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.

Thesis (MScAgric)--University of Stellenbosch, 2004. / ENGLISH ABSTRACT: Botrytis cinerea Pers.: Fr., a pathogen of grapevine (Vitis vinifera L.), moves mainly through conidia by air currents in vineyards which are deposited intermittently on the surfaces of leaves, inflorescences and bunches. Little is known about the relationship between the inoculum dosage in air and incidence of Botrytis bunch rot, and how the relationship is influenced by environmental and host factors. To better understand this relationship, information is needed on the period over which conidia have accumulated, the time they are able to survive and remain infectious, time of symptom expression in relation to conidium arrival at the infection court and host surface wetness. The aims of this study were (i) to estimate the amount of viable B. cinerea occurring in air in vineyards, and at different positions on leaves, inflorescences and bunches of grape at different phenological stages, (ii) to determine the relationships between the number of B. cinerea colonies recorded on spore traps placed in the bunch zone of vines and the incidence of B. cinerea recorded from the different tissues, and (iii) to compare the efficacy of fenhexamid on leaves and inflorescences carrying natural B. cinerea inoculum with those inoculated with dry, airborne conidia. Different techniques were used to detect viable Botrytis cinerea in air currents and on plant material obtained from table (cultivars Dauphine and Waltham Cross in Paarl- and Worcester-district) and wine grape (cultivars Chardonnay, Sauvignon Blanc and Merlot in Stellenbosch- and Malmesbury district) vineyards in the Western Cape province during 2001-02 and 2002-03. For four consecutive days during prebloorn, bloom, pea-size, bunch closure, veraison and harvest, sets of Petri dishes with freshly prepared Kerssies' B. cinerea selective medium (spore traps) were left overnight in the bunch zone of vines. Plant material was collected from the vines on the fourth day. Leaves, infloresence and bunches were treated with paraquat to terminate host resistance and to promote the development of the pathogen on the tissues. The B. cinerea inoculum dosage in air, and the incidence at which the pathogen was detected at various positions on leaves and in bunches normally differed between vineyards. However, the various tests revealed that the pathogen generally occurred in a consistent pattern in air in the bunch zone of vines, on leaves and in bunches from all vineyards. The inoculum dosage in air in the bunch zone of the vine was generally highest during prebioom or during bloom, it decreased at pea size and mostly remained at a very low level at the later growth stages. The estimations of viable B. cinerea residing naturally on leaves and in bunches, showed that their amounts depicted levels occurring in air in the bunch zone of the vine. Necrotic leaves occurring early season in vineyards were identified as an important source of secondary inoculum for dispersal to the developing bunches. Latent infections at the various positions in bunches were few at véraison and harvest. However, due to the necrotrophic ability of the pathogen, extensive berry rot (due to berry-to-berry contact) and thus severe bunch rot developed from a single berry that become symptomatic at the base of the pedicel/berry attachment zone. The B. cinerea occupation pattern explains why Botrytis bunch rot develops mostly from the inner bunch and why disease management strategies should concentrate on the bloom to pre-bunch closure stage and on inhibiting B. cinerea development in the inner bunch during the early part of the season. Thus, to effectively reduce B. cinerea in grapevine, preventative applications are recommended to reduce two primary infection events: (a) between budding and pre-bloom to counteract primary leaf infection; (b) during late bloom or early pea size stage, to reduce the amount of the pathogen on leaves and infloresences and to prevent colonisation of floral debris. A third spray can be applied at bunch closure to reduce the amount of B. cinerea at various positions of the inner bunch, especially for cultivars with tight bunches. The efficacy of fenhexamid on leaves and inflorescences carrying natural B. cinerea inoculum was compared with those inoculated with dry, airborne conidia. Shoots were obtained during late bloom from a vineyard (wine grape cultivar Merlot) in the Stellenbosch region. The shoots were divided into two main groups. One group of shoots was left uninoculated, the other shoots were inoculated by dusting with dry B. cinerea conidia in a settling tower. Before inoculation, equal numbers of shoots in each main group was sprayed with fenhexamid, or left unsprayed. Following inoculation and incubation, shoots of each treatment were divided in two equal groups. The one lot of shoots were rinsed in water. The other lot of shoots were immersed in paraquat solution to terminate host resistance and to promote the development of the pathogen from the tissues. For both uninoculated and inoculated shoots, irrespective of fungicide treatment, leaves remained asymptomatic at both the blade and petiole position for the water rinse treatment. No symptom of B. cinerea decay developed at any of the positions on leaves from shoots sprayed with fenhexamid. Spraying of shoots with fenhexamid completely suppressed B. cinerea infection and symptom expression on both uninoculated and inoculated inflorescens. For inoculated shoots, B. cinerea developed from approximately 50% of the laterals in the water rinse treatment. However, inflorescences rinsed in water remained asymptomatic. The laboratory studies showed that fungicides, if applied properly to shoots and bunches under controlled conditions, effectively reduced the amount of B. cinerea at the various positions on leaves and inflorescence, and prevented infection and symptom expression at bloom. However, these goals are not achieved in vineyards where the fungicides are applied by conventional spraying methods. Therefore, more work is needed to evaluate fungicide application techniques by conventional spraying methods for proper fungicide coverage, and the reduction of B. cinerea in bunches. / AFRIKAANSE OPSOMMING: Botrytis cinerea Pers.: Fr., 'n patogeen van druiwe (Vilis vinifera L.), beweeg hoofsaaklik deur middel van konidia in lugstrome deur die