitro antagonism. In a second pot experiment, the nitrite producing bacteria were isolated from root tissue below the crown (5 cm in length) every 2 weeks of continuous cropping of soils with soybean (a new crop was planted every 2 weeks). Plate-counting was carried out to determine the population of nitrite producing bacteria while a liquid culture MPN method was used for determination of NO, N2O and N2 producing bacteria. Linear regression analysis of the incidence of pre-emergence damping-off and soybean yields in seedling that did emerge showed a highly significant negative correlation between these parameters for both soils. ANOVA showed that there was a significant difference between soil type, with the Thai soil showing higher population densities of antagonistic bacteria on soybean roots. All nitrite producing bacterial isolates were screened for antifungal activity, but the plant pathogenic fungus, Pythium ultimum, was used in this experiment. The results showed that between 10 and 40% of nitrite producing bacteria showed in vitro antagonism. However, regression analysis showed that there was no significant increase or decrease in the nitrite producing antagonistic bacterial population with continuous soybean cropping. All 900 isolates of nitrite producing bacteria isolated from the soybean rhizoplane were screened for antagonistic activity towards Pythium ultimum based on a pot trial assay in the greenhouse. As expected, very low numbers of nitrite producing bacteria showed activity against P. ultimum and only one isolate gave a significant reduction in disease incidence in pot trials. The interactive effects of nitrite producing antagonist and an Arbuscular Mycorrhizal fungus (Glomus mosseae) and Bradryrhizobium japonicum, on control of the fungal pathogens, P. ultimum or M. phaseolina were investigated in the greenhouse. The results showed that improved plant growth was obtained with certain combined inocula involving nitrite producing bacterial antagonists, Glomus mosseae and Bradryrhizobium japonicum.

Recent demands for minimally-processed foods, has led to the exploration of plant-derived essential oil (EO) compounds as an alternative means of preservation. While some of these compounds are effective against foodborne pathogens, their strong aroma and "spicy" flavor are not compatible with the flavor of juice. The purpose of this research was to evaluate the antimicrobial activity of three EO compounds (thymol, eugenol, and trans-cinnamaldehyde) alone and in combination with three naturally-occurring apple aroma compounds (hexanal, trans-2-hexenal and 1-hexanol) in order to identify combinations that lower the concentrations needed to destroy foodborne pathogens in apple juice. The standard agar dilution method (SAD) and the Spiral Gradient Endpoint method (SGE) were compared for their abilities to determine minimum inhibitory concentrations (MIC) of the EO compounds. Both methods produced similar patterns of inhibition; however, the MICs produced by the SGE system were significantly higher than those produced by the SAD method of analysis (P<0.05). Since the results produced by the SAD method were more comparable with those published in literature, this method was selected for further testing. In general, the EO compounds were significantly more effective against the test pathogens (Listeria monocytogenes, Salmonella Typhimurium and Staphylococcus aurues) than were the apple aroma compounds (P<0.05). Cinnamaldehye exhibited the highest degree of activity, followed by thymol and eugenol. Eugenol was the only compound that acted synergistically with the apple aroma compounds. The most effective compounds (cinnamaldehyde, eugenol and trans-2-hexenal) were then used to inactivate L. monocytogens and S. Typhimurium in preservative-free apple juice. In most cases, treatment with 0.05% of each compound resulted in a 5 log CFU/ml reduction in bacterial numbers following one day of storage at 4°C or 25°C. Likewise, treatment with antimicrobial combinations (containing 0.025% of trans-2-hexenal in combination with 0.025% trans-cinnamaldehyde or eugenol) also resulted in a 5 log CFU/ml reduction in bacterial numbers, following one day of storage at 4°C or 25°C. Since these combinations contained half the effective concentration of the essential oil compounds, they may be used to preserve the microbial quality of apple juice, while reducing the likelihood of off flavors in the final juice product.
Ph. D.

[Truncated abstract] Weeds are a major limitation to agricultural and horticultural production and the main method of control is the use of herbicides. In addition to the resulting chemical pollution of the environment, the wide spread and continues use of herbicides have resulted in many weeds developing resistance to commonly used herbicides. This study investigated the potential of using green manures as a cultural method of control of weed invasion in agricultural fields. To understand the general mechanisms involved in the suppression of seed germination in green manure amended soils, seeds of crop species with little or no dormancy requirements were used in certain studies. Lettuce (Lactuca sativa) and cress (Lepidium sativum) seeds were sown to a sandy soil amended with green manures of lupin (Lupinus angustifolius), Brassica juncea, or oats (Avena sativa) to determine if the amendments affected seed germination and/or decay. It was hypothesised that the addition of plant material would increase the microbial activity of the soil thereby increasing seed decay, under laboratory and greenhouse conditions. Initial experiments used lettuce, cress and lupin seeds. Lettuce and cress are commonly used as standard test species for seed viability studies. Subsequent experiments used seeds of annual ryegrass (Lolium rigidum), silver grass (Vulpia bromoides), wild radish (Raphanus raphanistrum) and wild oat (Avena fatua) as these weed species are commonly found throughout agricultural regions in Western Australia. Amending the soil with lupin or Brassica green manure was established as treatments capable of developing environments suppressive to seed germination. Lupin residues as green manure showed the strongest inhibition of seed germination and seed decay. The decay of certain seeds was enhanced with changes to soil microbial activity, dissolved organic carbon and carbon and nitrogen amounts in lupin amended soil. Seeds of weed species were decayed in lupin amended soil, but showed varied degree of decay. Annual ryegrass and silver grass were severely decayed and wild oat and wild radish were less decayed, in lupin amended soil.

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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)
Insect symbionts may have unknown functions in the interaction between insect-plant and insect with microorganisms that co-inhabit the same space. The objective of this study was to investigate the antagonism potential of symbiont microbiota from oral secretion D. saccharalis collected in the field, against Fusarium verticillioides and Colletotrichum falcatum pathogens commonly found inside the cane. For this, 4° and 5° instar caterpillars were collected inside sugarcane varieties RB-835 054 and SP- 813 250, and brought to the lab inside the cane stalks. The microbiota of oral secretion was transferred to two selective media, NA (nutrient agar) for bacteria and DRBC (dicloran Rose Bengal Chloramphenicol) for yeast. Based on morphology and coloration of the colonies twenty colonies of bacteria and yeast were selected. Four culture media were tested in co-cultivation of F. verticillioides and C. falcatum versus bacteria or yeast isolates: PDA (potato, dextrose, agar), YEPD (yeast extract, peptone, dextrose), CCS (supplemented cane broth) and NA (Nutrient Agar). The most suitable culture medium for growth of most microorganisms was BDA. Antagonism potential of 82 bacterial isolates and 87 yeast isolates to C. falcatum and F. verticillioides was assessed using a visual scale of categories 1 to 4, with 4 being the maximum degree of antagonism. Isolates that allocated category greater than or equal to 2 were evaluated in co-culture with C. falcatum and F. verticillioides as the percentage of growth inhibition. It was possible to identify four isolates of bacteria which have the potential to inhibit growth of pathogens and 9 isolates with the same potential but with much lower percentages. These results demonstrate that some isolates of bacteria and yeast may influence the relationship between the bit-rot complex and sugarcane plant, may in future be used as a biological control of these pathogens or have some molecules of biotechnological interest extracted and purified.
Simbiontes de insetos podem ter funções desconhecidas na interação entre insetoplanta e do inseto com microrganismos que co-habitam o mesmo espaço. O objetivo desse estudo foi investigar o potencial de antagonismo da microbiota simbionte, presentes na secreção oral de Diatraea saccharalis, com os fitopatógenos Fusarium verticillioides e Colletotrichum falcatum que habitam o colmo de cana-de-açúcar. Para isso, foram coletadas, nas variedades de cana RB-835054 e SP-813250, lagartas de 4° e 5° instar e trazidas para o laboratório junto aos toletes de cana. A microbiota da secreção oral foi transferida para dois meios de cultura seletivos, NA (nutrient agar) para bactérias e DRBC (Dicloran Rosa-de-Bengala Cloranfenicol) para leveduras. Baseado na morfologia e coloração das colônias, foram selecionadas, vinte colônias de bactéria e também de levedura de 5 lagartas. Foram testados quatro meios de cultura: BDA (batata, dextrose, agar), YEPD (yeast extract, peptone, dextrose), CCS (caldo-de-cana suplementado) e NA para os testes de cultivo pareado. O meio de cultura mais adequado para o crescimento da maioria dos microrganismos foi o BDA. O potencial de antagonismo de 82 isolados de bactéria e 87 isolados de levedura a C. falcatum e F. verticillioides foi avaliado através de uma escala visual de categorias de 1 a 4, sendo 4 o grau máximo de antagonismo. Os isolados a que foi atribuída categoria maior ou igual a dois foram avaliados em co-cultivo com C. falcatum e F. verticillioides quanto à porcentagem de inibição do crescimento. Foi possível identificar 4 isolados de bactéria que tem o potencial de inibir o crescimento dos fitopatógenos e 9 isolados com o mesmo potencial, porém com porcentagens menores. Esses resultados demonstram que alguns isolados de bactérias e leveduras podem influenciar na relação existente entre o complexo broca-podridão e a planta de cana-de-açúcar, podendo, futuramente, serem utilizados como controle biológico desses fitopatógenos ou terem algumas moléculas de interesse biotecnológico extraída e purificada.

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Endophytic fungi are those living at least a part of their life cycle within plant tissue, apparently without causing any damage to their hosts. These microorganisms have been studied for their capacity to produce secondary metabolites of biotechnological interest, and also for their ability for biological control of phytopathogens. Endophytic fungi have been isolated from a wide variety of plant species, including crops such as common bean (Phaseolus vulgaris L.). Fungi from the genus Diaporthe were abundant in the endophytic community of this legume. The objectives of this study were to evaluate the antimicrobial activity and the potential use of endophytic fungi of P. vulgaris in the control of phytopathogens of bean, and to analyze phylogenetic relationships and genetic variability of endophytic fungi of the genus Diaporthe isolated from P. vulgaris. Dual culture assays were performed between ninety endophytic fungi and four phytopathogenic fungi (Colletotrichum lindemuthianum, Fusarium oxysporum, Rhizoctonia solani and Sclerotinia sclerotiorum) to assess for the ability of endophytic fungi inhibit the growth of phytopathogens. The isolates that were able to inhibit the four phytopathogens tested were selected for cultivation and obtention of metabolites crude extracts. These extracts were evaluated for antibacterial and antifungal activity. The sequences of the ITS region of rDNA of the isolates of the genus Diaporthe from common bean, along with the partial sequences of the genes encoding translation elongation factor 1-α, β-tubulin and calmodulin were used for the study of the phylogenetic relationships of these isolates. Molecular markers based on sequences of retrotransposons, IRAP (Inter-Retrotransposon Amplified Polymorphism) and REMAP (Retrotransposon-Microsatellite Amplified Polymorphism), were used for the analysis of genetic variability. It was found that endophytic fungi of common bean are able to inhibit the in vitro growth of phytopathogenic fungi and that metabolites crude extracts of endophytic exhibit significant antimicrobial activity especially regarding the inhibitory activity against Gram-positive bacteria (Listeria monocytogenes, Bacillus cereus, B. subtilis and Staphylococcus aureus). Diaporthe sp., D. infecunda, D. melonis and D. phaseolorum are members of the endophytic fungal community in the common bean as verified by multilocus phylogenetic analysis. Cluster analysis, conducted with pooled data from IRAP and REMAP markers, was consistent regarding what was observed in multilocus phylogeny and revealed the existence of high genetic variability, particularly among isolates of D. infecunda. It was concluded that the metabolites crude extracts of endophytic fungi of common bean exhibit promising antimicrobial activity; that endophytic fungi used in this study have the potential to control phytopathogens that affect the bean crop; the multilocus phylogenetic approach was more effective than individual analysis of ITS sequences in the study of phylogenetic relationships of endophytic fungi of the genus Diaporthe from common bean and that IRAP and REMAP markers can be employed in the study of genetic variability of this genus of fungi.