wingerd, en word dan afwisselend op die oppervlakte van die blare, bloeiwyses en trosse gedeponeer. Daar is nog min bekend oor die verhouding tussen die hoeveelheid inokulum in die lug en die voorkoms van Botrytis op die trosse, en hoe die verhouding deur omgewings- en gasheerfaktore beïnvloed word. Ten einde hierdie interaksie beter te verstaan, word inligting benodig oor die tydperk waarin die konidia akkumuleer, die tyd wat hulle oorleef en virulent bly, en die tyd van simptoom-uitdrukking in verhouding tot die verspreiding van die konidia by die infeksie-setel en benatbaarheid van die gasheer-oppervlakte. Die doel van hierdie studie was (i) om die hoeveelheid lewensvatbare B. cinerea wat in die lug voorkom, asook by verskeie posisies op blare, bloeiwyses en trosse by verskillende fenologiese stadiums te kwantifiseer, (ii) om die verhouding tussen die aantal aangetekende B. cinerea kolonies op spoorvangers wat in die trossone van die wingerd geplaas is, en die voorkoms van B. cinerea, aangeteken van verskeie weefsels, te bepaal, en (iii) om die effektiwiteit van fenhexamid op blare en bloeiwyses wat natuurlike B. cinerea inokulum dra, te vergelyk met dié wat met droë, luggedraagde konidia geïnokuleer is. Verskillende tegnieke is gebruik om lewensvatbare Botrytis cinerea in lugstrome en op plantmateriaal van tafeldruiwe (kultivars Dauphine en Waltham Cross In Paarl- en Worcester-distrik) en wyndruiwe (kultivar Chardonnay, Sauvignon Blanc en Merlot in Stellenbosch- en Malmesbury distrik) in wingerde van die Wes-Kaap provinsie gedurende 2001-02 en 2002-03 te kwantifiseer. Petri bakkies met vars voorbereide Kerssies medium, selektief vir B. cinerea (spoorvangers), is vir vier agtereenvolgende dae gedurende vóórblom, blom, ertjiekorrel, trostoemaak, kleurbreek en oes, oornag in die trossone van wingerdstokke in betrokke wingerde, gelaat. Plantmateriaal is op die vierde dag versamel. Blare, bloeiwyses en trosse is met paraquat behandel ten einde die gasheerweerstand af te breek en ontwikkeling van die patogeen op die weefsel te bevorder. B. cinerea inokulum in die lug, en die frekwensie waarby die patogeen op verskeie posisies op blare en in die trosse voorgekom het, het normaalweg tussen wingerde verskil. Die verskeie toetse het getoon dat die patogeen normaalweg in 'n vaste patroon in die lug en die trossones van wingerde, asook op blare en in trosse van alle wingerde voorkom. Die inokulumkonsentrasie in die lug in die trossones van wingerdstokke was normaalweg die hoogste gedurende vóórblom of gedurende blom. Die inokulumdruk het by ertjiekorrel verminder en meestal by 'n 'n baie lae vlak tydens die latere groeistadia gebly. Die bepaling van lewensvatbare B. cinerea wat natuurlik op blare en in trosse gedeponeer is, het getoon dat hul hoeveelhede ooreenstem met vlakke wat in die lug in die trossone van die wingerd voorkom. Nekrotiese blare vroeg in die seisoen is 'n belangrike bron van sekondêre inokulum en speel dus 'n belangrike rol by die verspreiding van Botrytis tussen die ontwikkelende trosse. Latente infeksies by die verskeie posisies in trosse was laag by kleurbreek en oes. Weens die saprofitiese vermoëns van die patogeen, kan uitgebreide korrelvrot (a.g.v. korrel-tot-korrel kontak) en dus ernstige trosvrot, ontwikkel. 'n Enkele korrel kan by die basis van die pedisel/korrel vashegtingsone simptomaties raak, en vandaar na aangrensende korrels versprei. Die B. cinerea kolonisasiepatroon verduidelik waarom Botrytis trosvrot meestal vanaf die binneste tros ontwikkel en waarom siektebeheerstrategieë op die vóórblom- tot blomstadium gekonsentreer moet word, en op die inhibering van B. cinerea ontwikkeling in die binneste tros gedurende die vroeë stadia van die seisoen. Dus, om B. cinerea effektief tydens die twee primêre infeksie stadiums in wingerde te verminder, kan voorkomende toedienings aanbeveel word: (a) tussen knopvorming en vóórblom om primêre blaarinfeksie te verhoed; (b) gedurende láátblom en vroeë ertjiekorrel om die hoeveelheid inokulum op die blare en bloeiwyses te verminder, en die kolonisasie van blomdebris te voorkom. 'n Derde toediening kan tydens trostoemaak aangewend word om B. cinerea by verskeie posisies in die binneste tros te verminder, veral by kultivars met digte trosse. Die effektiwitiet van fenhexamid op blare en bloeiwyses waarop natuurlike B. cinerea inokulum voorkom is vergelyk met dié wat met droë, luggedraagde konidia geïnokuleer is. Lote is vanaf 'n wingerd (wyndruif kultivar Merlot) in die Stellenbosch distrik tydens láátblom verkry en in twee hoofgroepe verdeel. Die een groep lote is geïnokuleer deur droë B. cinerea konidia in 'n afsettingstoring te strooi, terwyl die ander groep nie geïnokuleer is nie. Vóór inokulasie, is die helfte van die lote in elke groep met fenhexamid behandel, terwyl die ander helfte onbehandeld gelaat is. Ná inokulasie en inkubasie, is lote van elke behandeling verder in twee eweredige groepe verdeel. Die een groep lote is in water gespoel, terwyl die ander groep lote in 'n paraquatoplossing gedompel is om die gasheerweerstand te verwyder, en die ontwikkeling van die patogeen vanuit die weefsels te bevorder. Vir die waterspoelbehandeling van beide ongeïnokuleerde en geïnokuleerde lote, ongeag van die fungisiedbehandeling, het die blare asimptomaties by beide die bladoppervlakte en blaarsteelposisie gebly. Geen simptome van B. cinerea verrotting het by emge van die blaarposisies van die lote, met fenhexamid gespuit, ontwikkel nie. Die spuit van die lote met fenhexamid het die B. cinerea infeksie en die simptoomontwikkeling op beide die ongeïnokuleerde en geïnokuleerde bloeiwyses heeltemalonderdruk. By die geïnokuleerde lote, het B. cinerea vanaf ongeveer 50% van die laterale in die waterspoelbehandeling ontwikkel, alhoewel, bloeiwyses wat in water afgespoel is, heeltemal asimptomaties gebly het. Laboratoriumstudies het getoon dat fungisiedes, indien korrek toegedien op lote en trosse onder gekontroleerde toestande, tot effektiewe vermindering van B. cinerea getalle by die verskillende posisies op blare en bloeiwyses lei, en infeksie en simptoomuitdrukking tydens blom voorkom. Weens die feit dat die doelwitte nie behaal kan word in wingerde waar die fungisiede deur konvensionele spuitmetodes toegedien is nie, moet meer studies gedoen word om fungisied toedieningstegnieke, by konvensionele spuitmetodes, VIr deeglike fungisiedbedekking en die vermindering van B. cinerea in trosse, te evalueer.