Fungos endofíticos são aqueles que vivem, em pelo menos uma parte de seu ciclo de vida, no interior de tecidos vegetais, sem causar aparentemente qualquer dano a seus hospedeiros. Esses micro-organismos têm sido estudados em relação à capacidade de produzirem metabólitos secundários de interesse biotecnológico e quanto ao potencial de controle biológico de fitopatógenos. Fungos endofíticos já foram isolados de uma ampla variedade de espécies vegetais, incluindo culturas agrícolas como o feijoeiro comum (Phaseolus vulgaris L.). O gênero Diaporthe foi um dos mais abundantes na comunidade endofítica dessa leguminosa. Os objetivos do presente estudo foram avaliar a atividade antimicrobiana e o potencial de uso de fungos endofíticos de P. vulgaris no controle de fitopatógenos da cultura do feijoeiro e analisar as relações filogenéticas e a variabilidade genética de fungos endofíticos do gênero Diaporthe isolados de P. vulgaris. Ensaios de cultura dupla entre 90 isolados endofíticos e quatro fungos fitopatogênicos (Colletotrichum lindemuthianum, Fusarium oxysporum, Rhizoctonia solani e Sclerotinia sclerotiorum) foram realizados para verificar a capacidade dos isolados endofíticos inibirem o crescimento dos fitopatógenos. Os isolados que foram capazes de inibir os quatro fitopatógenos testados foram selecionados para cultivo e obtenção de extratos brutos de metabólitos. Esses extratos foram avaliados quanto à atividade antibacteriana e antifúngica. As sequências da região ITS do rDNA dos isolados do gênero Diaporthe do feijoeiro, juntamente com as sequências parciais dos genes codificam o fator de elongação da tradução 1-α, β-tubulina e calmodulina, foram utilizadas para o estudo das relações filogenéticas desses isolados. Marcadores moleculares baseados em sequências de retrotransposons, IRAP (Inter-Retrotransposon Amplified Polymorphism) e REMAP (Retrotransposon-Microsatellite Amplified Polymorphism), foram utilizados para a análise de variabilidade genética. Foi constatado que os fungos endofíticos do feijoeiro comum são capazes de inibir o crescimento in vitro de fungos fitopatogênicos e, que extratos brutos de metabólitos de isolados endofíticos exibem atividade antimicrobiana significativa, principalmente em relação à atividade inibitória sobre bactérias Gram-positivas (Listeria monocytogenes, Bacillus cereus, B. subtilis e Staphylococcus aureus). Por meio de análise filogenética multilocus, verificou-se que Diaporthe sp., D. infecunda, D. melonis e D. phaseolorum são integrantes da comunidade fúngica endofítica do feijoeiro comum. A análise de agrupamento, realizada com os dados dos marcadores IRAP e REMAP em conjunto, foi coerente em relação ao que foi verificado na filogenia multilocus e, revelou a existência de grande variabilidade genética, sobretudo entre os isolados de D. infecunda. Concluiu-se que extratos brutos de metabólitos de fungos endofíticos do feijoeiro exibem atividade antimicrobiana promissora; que fungos endofíticos utilizados neste estudo apresentam potencial para controle de fitopatógenos que acometem a cultura do feijoeiro; que a abordagem filogenética multilocus foi mais efetiva que análise individual de sequências de ITS no estudo das relações filogenéticas de fungos endofíticos do gênero Diaporthe do feijoeiro e que marcadores IRAP e REMAP podem ser empregados no estudo de variabilidade genética de fungos desse gênero.

Sclerotinia sclerotiorum, a fungal pathogen of over 400 plant species has been estimated to cost UK based farmers approximately £20 million per year during severe outbreak (Oerke and Dehne 2004). S. sclerotiorum disease incidence is difficult to predict as outbreaks are often sporadic. Ascospores released from the fruiting bodies or apothecia can be dispersed for tens of kilometres. This makes disease control problematic and with no S. sclerotiorum resistant varieties available, growers are forced to spray fungicides up to three times per flowering season in anticipation of the arrival of this devastating disease. This thesis reports the development of the first infield S. sclerotiorum biosensor which aims to enable rapid detection of airborne ascospores, promoting a more accurate disease risk assessment and fungicide spraying regime. The sensor is designed to detect the presence of oxalic acid, the main pathogenicity factor secreted during early S. sclerotiorum ascospore germination. Upon electrochemical detection of this analyte in the biosensor, a binary output is relayed to farmer to warm him of a disease risk. This project focused on the development of a nutrient matrix which was designed to be contained within the biosensor. The role of this matrix was to promote the growth of captured airborne S. sclerotiorum ascospores and induce high levels of oxalic acid secretion. The use of the designed biological matrix to promote oxalic acid production was tested during three field trials in S. sclerotiorum artificially inoculated fields. This thesis describes the use of contemporary pathogenomics technologies to further investigate candidate genes involved in pathogenicity alongside the secretion of oxalic acid. A pre-described bioinformatics pipeline was used to predict the S. sclerotiorum secretome to identify potential effector proteins as well as explore proteins which are unique to S. sclerotiorum to be used as other novel targets for detection. GFP tagged constructs were designed to investigate the expression of the putative targets for S. sclerotiorum detection. The transcriptomes of wild type and oxalic acid deficient S. sclerotiorum strains during infection as well as during a saprotrophic stage were investigated. This study provided expression support for not only some of the unannotated genes identified in the putative secretome, but some candidate genes speculated to be involved in infection.

Il est connu que les ‘pesticides’ (produits phytopharmaceutiques) utilisés depuis plus de 100 ans en agriculture sont néfastes, autant pour les vivants que pour l’environnement. Il est urgent de développer un remplacement de ces produits toxiques à l’homme et à son entourage. Le but de ce travail est double : d’abord, trouver les micro-organismes non pathogènes dans les vignobles et, ensuite, les mettre en concurrence avec les parasites de la vigne. Nous envisageons de tirer bénéfice sur deux fronts : 1. Lutte biologique contre les maladies cryptogamiques de la vigne, 2. Déclenchement de la résistance chez la vigne contre les maladies. Les micro-organismes utilisés deviennent ainsi, des ‘bio-fongicides’ qui remplacent les produits chimiques. La vigne est attaquée par plusieurs parasites; les plus importants en Bourgogne sont : le mildiou par Plasmopara viticola, l’oïdium par Uncinula necator, et la pourriture grise par Botrytis cinerea. Pour cette thèse nous avons choisi de nous concentrer sur la « pourriture grise » de la vigne et d’autres végétaux, maladie provoquée par le redoutable champignon, Botrytis cinerea. Nous avons isolé quelques espèces de Pythium non pathogènes à partir du sol de vignobles ainsi que d’autres sols agricoles et forestiers. .
The pesticides used in agriculture for controlling phyto-pathogens have hazardous health effects on plants, animals and humans. Massive application of toxic pesticides is a serious problem today in almost all developing countries. Developing safer, environment-friendly biological products may help in overcoming the risks posed by pesticides and may also help in protecting public health, thereby promoting safer means of pest control. This thesis aims at the Biological control of plant diseases by non-phytopathogenic microorganisms, isolated from soil of vineyards and rhizosphere of crop fields. Introduction of these microorganisms leads to a twofold benefit 1. Biological control of the disease by the antagonist effect of the micro-organisms; 2. Enhanced disease resistance in plants to phytopathogens. Hence, these microorganisms could serve as an alternative to chemical control of plant diseases. Grapevine is challenged every year by fungal and viral and bacterial parasites. Among the fungal, the major ones are downy mildew by Plasmopara viticola, powdery mildew by Uncinula necator, black rot by Guignardia bidwellii, phomopsis leaf, cane spot and fruit rot disease by Phomopsis viticola, Eutypa Dieback by Eutypa armeniaceae and grey mould by Botrytis cinerea. Crown gall by bacterial parasite Agrobacterium tumefaciens, and viral diseases includes Peach Rosette mosaic virus disease, tomato ringspot and tobacco ringspot by nematode Xiphinema americanum. .

Colletotrichum coccodes strain DAOM 183088 is considered a potential bioherbicide for velvetleaf (Abutilon theophrasti), a devastating weed in North American corn and soybeans. Risk assessment studies have created a demand for an accurate and robust method to monitor this strain, and to distinguish it from indigenous background population of microorganisms present in the field. Safe biological control management of velvetleaf also requires comprehensive understanding of the pathogenicity determinants employed by this host-specific fungus to establish infection on velvetleaf, an aspect central to a safe biocontrol strategy task. In this study, molecular markers were designed that allow strain specific identification of the bioherbicide strain of C. coccodes and its identification within complex plant and soil matrices. An assay was developed to quantify C. coccodes from deliberate release field soil samples, in which biases caused by soil-originating PCR inhibitors were monitored on a sample per sample basis. The developed external control assay allowed for the estimation of target C. coccodes DNA quantities with normalization for the presence of PCR inhibitory compounds. Kinetic growth curves of disease development were performed for C. coccodes wild-type and T20-a (genetically engineered for hypervirulence with the NEP1 (necrosis and ethylene inducing peptide) gene) strains on velvetleaf leaves over a period of 14 days after C. coccodes infection. The wild-type strain was more efficient at infecting velvetleaf than the transgenic T-20a strain, while expression of NEP1 could not be detected suggesting that the introduced gene may not be transcriptionally active in the transformed strain, a result in conflict with previous observations. Velvetleaf and C. coccodes genes specifically upregulated at 12 and 24 h after fungal infection were cloned and differentially screened by microarrays. The resulting EST collection was sequenced and assigned to putative functions. Early gene up-regulation was confirmed by QRT-PCR analysis for type 3 metal lothionein, EREB, WRKY, and bZIP transcription factors, reticuline oxidase, ascorbate peroxidase, and ACC oxidase gene candidates. In addition, type 2, type 3 metallothionein, and bZIP gene expression profiles were investigated over a period of 14 days after C. coccodes infection, and the results indicated that C. coccodes altered the expression of all three gene analyzed.

The antibiotic, 2,4-diacetylphloroglucinol (Phl), is produced by a range of naturally isolated fluorescent pseudomonads, found in disease suppressive soils. The natural isolate, P. fluorescens F113, protects pea plants from the pathogenic fungus, Pythium ultimum, by reducing the number of pathogenic lesions on the plant's roots. This beneficial effect was however, outweighed by the F113 causing an overall reduction in the emergence of the pea plants in the infected soil. The gene locus responsible for the Phl production was shown to be functionally conserved between the P. fluorescens F113 and another Phl producing organism, P. fluorescens Q2-87. Following identification of this functional sequence homology, the genes were isolated from F113, by optimised, long PCR. The 6.7-kb gene cluster was inserted into the chromosome of a non-pathogenic P. fluorescens, SBW25, which can effect biological control against the plant pathogen, Pythium ultimum through competitive exclusion of the fungus, by means of its strong colonising competence. The insertion was a targeted, homologous recombination designed to insert the Phl coding genes, from the F113, into a non-essential, lacZY coding region of the SBW25 chromosome. The transformed strains of SBW25 assumed two different morphological appearances. The morphological changes were noted at a ratio of 1:1 of normal morphology and altered morphology. Transformation of SBW25 with the Phl locus without this repressor element led to transformants with only normal morphology. All transformants were able to suppress P. ultimum through antibiotic production following the Phl transformation. However, the fitness of the transformants was reduced in flask culture, at 30°C, against the un-transformed SBW25. The organisms transformed with the entire Phl locus were seen to clump together in the culture media. The strain transformed with the Phl locus lacking the repressor element behaved normally. When inoculated on pea seedlings, the strain containing no repressor element behaved similarly to the F113, causing lower pea seed emergence. A transformant containing the entire Phl genetic locus had not lost its environmental competence on the pea roots, maintaining a high population, but was unable to maintain a high population in the surrounding soil.