Thesis (MScAgric)--University of Stellenbosch, 2002. / ENGLISH ABSTRACT: Historically root diseases have been a production-limiting problem for the strawberry industry worldwide. In the Western Cape Province of South Africa the most serious root disease is black root rot, which causes losses of up to 30%, annually. The aims of this study were to investigate aspects of the etiology and epidemiology of this disease in the Western Cape, and to provide information that can be incorporated in an integrated disease management strategy. In Chapter I a summary of published information on this disease is presented. Disease symptoms include severe stunting of plants, which have black, rotted, reduced root systems. Even though this disease is of great economic importance, the etiology remains unresolved. However, soilborne fungal root pathogens, particularly Pythium and Rhizoctonia spp. have been implicated as major role players. Control of this disease, as well as the other root diseases affecting strawberries, has relied on soil fumigation with broad spectrum chemical fumigants, in particular methyl bromide. However, due to the ozone depleting action of methyl bromide it was decided at the signing of the Montreal Protocol to remove this chemical from the market. This action has caused great demand for alternative measures to control root diseases on many crops including strawberries. Development of integrated disease management strategies is dependent upon a more complete understanding of the etiology, biology and ecology of the disease complex. In Chapter 2 fungal pathogens associated with diseased plants were isolated and Koch's postulates were carried out. The most frequently isolated fungal pathogens were Pythium irregulare, Rhizoctonia spp. and Cylindrocarpon destructans. Two morphotypes of Rhizoctonia were isolated viz. a brown and a white type. Pythium irregulare was isolated more frequently in July than in September, and was not isolated at all in November. Rhizoctonia spp. were present at all sampling dates but were more frequently isolated in November than at the other times. All the fungi that were tested were pathogenic and caused root lesions. Cylindrocarpon destructans and Coniella fragariae did not have a stunting effect on the plants. These results confirm a major role for Pythium and Rhizoctonia in the black root rot complex and suggest that there is a complimentary seasonal variation in occurrence between these two pathogens. The Rhizoctonia species and anastomosis groups of isolates obtained from diseased strawberries in the Western Cape Province were determined, and their pathogenicity and relative virulence assessed. Both binucleate and multinucleate types were recovered from diseased roots and identified as R. fragariae and R. so/ani, respectively. All isolates of R. solani were members of anastomosis group (AG) 6, whereas three AG types were identified among isolates of R. fragariae, viz. AG-A, AG-G and AG-I at a relative occurrence of 69%, 25%, 6% respectively. All Rhizoctonia isolates were pathogenic to strawberry, but R. solani (AG 6) was the most virulent causing severe stunting of plants. This is the first species confirmation and AG type identification of Rhizoctonia taxa causing root rot of strawberries in South Africa. An assessment of the presence and quantity of black root rot pathogens associated with soils prior to fumigation and post fumigation with methyl bromide, was made in Chapter 4. Isolations were also made from nursery plants to determine whether any black root rot pathogens were in the plants before transplanting. Results demonstrated that after fumigation the soil was free of all pathogenic fungi associated with the disease. However, the main pathogens involved in black root rot, viz. Rhizoctonia fragariae, R. solani and Pythium spp. were isolated from nursery plants. The fact that the plants are already infected with these pathogens renders the prospects for control of this disease difficult. Further studies are urgently required to develop production practices that can be included in disease management programmes. In vitro studies were carried out to determine the ECso values of different fungicides for isolates of Pythium irregulare, Rhizoctonia fragariae AG-A, AG-G and AG-I and R. solani AG 6. Benomyl, fludioxonil and tolc1ofos-methyl were used in these tests. Field trials were also conducted using these fungicides. In Chapter 5 it is shown that in general application of fungicides improved the yield and did not affect the survival rate of strawberry plants. Fludioxonil showed potential for short-term use. Applications of fungicides that targeted specific fungal genera were not sufficient to control the disease. Seasonal fluctuation of Pythium and Rhizoctonia spp. became apparent with the occurrence of Pythium being relatively high early in the season but low late in the season. Conversely, the occurrence of Rhizoctonia was low at the beginning of the season but high late in the season. In the short-term there is potential for fungicide applications as part of an integrated disease management strategy, but the economic feasibility of this practice needs to be assessed. In this study the major pathogens causing black root rot were identified in the Western Cape Province of South Africa, and important information regarding the epidemiology of the disease was reported. These results can be incorporated in an integrated management strategy to reduce losses of strawberry production, attJibutable to black