Sclerotinia sclerotiorum is a necrotrophic fungal pathogen with a worldwide distribution and a wide host range, including many economically important crops. The control strategies for this pathogen and related species include using fungicides, biological control agents and cultural practices such as crop rotations. However, the genetic diversity and the long term survival structures (sclerotia) of this pathogen, combined with the recent discovery of the related species S. subarctica in England and the need for growers to implement integrated disease management strategies means that new control measures need to be sought. Biofumigation, using green manures which are macerated and ploughed into the soil, may be a useful new control approach in an integrated programme. Microcosm and in vitro experiments clearly showed that volatiles released from biofumigation crops have a direct inhibitory effect on the mycelial growth and carpogenic germination of S. sclerotiorum sclerotia. The most effective biofumigation crop for inhibiting carpogenic germination varied depending on whether the volatiles released from the biofumigant crops were in direct contact with the sclerotia when the most effective crop was Raphanus sativus ‘Terranova’, or in the vapour phase when the most effective crop was B. juncea ‘Vittasso’. Carrot root inoculations showed that the number of sclerotia produced on carrot roots was significantly affected by S. sclerotiorum isolate. However, the results also showed that the weight of individual sclerotia produced by different isolates was influenced by carrot accession, but not by S. sclerotiorum isolate. Additionally, the carrot plant and detached leaf inoculations showed significant differences in the rate of lesion progression of S. sclerotiorum on different carrot accessions, indicating differences in susceptibility to the pathogen. S. subarctica microsatellite haplotypes identified in this research were shown to be shared between Scotland and Norway, and between crop plants and meadow buttercup. However, the English population did not share any microsatellite haplotypes with any other population, and analysis indicated that this S. subarctica population in England may be isolated and inbred.

Sclerotinia sclerotiorum is a widespread plant pathogen that produces structures known as sclerotia. When sclerotia germinate they give rise to infective hyphae, myceliogenic germination, or they produce ascocarps, carpogenic germination. Biological control has usually targeted sclerotia or ascospores. The main objectives of the research presented herein were to observe the effect of a mycoparasite and fungus gnats (Bradysia coprophila) on the survival of sclerotia in vitro and in field conditions, and to study the enzymatic activity of the mycoparasite when in contact with sclerotia damaged by fungus gnats.
In this research several mycoparasites were evaluated for their efficacy to degrade sclerotia in soil. From these tests, an isolate of Trichoderma hamatum, TMCS 3 proved to be the most effective. Larvae of fungus gnats have also been reported to feed on sclerotia. When both organisms were combined in laboratory tests, fewer sclerotia survived than when the organisms acted alone. Sclerotia recovered from this treatment contained fewer viable cells when compared to sclerotia recovered from treatments with TMCS 3 or fungus gnats alone. The results obtained from field trials showed that TMCS 3 was effective at degrading sclerotia. Unfortunately environmental conditions were not always optimal for the establishment of high populations of fungus gnats. Few larvae were observed feeding on sclerotia and no significant differences were found among treatments.
Growth of TMCS 3 was studied using different carbon sources as substrates, including sclerotia of S. sclerotiorum. Biomass obtained from this latter treatment was significantly larger than on the other carbon sources tested. Enzymatic activity was also induced by the presence of sclerotia. In many cases, sclerotial exudates from mechanically damaged sclerotia or sclerotia damaged by larval feeding showed that the concentration of amino acids, carbohydrates, and electrolytes was increased as damage to the sclerotia has increased. Exudation of protein was not different when damaged and undamaged sclerotia were compared. Exudates from sclerotia with the melanized rind completely removed by fungus gnats feeding accelerated the germination of conidia of TMCS 3. These heavily damaged sclerotia also enhanced the growth of TMCS 3 when both organisms were grown together. However enzymatic (i.e. glucanase and chitinase) activity of TMCS 3 was not increased by the damage to the sclerotia. When damaged sclerotia were buried in soil infested with TMCS 3 they were degraded faster when the medulla of sclerotia was completely uncovered by larval feeding.

Thesis (MSc)--University of Stellenbosch, 2002.