root rot. / AFRIKAANSE OPSOMMING: Wortelsiektes is wêreldwyd 'n produksie-beperkende probleem vir die aarbeibedryf. . Swartwortelvrot, wat jaarliks verliese van tot 30% veroorsaak, is die belangrikste wortelsiekte in die Wes-Kaap Provinsie van Suid-Afrika. Die doelwitte van hierdie studie was om aspekte van die etiologie en epidemiologie van die siekte in die Wes- Kaap te ondersoek en inligting wat in geïntegreerde siektebestuurstrategië ingesluit kan word, te voorsien. In Hoofstuk 1 word 'n opsomming van gepubliseerde inligting aangaande die siekte uiteengesit. Siektesimptome sluit ernstige verdwerging van plante met swart verotte en verkleinde wortelstelsels in. Alhoewel die siekte van groot ekonomiese belang is, is die etiologie grootliks onbekend. Grondgedraagde wortelpatogene swamme, spesifiek Pythium en Rhizoctonia spp., is egter as belangrike rolspelers geïdentifiseer. Tot dusver het die beheer van hierdie siekte sowel as ander wortelsiektes van aarbeie berus op grondberoking met breë spektrum chemiese berokingsmiddels, spesifiek metielbromied. As gevolg van die osoonafbrekende aksie van metielbromied is daar egter tydens die ondertekening van die Montreal Protocol besluit om dié middel van die mark te verwyder. Hierdie besluit het 'n groot aanvraag na alternatiewe beheermaatreëls vir wortelsiektes van verskeie gewasse, insluitende aarbeie, veroorsaak. Die ontwikkeling van geïntegreerd siektebestuurstrategieë is egter afhanklik van 'n meer volledige begrip van die etiologie, biologie en ekologie van die siektekompleks. In Hoofstuk 2 is die patogene swamme wat met die siekte geassosieer word, geïsoleer, en is Koch se postulate uitgevoer. Die mees algemeen geïsoleerde patogene swamme was Pythium irregulare, Rhizoctonia spp. en Cylindrocarpon destructans. Twee morfotipes van Rhizoctonia is geïsoleer, nl. 'n bruin tipe en 'n wit tipe. Pythium irregulare is meer dikwels in Julie as in September geïsoleer, maar glad nie in November nie. Rhizoetonia het tydens alle monstertye voorgekom, maar is meer dikwels in November geïsoleer. Al die swamme wat getoets is, was patogenies en het letsels op die wortels veroorsaak. Cylindroearpon des true tans en Coniella fragariae het nie'n verdwergingseffek op plante gehad nie. Hierdie resultate bevestig die dominante rol van Pythium en Rhizoctonia in die swartwortelvrot kompleks en dui op 'n komplementêre seisoenale variasie in die voorkoms van hierdie twee patogene. Die Rhizoctonia spesies en anastomose groepe (AG) van die isolate geisoleer vanaf siek aarbeiplante in die Wes-Kaap Provinsie is bepaal, en die patogenisiteit en relatiewe virulensie is beraam. Sowel tweekernige as multikernige tipes is vanaf siek wortels geïsoleer en respektiewelik as R. fragariae en R. so/ani geïdentifiseer. Alle isolate van R. so/ani was lede van anastomose groep 6, terwyl drie AG tipes, nl. AGA, AG-G en AG-I onder die R. fragariae isolate geïdentifiseer is met relatiewe voorkomste van 69%, 25%, 6% respektiewelik. Alle Rhizoctonia isolate was patogenies op aarbeie, maar R. so/ani (AG 6) was die mees virulente en het ernstige verdwerging van plante veroorsaak. Hierdie is die eerste bevestiging van spesies en identifisering van AG tipes van Rhizoctonia taksa wat wortelvrot van aarbeie in Suid Afrika veroorsaak. In Hoofstuk 4 is 'n beraming van die voorkoms en hoeveelheid swartwortelvrot patogene geassosieer met grond voor, en na beroking met metielbromied, gemaak. Isolasies is ook vanaf kwekeryplante gemaak om te bepaal of enige swartwortelvrot patogene voor oorplanting in die plante teenwoordig was. Die resultate het getoon dat grond na beroking vry was van alle patogeniese swamme geassosieër met die siekte. Die hoof patogene betrokke in die swartwortelvrot kompleks, nl. Rhizoctonia fragariae, R. so/ani en Pythium spp. was egter in die kwekery plante teenwoordig. Die feit dat plante reeds met hierdie patogene geïnfekteer is, maak die vooruitsigte vir die beheer van hierdie siekte moeilik. Verdere studies word dringend benodig vir die ontwikkeling van produksiepraktyke wat by siektebestuursprogramme ingesluit kan word. In vitro studies om die ECso waardes van die isolate van Pythium irregulare, Rhizoctonia fragariae AG-A, AG-G en AG-I en R. so/ani AG 6 vir die fungisiedes benomyl, fludioxonil en tolclofos-metiel te bepaal, is uitgevoer. Hierdie fungisiedes is ook in veldproewe getoets. In Hoofstuk 5 is getoon dat aanwending van fungisiedes die opbrengs verbeter het en nie die oorlewing van aarbeiplante beïnvloed het nie. Fludioxonil het potensiaal vir korttermyn gebruik getoon. Die aanwending van fungisiedes wat spesifieke swamgenera teiken, was nie voldoende om die siekte te beheer nie. Seisoenale fluktuasies van Pythium en Rhizoctonia spp. het duidelik geword met die relatief hoë voorkoms van Pythium vroeg in die seisoen, maar lae voorkoms laat in die seisoen, terwyl die voorkoms van Rhizoctonia laag was aan die begin van die seisoen, maar hoog later in die seisoen. In die korttermyn is daar potensiaal vir fungisiedtoedienings as deel van 'n geïntegreerde siektebestuurstrategie, maar die ekonomiese haalbaarheid van hierdie praktyk moet bepaal word. In hierdie studie is die hoof patogene wat swartwortelvrot van aarbeie in die Wes-Kaap Provinsie van Suid-Afrika veroorsaak geïdentifiseer, en belangrike inligting rakende die epidemiologie van die siekte is aangeteken. Hierdie resultate kan in 'n geïntegreerde bestuurstrategie geïnkorporeer word om verliese van aarbeiproduksie, toeskryfbaar aan swartwortelvrot te, verminder.