ENGLISH ABSTRACT: Seed germination is the most vulnerable time in a plant's life cycle, since the thick protective seed coat ruptures and the moist and humid soil environment not only favours seed germination, but also the growth and development of plant pathogens. Infection of plant seeds during germination, however, is the exception rather than the rule. Plant seeds have - - -developed a--cemplex preformed defense mechanism that includes anttfungal agents thatdiffuse into the surrounding environment to form a protective layer around the seed. This protective layer prevents fungal and bacterial pathogens from infecting the young seedling. Over the last decade, scientists have studied the defense mechanisms of different seeds in an effort to understand and ultimately to introduce and/or manipulate these mechanisms in plants as part of the plant's endogenous disease resistance to pathogens. Various chemical compounds, peptides and proteins that showed strong in vitro activities against various fungi were isolated in these efforts. The mere demonstration of in vitro activity alone, however, is not sufficient to assign a defense role to these antifungal agents. Typically, mutant plants that have lost the ability to produce the antifungal agent, or mutants that are overproducing the agent, have been used to correlate the mutant phenotype to either a decline or increase in disease resistance respectively. Genetic transformation and the subsequent development of transgenic plants have made an unprecedented impact in this regard, specifically in understanding the role of specific defense-related proteins and their interaction with plant pathogens. In this study, the antifungal peptide, Hs-AFP1, from Heuchera sanguinea, a plant defensin, was evaluated in a heterologous in planta environment as a defense protein with potential for engineering disease resistant crops. The in vitro assays performed with Hs-AFP1 against Botrytis cinerea showed antifungal activities of 88% growth inhibition at a concentration of 8 J,lg/ml of the purified peptide, while inducing a characteristic hyperbranching effect on the Botrytis hyphae. Tobacco was subsequently transformed with a construct, pFAJ3068, expressing Hs-AFP1 under the strong constitutive 35S promoter. The peptide was targeted to the apoplastic region with the signal peptide from Mj-AMP2, an antimicrobial peptide from Mirabilis jalapa. Due to reports of peptide instability in transgenic plant systems, two additional constructs were prepared and transformed into tobacco to anticipate possible Hs-AFP1 instability in the heterologous tobacco environment. A putative peptide stabilization construct, pHs-EXG1, consisted of a fusion between Hs-AFP1 and the antifungal exo-glucanase (encoded by EXG1) from Saccharomyces cerevisiae. A control construct, pMj-EXG1, expressing EXG1 targeted to the apoplastic region with the Mj-AMP2 signal peptide, was also prepared and transformed into tobacco to normalize the background antifungal activity as a result of the exoglucanase in the fusion construct lines. Tobacco was successfully transformed with pFAJ3068, pHs-EXG1 and pMj-EXG1, resulting in transgenic tobacco lines designated THs, THE and TME respectively. Transgene expression was confirmed for the THs and THE transgenic lines. The translation of these transcripts into proteins was also confirmed with Western blot analysis. Moreover, the heterologous production of Hs-AFP1 in tobacco led to an increase in disease resistance to B. cinerea in the THs lines in comparison with the untransformed tobacco controls. An increase of up to 42% in disease resistance was observed in an in planta detached leaf assay. Crude protein extracts from the THs lines were also analyzed in an in vitro quantitative fungal growth assay. This assay confirmed the results obtained with the disease resistance assay, with crude protein extracts exhibiting up to 40% fungal growth inhibition. The incubation of B. cinerea in the presence of crude protein extracts from THs lines resulted in hyperbranching of the fungal hyphae, which is characteristic of Hs-AFP1 activity. From these analyses it was clear that the heterologously expressed Hs-AFP1 was quite stable in the transgenic environment. The fusion between Hs-AFP1 and EXG1 did not increase the stability of Hs-AFP1, but rather led to a loss of the Hs-AFP1 activity. All the analyses performed showed the THE lines to be reduced in their ability to inhibit fungal infection in comparison to the THs line. Also, microscopic analysis of the effects of the crude THE extracts on B. cinerea growth showed no hyperbranching activity, again confirming the loss of peptide activity due to the fusion to EXG1. This is in agreement with previous work, in which sarcotoxin 1A was fused to a reporter gene and also lost activity. Although integration of the Mj-EXG1 expression cassette was confirmed, no mRNA levels could be detected with Northern blot or RT-PCR analysis of the TME lines. These lines also did not show any in vitro antifungal activities, probably indicating post-transcriptional gene silencing. This silencing was overcome in the fusion constructs that were expressed in the THE plant lines. These lines also showed EXG1 protein activity, as measured by ~-glucosidase assays. Although the THE lines did not serve the functions originally envisaged, they fortuitously showed that a fusion strategy might stabilize glucanase expression in a transgenic environment. A variety of glucanases have been shown to be prone to gene silencing when overexpressed in a plant environment and the yeast glucanase can now be added to that list if it is not present as a fusion protein. Overall, this study confirmed that Hs-AFP1 is involved in plant defense systems and provided valuable information on the stability of small peptides in a heterologous environment. The positive results obtained with overexpressed Hs-AFP1 on fungal inhibition in this study merits further investigations into the use of this peptide in the engineering of disease-resistant crops.