Thesis (MScAgric)--Stellenbosch University, 2003. / ENGLISH ABSTRACT: The resistance of plants to infection by phytopathogenic microorganisms is the result of multiple defence reactions comprising both constitutive and inducible barriers. While disease is the exception, such exceptions can be costly and even devastating. In particular, fungal diseases remain one of the major factors limiting crop productivity worldwide, with huge losses that need to be weighed up against massive cash inputs for pesticide treatments. Part of the defence reactions of plants is the synthesis of pathogenesis-related proteins, such as the plant hydrolases, glucanases and chitinases. In recent years, attention has been paid to the implementation of these proteins in plant transformation schemes. The rationale for this approach was that these antimicrobial agents not only degrade the main cell wall components of fungi, but also produce glucosidic fragments that act as elicitors of the biosynthesis of defence metabolites by the host. Furthermore, since these active antimicrobial agents are individually encoded by single genes, these defence systems should and have been shown to be highly amenable to manipulation by gene transfer. In this study, yeast glucanases from Saccharomyces cerevisiae were evaluated for their potential as antifungal proteins. The glucanases tested for their antifungal activity against Botrytis cinerea were the yeast EXG1 and BGL2 genes, encoding an exoglucanase and an endoglucanase respectively. An in vitro assay performed on these glucanases indicated that exoglucanase had a more detrimental effect on B. cinerea hyphal development and growth than the endoglucanase; the former caused typical disruption of the cells and leakage of cell material. The yeast exoglucanase was subsequently subcloned into a plant expression cassette containing the strong constitutive 358 promoter, yielding plasm ids pEXG1 and pMJ-EXG1. The pMJ-EXG1 construct targeted the exoglucanase to the apoplastic region with a signal peptide from an antimicrobial peptide from Mirabilis jalapa, Mj-AMP2. The pEXG1 and pMJ-EXG1 constructs were mobilised into Agrobacterium tumefaciens to facilitate the subsequent tobacco transformation, which yielded transgenic tobacco lines designated E and MJE respectively. Transgene integration was confirmed with southern blot and PCR analyses for both the E and MJE lines. The expression and heterologous production of the EXG1-encoded exoglucanase in the E-transgenic lines was shown with northern blots and activity assays respectively. Moreover, the high level of expression of the yeast exoglucanase led to a decrease in susceptibility of the E lines to B. cinerea infection in comparison to the untransformed tobacco controls. An average decrease in disease susceptibility of 40% was observed in an in planta detached leaf assay. Crude protein extracts from the E lines were also analysed in an in vitro quantitive fungal growth assay, inhibiting in vitro fungal growth by average 20%, thus further confirming the antifungal nature of the yeast exoglucanase. 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 MJE lines. These lines also did not show any in vitro antifungal activities or a decrease in susceptibility to B. cinerea infection in the detached leaf assay. It is suspected that this result is possibly linked to gene silencing, a phenomenon quite frequently associated with heterologous and/or overexpression of glucanases in plant hosts. It appears as if the targeted overexpression to the apoplastic space triggered the gene silencing response, since the intracellularly overexpressed product was produced and shown to display activity. The yeast exoglucanase thus joins the list of silenced glucanases in overexpression studies in plants. Overall, this study confirmed the antifungal characteristics of the Saccharomyces exoglucanase and provides valuable information of the possibility of utilising yeast glucanases in a transgenic environment. A decrease in the susceptibility of tobacco to B. cinerea infection, as shown by the overexpressed EXG1-encoded exoglucanases, merits further investigation into the use of this gene in the engineering of disease-resistant crops. / AFRIKAANSE OPSOMMING: Die weerstand van plante teen infeksie deur fitopatogeniese mikroórganismes is die resultaat van verskeie meervoudige verdedigingsreaksies wat beide konstitutiewe en induseerbare versperrings behels. Terwyl siekte die uitsondering eerder as die reël is, kan sulke uitsonderinge duur en selfs verwoestend wees. In die besonder is swamsiektes een van die vernaamste faktore wat gewasproduksie wêreldwyd beperk, met enorme verliese wat teen kontantinsette vir plaagdoders opgeweeg moet word. Deel van die verdedigingsreaksie van plante is die sintese van patogeen-verwante proteïene, soos die planthidrolases, -glukanases en -chitinases. In die onlangse tyd is aandag geskenk aan die implementering van hierdie proteïene in plant transformasieskemas. Die grondrede hiervoor was dat hierdie antimikrobiese agente nie net die hoof selwandkomponente van swamme kan afbreek nie, maar ook glukosidiese fragmente produseer wat as ontlokkers van metabolietbiosintese vir die verdediging van die gasheer kan optree. Aangesien hierdie aktiewe antimikrobiese agente individueel deur enkele gene enkodeer word, blyk hierdie verdedigingsisteme om hoogs ontvanklik vir manipulasie deur geenoordrag te wees. In hierdie studie is die gisglukanase van Saccharomyces cerevisiae vir hul potensiaal as antifungiese proteïene geëvalueer. Die glukanases wat vir hul antifungiese aktiwiteit teen Botrytis cinerea getoets is, was die gis EXG1- en -BGL2-gene, wat onderskeidelik vir "n eksoglukanase en 'n endoglukanase enkodeer. "n In vitro toets wat op hierdie glukanases uitgevoer is, het aangedui dat die eksoglukanase 'n meer skadelike effek op die hife-groei en -ontwikkeling van B. cinerea as die endoglukanase gehad het; eersgenoemde het die tipiese ontwrigting van die selle en die uitlek van selmateriaal tot gevolg gehad. Die gis-eksoglukanase is gevolglik in 'n plant uitdrukkingskasset wat die sterk konstitutiewe 35S promotor bevat, gesubkloneer, wat plamiede pEXG1 en pMJ-EXG1 opgelewer het. Die pMJ-EXG1-konstruk het die eksoglukanase na die apoplastiese gebied geteiken deur 'n seinpeptied vanaf "n antimikrobiese peptied van Mirabilisjalaba, Mj-AMP2. Die pEXG1- en pMJ-EXG1-konstrukte is in Agrobacterium tumefaciens gemobiliseer, wat die gevolglike tabaktransformasies gefasiliteer het wat die E en MJE transgeniese tabaklyne onderskeikelik gelewer het. Transgeen-integrasie is deur suidelike klad- en PKR-analises vir beide die E en MJE lyne bevestig. Die uitdrukking en heteroloë produksie van die EXG1-enkodeerde eksoglukanase is in die transgeniese E lyne deur noordelike klad en aktiwiteitstoetse onderskeidelik aangetoon. Verder het die hoë uitdrukkingsvlak van die gis-eksoglukanase tot 'n vermindering in die vatbaarheid van die E lyne vir B. cinerea-infeksie relatief tot die ongetransformeerde tabakkontroles gelei. 'n Gemiddelde vermindering in siektevatbaarheid van 40% is in 'n in planta verwyderde-blaartoets waargeneem. Ru proteïen-ekstrakte van die E lyne is ook in 'n in vitro kwantitatiewe swamgroeitoets geanaliseer en het in vitro swamgroei met tot gemiddeld 20% geïnhibeer, wat dus verder die antifungiese aard van die gis-eksoglukanase bevestig het. Alhoewel die integrasie van die pMJ-EXG1 uitdrukkingskasset bevestig is, kon geen mRNA-vlakke met die noordelike klad- of RT-peR-analises van die MJE-Iyne waargeneem word nie. Hierdie lyne het ook geen in vitro antifungiese aktiwiteite of 'n vermindering in die vatbaarheid vir B. cinerea-infeksie getoon nie, soos in die verwyderde-blaartoets uitgevoer is nie. Dit word vermoed dat hierdie resultaat moontlik aan geenstilmaking gekoppel is, 'n verskynsel wat gereeld met heteroloë- en/of ooruitdrukking van glukanases in plantgashere gekoppel word. Dit blyk dat die ooruitdrukking wat tot die apoplastiese ruimte geteiken is, tot die geenstilmaking-respons aanleiding gegee het, aangesien die intrasellulêre ooruitgedrukte produk gemaak is en aktiwiteit getoon het. Die gis-eksoglukanase word dus deel van die lys van stilgemaakte glukanases in die ooruitdrukkingstudies van plante. In die algemeen het hierdie studie dus die antifungiese kenmerke van die Saccharomyces eksoglukanase bevestig en waardevolle inligting oor die moontlike gebruik van gis-glukanases in 'n transgeniese omgewing verskaf. 'n Afname in die vatbaarheid van tabak vir infeksie deur B. cinerea, soos deur die ooruitdrukking van EXG1-enkodeerde eksoglukanase getoon is, verdien dus verdere ondersoek van die gebruik van hierdie geen in die skepping van siekteweerstandbiedende gewasse.

Thesis (MScAgric)--Stellenbosch University, 1998. / ENGLISH ABSTRACT: Eyespot is an important disease of spring wheat (Triticum aestivum L.). Four species of Ramulispora are associated with this disease, of which Tapesia yallundae and T. acuformis. are common. This thesis investigates the broader subjects of genetic variability, reproductive dynamics and fungicide resistance in Tapesia yallundae. Each of the chapters treats specific but related topics. T. yallundae, which is the only species thus far reported from South Africa, has been associated with yield losses of up to 50%. To enable the implementation of more accurate and effective control measures, understanding the dynamics of reproduction and the genetics of the pathogen is of utmost importance. Of the many plant disease control measures such as cultural practices, sanitation, biological control, etc., fungicide application is the most commonly resorted to measure in eyespot control. This thesis investigates the broader subjects of genetic variability, reproductive dynamics and fungicide resistance of Tapesia yallzll7dae. Fungicide application, however, is not without problems. The pathogen can build up resistance to fungicides. The most commonly used fungicides in eyespot control include the benzimidazole carbendazim, triazoles such as flusilazole, tebuconazole, propiconazole, bromuconazole, flutriafol, fenbuconazole, triademinol, and the imidazole, prochloraz. Cases of resistance to the groups listed above have been reported. Frequent monitoring for resistance is thus crucial to prevent wastage of fungicide and unnecessary impregnantation of the environment with potentially ineffective chemicals. In chapter 2 of this thesis 300 isolates of T. yallundae from 15 fields were evaluated for resistance against carbendazim, flusilazole, tebuconazole, propiconazole, bromuconazole, flutriafol and fenbuconazole. These results indicated that to some triazoles, such as fenbuconazole, a high level of resistance was already present in field populations. In a sexually reproducing fungus such as T. yallundae, knowledge pertaining to its ability to pass resistance factors to offspring is equally important. Mating studies were, therefore, also conducted with parental strains that showed signs of triazole resistance. Three generations were subsequently tested for resistance to five triazoles, namely flusilazole, tebuconazole, propiconazole, bromuconazole and flutriafol. Results of this study showed variable sensitivity in progeny, which indicated quantitative inheritance of resistance to triazoles. Although the sexual stage has not yet been observed in the field in South Africa, this knowledge lays the foundation for the long-term understanding of the population dynamics of the fungus. The ability of a heterothallic ascomycete population to reproduce