AFRIKAANSE OPSOMMING: Saadontkieming is die mees vatbare tyd vir siekteontwikkeling gedurende 'n plant se lewenssiklus. Die saadhuid bars en die vogtige grondkondisies bevoordeel nie net saadontkieming nie, maar ook die groei en ontwikkeling van plantpatogene. Infeksie van plantsade tydens ontkieming is egter die uitsondering eerder as die reël. Plantsade besit komplekse -veraeaigingsfueganfsmes-reen moontlike - patoqeeninteksies. Die meqanismes sluit die produksie van antifungiese agense, wat tydens saadontkieming na die omliggende omgewing diffundeer om 'n beskermende sone om die ontkiemende saad te vorm, in. Die gevolglike antifungiese sone beskerm die saad teen infeksie deur bakterieë en swamme. Gedurende die laaste dekade het navorsers baie aandag aan die bestudering van plantsaadverdedigingsmeganismes gegee. Dié kennis word gebruik om die verdedigingsmeganismes beter te verstaan, asook om dié meganismes te manipuleer en/of oor te dra aan plantspesies met inherente swak weerstandsmeganismes wat gereeld aan plantpatogeeninfeksies onderhewig is. Navorsing op plantsade het tot die isolasie van verskeie chemiese agense, peptiede en proteïene, wat sterk in vitro aktiwiteite teen 'n wye reeks swampatogene vertoon, gelei. Die vermoë van dié agense om swamme in 'n in vitro omgewing te inhibeer, is alleen egter nie 'n bewys dat hulle 'n rol in plantverdeging speel nie. Studies waar mutante gebruik word, is gewens om addisionele bewys te lewer dat die substanse 'n rol in plantverdediging vervul. Sodanige mutante sluit plantlyne, waarin die geen van belang gemuteer is of ooruitgedruk word om so die rol van die geen in 'n in planta omgewing te bepaal in. In hierdie toepassings het genetiese transformasie en die daarstelling van transgeniese plante 'n ongeëwenaarde bydrae gelewer. In dié studie is die antifungiese peptied, Hs-AFP1, wat aan die peptiedgroep van plant- "defensins" behoort en van Heuchera sanguine a afkomstig is, in 'n heteroloë in planta omgewing geëvalueer as 'n verdedigingspeptied met die potensiaal om in die generering van transgeniese siektebestande gewasse gebruik te word. Die antifungiese aktiwiteit van Hs-AFP1 is teen Botrytis cinerea in 'n in vitro reaksie geëvalueer, waar die toediening van 8 ,",g/mlgesuiwerde Hs-AFP1 peptied aanleiding gegee het tot 'n 88% afname in hifegroei van B. cinerea. Hipervertakkings van swamhifes, 'n kenmerkende eienskap van Hs-AFP1 aktiwiteit, kon duidelik waargeneem word. Tabakplante is voorts getransformeer met 'n konstruk, pFAJ3068, wat die koderende geen van Hs-AFP1 onder die sterk konstitutiewe CaMV 35S promotor bevat het. Die peptied is met behulp van die seinpeptied wat afkomstig is van die Mirabilis jalapa antimikrobiese peptied, Mj-AMP2, na die apoplastiese omgewing geteiken. Voorheen is gerapporteer dat transgeniese peptiede in die heteroloë omgewing soms onstabiel is. Dit het gelei tot die generering van twee addisionele konstrukte om die moontlikheid van peptiedonstabiliteit te ondervang. 'n Stabiliseringskonstruk, pHs-EXG1, bestaande uit In versmelting tussen Hs-AFP1 en In antifungiese eksoglukanase van Saccharomyces cerevisiae, gekodeer deur EXG1, is in tabakplante getransformeer. In Kontrolekonstruk, pMj-EXG1, met die EXG1-geen saam met die Mj-AMP2-seinpeptied, is ook voorberei en in tabakplante getransformeer. Dit is gebruik om die antifungiese aktiwiteit van die eksoglukanase in die antifungiese aktiwiteitstoetse van die stabiliseringskonstruk te kwantifiseer en te normaliseer. Tabak is suksesvol met pFAJ3068, pHs-EXG1 en pMj-EXG1 getransformeer, wat onderskeidelik gelei het tot die sogenaamde THs, THE en TME transgeniese tabaklyne. Transgeentranskripsie en -translasie in die THs en THE tabaklyne is onderskeidelik deur Noordelike- en Westelike-kladanalises bevestig. Die aktiewe uitdrukking van Hs-AFP1 het die vermoë van tabakplante om B. cinerea infeksies te weerstaan, met tot 42% verhoog in vergelyking met ongetransformeerde kontrole tabakplante tydens 'n in planta siekteweerstandstoets. Totale proteïenekstrakte van THs tabaklyne is voorts ook in In in vitro inhibisietoets geëvalueer, wat gelei het tot resultate wat goed met dié van die in planta toetse ooreenstem. Die totale proteïenekstrakte het swamgroei met 40% geïnhibeer en die kenmerkende hipervertakking van Hs-AFP1-aktiwiteit is ook mikroskopies waargeneem. Resultate wat verkry is vanaf al die analises wat op die transgeniese THs tabaklyne uitgevoer is, het aangedui dat Hs-AFP1 baie stabiel in die heteroloë tabakomgewing is en peptiedstabiliteit was dus nie In probleem, soos verwag is nie. Die fusie tussen Hs-AFP1 en EXG1 het dus nie die stabiliteit van die reeds stabiele Hs-AFP1 peptied verder verbeter nie, maar het wel tot die verlies van Hs-AFP1 aktiwiteit gelei. Die antifungiese analises van die THE tabaklyne het verder bevestig dat dié lyne selfs swakker inhibisie van B. cinereainfeksies tot gevolg gehad het, as ongetransformeerde tabakplante. Mikroskopiese analises van totale THE proteïenekstrakte het voorts ook geen kenmerkende hipervertakkings in die swamhifes vertoon nie, wat alles daarop dui dat die Hs-AFP1-deel van die fusieproteïen as gevolg van die fusie met EXG1 geïnaktiveer is. Dié resultaat is in lyn met vorige navorsing, wat getoon het dat In ander peptied, sarcotoxin 1A, sy antifungiese aktiwiteit verloor indien dit met In verklikkergeen versmelt word. Alhoewel integrasie van die pMj-EXG1-konstruk in die TME-tabaklyne bevestig is, kon geen mRNA met Noordelike-klad- of trutranskriptase-PKR (RT-PKR)-analises waargeneem word nie. Die TME plant het ook geen antifungiese aktiwiteit in in vitro toetse getoon nie en dit het geblyk dat die pMj-EXG1-konstruk aan geenafskakeling in die heteroloë tabakomgewing onderworpe was. Dié afskakelingseffek is egter in die THE plante oorkom, aangesien laasgenoemde sterk EXG1 proteïenaktiwiteit met J3-glukosidase aktiwiteitstoetse vertoon het. Alhoewel die THE plante nie die stabiliteit van Hs-AFP1 verbeter het nie, het dit onwerwags tot die stabilisering van EXG1 in In heteroloë omgewing gelei. Versmeltingstegnologie kan dus moontlik gebruik word as 'n strategie om ander glukanases, wat bekend is vir geenafskakeling in transgeniese omgewings, heteroloog uit te druk. In die geheel gesien, het dié studie getoon dat Hs-AFP1 'n onbetwiste rol in plantverdedigingsmeganismes speel en daar is voorts ook meer kennis oor die stabiliteit van peptiede in 'n heteraloë plantomgewing ingewin. Die positiewe resultate t.o.v. die verhoogde siekteweerstand in die transgeniese THs plantlyne regverdig ook die verdere bestudering van dié peptied om transgeniese siekteweerstand in gewasse te bewerkstellig.

Non-salt tolerant cultivars of rice, lettuce and radish as well as salt tolerant varieties of alfalfa, barley, and wheat were screened in the greenhouse and laboratory to determine if Labyrinthula terrestris, a new turfgrass pathogen, could infect plants other than turfgrasses. Wheat, barley and rice plants were infected, symptomatic and died. Radish and lettuce were infected but nonsymptomatic. Alfalfa was not infected and exhibited no symptoms. Results indicate that L. terrestris is capable of infecting and causing symptoms in plants other than cool season turfgrasses.

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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior
Weed plant invasions, in natural as well as in anthropic modified environments, results in significant economical losses or ecosystem disequilibrium. Some ecosystems are more susceptible than others to biological invasions, once they have disharmonic fauna and flora (with empty ecological niches or niches occupied by low adapted species to the ecological functions required), due to its geographic isolation, as in oceanic islands (Hawaii, French Polynesia, Fernando de Noronha and other archipelagos), or due to its separation form the continental bulk during the process of continental derivation in a remote past, as in the case of Australian continent. In such places, the introduction of exotic vegetal or animal species may be a disaster for the ecosystem. A exemple is the introduction of Tibouchina herbacea in the Hawaiian archipelago resulted in environmental invasion. This plant belongs to the Melastomataceae family and it is native that South America. A biological program involving entomologists and plant pathologists on origin center of T. herbacea will be developing, searching natural enemies. With the purpose of future development of control programs using phytopathogenic fungi, the mycological diversity associated with this plant was assessed. A total of eighty one samples originated from Brazil, Dominican Republic and Costa Rica were analyzed in this study. This work was limited to the taxonomic treatment of the fungi obtained from samples and represents the first step for the development of a future biological control program using phytopatogenic fungi to control the weed plant Tibouchina herbacea. In present work 16 fungi species were found: 6 Hyphomycetes Cladosporium, Passalora, Cercospora and three Pseudocercosporas; 4 Coelomycetes Septoria, Hainesia, Chaetophiophoma, Pestalotiopsis; and 6 Ascomycetes Asteridiella, Mollisia, Asterina, Perisporiopsis, Gnomonia, Leptosphaeria. There are recognized as new to science and described here: Cladosporium tibouchinensis, Mollisia tibouchinae, Passalora tibouchinae, Pseudocercospora subsinematosa, Pseudocercospora tibouchinensis, Pseudocercospora tibouchinicola and Septoria tibouchinensis Among the species of fungi founded in this study, three have potential for use in biological control programs causing severe disease in T. herbacea: S. tibouchinensis, P. tibouchinae and M. tibouchinae. Although the specificity of these fungi has not yet been tested, the first two genres belong to fungi that include species considered quite specific restricted to a single botanical family.
As invasões causadas por plantas tanto em ambientes naturais como em ambientes modificados pela ação antrópica resultam em grandes perdas, de natureza econômica ou no desequilíbrio de ecossistemas. Alguns ecossistemas são mais suscetíveis a invasões biológicas, pois têm fauna e flora endêmicas desarmônicas (com nichos ecológicos vazios ou ocupados por espécies pouco adaptadas às funções ecológicas ali desempenhadas) devido ao seu surgimento em isolamento geográfico, como as ilhas oceânicas dos arquipélagos Havaiano, Polinésia Francesa, Fernando de Noronha e outros, ou à sua separação da massa continental no processo de deriva dos continentes em passado evolutivo remoto, como é o caso do Continente Australiano. Nesses locais a introdução de uma espécie exótica vegetal ou animal pode ser desastrosa para o ecossistema. Um exemplo é a introdução de Tibouchina herbacea no arquipélago Havaiano, que resultou em invasão de ecossistemas nativos. Esta planta pertencente à família Melastomataceae, e é nativa da América do Sul. Ela tem sido tratada como alvo de um programa de controle biológico utilizando-se insetos e fungos provenientes do seu centro de origem. Para este fim foi feito um levantamento da micodiversidade associada a T. herbacea. Deste levantamento resultaram 81 amostras, coletadas em três países: Brasil, República Dominicana e Costa Rica. No presente trabalho a ênfase foi dada ao esclarecimento da identidade dos fungos obtidos, como um primeiro passo para uma futura utilização de fungos fitopatogênicos no controle de T. herbacea. 16 espécies de fungos foram obtidas, sendo: seis hifomicetos Cladosporium, Passalora, Cercospora apii e três espécies do gênero Pseudocercospora; quatro coelomicetos Septoria, Hainesia, Chaetophiophoma e Pestalotiopsis; e seis ascomicetos Asteridiella, Mollisia, Asterina, Perisporiopsis, Gnomonia, Leptosphaeria. Foram reconhecidas como taxa novos para a ciência e são aqui descritos: Cladosporium tibouchinensis, Mollisia tibouchinae, Passalora tibouchinae, Pseudocercospora subsinematosa, Pseudocercospora tibouchinensis, Pseudocercospora tibouchinicola and Septoria tibouchinensis. Dentre as espécies de fungos encontradas no presente trabalho, três parecem ter potencial para uso em programas de controle biológico por causarem doenças severas em T. herbacea: S. tibouchinensis, P. tibouchinae e M. tibouchinae. Embora a especificidade destes fungos não tenha ainda sido testada, os dois primeiros fungos pertencem a gêneros que incluem espécies tidas como bastante específicas (pelo menos restritas a uma única família botânica).