sexually is dependent on the availability of its two mating types, MATI-I and MATI-2, their distribution, and female fertility amongst other factors. In the UK. the teleomorph is commonly observed in the field, which is in contrast to the situation in South Africa, where it has only been induced in the laboratory. A comparative study between the South African and the UK. populations was therefore undertaken. Isolates representative of the two populations were mated with tester strains as both sperm recipients and as sperm donors. This allowed the percentage of hermaphrodites to be determined. No difference in terms of female fertility was observed between the South African and the UK. populations, with both populations showing low effective population numbers. These data suggested, therefore, that the teleomorph would also occur more frequently in South Africa if the climate was more indusive to its development. The overall results of this study indicated that eyes pot could still be controlled by means of fungicide application in South Africa. Although a shift in sensitivity was observed towards fenbuconazole and flusilazole, no resistance was detected towards carbendazim. The latter might be due to the absen<.:eof the sexual stage in the field, coupled by the monocyclic nature of the pathogen and sensible fungicide regimes. The absence of T. acujormis makes the disease situation less complicated in terms of fungicide application and management. Continuous surveys will have to be conducted, however, to monitor this situation in future. / AFRIKAANSE OPSOMMING: Hierdie studie ondersoek die genetiese variasie, reproduksie dinamika en fungisied weerstand in Tapesia yallundae. Elke hoofstuk handel oor spesifieke maar verwante onderwerpe. Oogvlek is 'n belangrike siekte van lentekoring (Triticum aestivum L.). Vier spesies van Ramulispora word geassosieer met die siekte, waarvan Tapesia yallundae en T. acuformis mees algemeen voorkom. T. yallundae, wat tans die enigste spesie is wat in Suid-Afrika aangeteken is, het al verliese van tot 50% veroorsaak. Om meer akkurate en effektiewe beheermaatreels te implementeer, is dit noodsaaklik om die oorlewingsdinamika van die patogeen te verstaan. Van al die siektebeheermaatreels soos kulturele praktyke, sanitasie, biologiese beheer ens., bly fungisiedbehandeling die mees algemene maatreel vir die beheer van oogvlek. Fungisiedtoediening het egter ook verskeie probleme. Die patogeen kan weerstand opbou teen die fungisied. Die mees algemene fungisiedes wat vir oogvlekbeheer aangewend word sluit onder meer die benzimidasool karbendazim in, triasole soos flusilasool, tebukonasool, propikonasool, bromukonasool, flutriafol, fenbukonasool, triadimenol, en die imidasool, prochloraz. Weerstand is egter reeds teen hierdie middels bekend. Gedurige monitering vir weerstand is dus krities om die vermorsing van fungisied en besoedeling van die omgewing met oneffektiewe middels te beperk. In hoofstuk 2 van hierdie manuskrip word 300 isolate van T. yallundae van 15 lande geevalueer vir weerstand teenoor karbendazim, flusilasool, tebukonasool, propikonasool, bromukonasool, flutriafol en fenbukonasool. Resultate dui daarop dat teen sommige van hierdie triasole, soos bv. fenbukonasool, daar reeds 'n hoe vlak van weerstand teenwoordig was in veldpopulasies. In 'n seksueel reproduserende fungus soos T. yalluJ1dae, is dit noodsaaklik om te bepaal wat sy vermoe is om weerstandbiedenheid aan die nageslag oor te dra. Om die rede is paringstudies ook op ouers wat tekens van weerstand teenoor triasole getoon het uitgevoer. Drie generasies was gevolglik getoets vir weerstand teenoor vyf triasole, naamlik flusilasool, tebuconasool, propikonasool, brumukonasool en flutriafol. Resultate van die studie het 'n variasie in sensitiwiteit van die nageslag getoon, wat op 'n kwantitatiewe oorerwing van weerstand teen £riasole dui. Alhoewel die teleomorf nog nie in lande in Suid-Afrika opgemerk is nie, Ie hierdie kennis die fondament vir die langtermyn vertolking van die populasie dinamika van hierdie fungus. Die vermoe van 'n heterotalliese askomiseet populasie om seksueel voort te plant is afhanklik van die beskikbaarheid van sy twee paringstipes, MATI-I en MATl-2, hul verpreiding, vroulike vrugbaarheid en ander faktore. Alhoewel die teleomorf algemeen in lande in die Verenigde Koninkryk opgemerk word, is dit in kontras met die situasie in Suid-Afrika, waar hierdie stadium nog slegs in die laboratorium gelnduseer kon word. 'n Studie is dus onderneem om die Suid-Afrikaanse en V.K. populasies met mekaar te vergelyk. Isolate van die twee populasies is dus gepaar met paringsisolate as beide sperm ontvangers en sperm donors. Hierdie prosedure het dit moontlik gemaak om die persentasie hermafrodiete te bepaal. Geen verskille in vroulike fertiliteit is tussen die Suid-Afrikaanse en V.K. populasies bespeur nie, en beide populasies het ook 'n lae effektiewe populasie getal getoon. Hierdie data het dus voorgestel dat die teleomorf ook meer algemeen in Suid-Afrika sou voorkom as die klimaat meer geskik was vir teleomorf vormmg. Die resultate van hierdie studie het tot die slotsom gelei dat oogvlek steeds deur fungisiedbehandeling in Suid-Afrika beheer kan word. Alhoewel daar 'n merkbare verskuiwing in sensitiwiteit teenoor fenbukonasool en flusilasool was, was geen weerstand teenoor karbendazim waargeneem nie. Laasgenoemde kan dalk toegeskryf word aan die afwesigheid van die teleomorf in die veld, gekombineer met die monosikliese natuur van die patogeen en gebruik van alternerende fungisiedes. Die afwesigheid van T. acuformis maak die plaaslike siektetoestand minder gekompliseerd in terme van fungisied aanwending en bestuur. Voortdurende opnames sal egter uitgevoer moet word om hierdie situasie ook in die toekoms te monitor.

Thesis (PhDAgric)--University of Stellenbosch, 2000. / ENGLISH ABSTRACT: Barley leaf scald, caused by Rhynchosporium secalis, is the most important disease of barley (Hordeum vulgare) in the Western Cape province of South Africa. The disease was first reported from South Africa in 1937. The present study is the first attempt to characterise the South African R. secalis population. Topics such as pathogenesisrelated proteins, virulence spectra, variability of pathotypes, sources of variation, host resistance, breeding strategies, molecular characterisation and fungicide sensitivity are summarised in Part 1 of this dissertation. In succeeding Parts the focus is on the characteristics of the local R. secalis population regarding virulence spectrum, DNA polymorphisms, in vitro as well as in vivo fungicide sensitivity. These aspects are treated as separate entities, leading to some duplication which is unavoidable. In Part 2 the virulence spectra of 50 R. secalis isolates from a population in the. Western Cape province were determined. Twenty-one races were detected using 17 differential barley cultivars. The two most prevalent races, namely races 4 and 7 had three and four virulence genes respectively. Both race 4 and 7 were virulent on the most susceptible cultivars, namely West China, Steudelli, C.I.8618 and C.I.2226. Considering the resistance genes reported for cultivars Atlas 46, Turk, and C.I.3515 which showed no susceptible cultivar-pathogen interaction, it would appear that the Rh- Rh3-Rh4 complex is primarily involved in conferring resistance to the local R. secalis isolates. A total of 20 races (47 isolates) characterised in Part 2 were selected for further characterisation by means of DNA fingerprinting. In Part 3 an anonymous multilocus DNA probe was used to characterise the genotypic structure of these isolates by means of RFLP analysis. No correlation between any particular fingerprint pattern, race, district, field or lesion was found. The two most prevalent races, 4 and 7, did not share the same genotypes, even when isolated from the same field or lesion. The genotypic diversity of the isolates studied was 46.5% of the theoretical maximum diversity. The high level of genotypic variation observed in the South African R. secalis population resembled the genotypic diversity observed in other cereal pathogens with known sexual structures. Although no teleomorph has yet been observed, these data suggest that sexual recombination may operate within the local population of R. secalis. In South Africa barley scald is primarily controlled by means of fungicides. The continued use of fungicides on cereal crops results in the build-up of fungicide resistance in the population, which could lower the efficacy of these compounds. These aspects were investigated in Part 4, where isolates (collected during 1993 to 1995) were evaluated in vitro for sensitivity to triadimenol, tebuconazole, flusilazole and propiconazole. The sensitivity fluctuated but in 1995 isolates were significantly less sensitive towards triadimenol than in the previous two years. In a second experiment, isolates collected from two fields with a 5-6 year-history of triadimenol seed treatments and tebuconazole applications, were evaluated for their fungicide sensitivity. A significant positive correlation was observed between tebuconazole and triadimenol sensitivity among,R. secalis populations from these fields. However, such a correlation was not found within the R. secalis population collected during 1993-1995 where shorter crop rotation patterns and a range of fungicides was applied. In a third experiment, the fungicide sensitivity of local R. secalis isolates was evaluated towards two new triazole fungicides, namely bromuconazole and triticonazole. Correlation coefficients observed between these new triazoles and those previously applied in South Africa were not significantly positive. The lack of significant cross-resistance has important practical implications regarding the management of fungicide resistance. In Part 5, isolates with different minimum inhibitory concentration (MIC) towards tebuconazole in vitro (1, 3 and 10 ug/ml) were compared in vivo. The aim of this study was to determine how MIC values would influence virulence (leaf area affected) and sporulation. Results indicated that all isolates were equally fit to induce lesions and sporulate in the absence of tebuconazole. Thus no fitness cost was associated with the degree of tebuconazole sensitivity in the present study. All R. secalis isolates were able to induce lesions on tebuconazole treated leaves, but differed significantly with respect to the percentage leaf area affected. Isolates, least sensitive (MIC = 10 ug/rnl) towards tebuconazole were more adapted on tebuconazole treated leaves, being able to repeatedly cause larger lesions than sensitive R. secalis isolates (MIC = 1 ug/rnl), Sporulation was not significantly different between isolates on lesions of untreated or tebuconazole treated leaves. Larger leaf areas affected and adequate sporulation suggest that a less sensitive population would result in more disease in tebuconazole treated fields. In conclusion, this study revealed the variability associated with the South African R. secalis population regarding virulence spectrum and genotypic structure. The data in this study suggest that it is likely that the local population will easily adapt to newly introduced, single gene resistance. For more durable resistance, higher levels of quantitative resistance should be introduced. This type of resistance is, however, more difficult to identify and incorporate than single gene resistance. Consequently, barley scald control will remain dependent on the efficacy of fungicide applications. Furthermore, the lack of cross-resistance and low frequency of resistant isolates indicates a low risk for the development of fungicide resistance in the local R. secalis population. Other factors such as current crop rotation practices and the range of fungicides being ~pplied also contribute to this low risk level. However, the status of these factors can change over time. The in vivo tebuconazole sensitivity study has indicated that a resistant field population of R. secalis may be able to build-up. It is, therefore, necessary to monitor the fungicide sensitivity of R. secalis isolates at timely intervals with view to successful barley cultivation in the future. / AFRIKAANSE OPSOMMING: Blaarvlek op gars (Hordeum vulgare), veroorsaak deur Rhynchosporium secalis, is die belangrikste siekte van gars in die Wes-Kaap provinsie van Suid-Afrika. Die voorkoms van R. secalis op gars is in Suid-Afrika vir die eertse keer in 1937 gerapporteer. Hierdie studie is die eerste poging tot karakterisering van die plaaslike R. secalis-populasie. Aspekte soos proteïene betrokke by patogenese, virulensiespektra, variabiliteit van patotipes, bronne van variasie, gasheerweerstand, teeltprogramme, molekulêre karakterisering en swamdodersensitiwiteit word in Deel I van die tesis opgesom. In die daaropvolgende gedeelte is die fokus op die karakterisering van die R. secalis-populasie en behels DNA karakterisering, virulensiespektrum, en swamdodersensitiwiteit in vitro asook in vivo. .. In Deel 2 is die virulensiespektra van 50 R. secalis isolate van 'n populasie in die. Wes-Kaap geëvalueer teenoor 17 differensiëel weerstandbiedende gars kultivars en hieruit is 21 rasse geïdentifiseer. Die twee mees algemene rasse (rasse 4 en 7), met onderskeidelik drie en vier virulensie gene, het virulent vertoon teenoor die mees vatbare kultivars soos West China, Steudelli, C.I.8618 en C.I.2226. Geen vatbare kultivar-patogeen interaksies is met kultivars Atlas 46, Turk en C.I.3515, wat al drie die Rh-Rh3-Rh4 kompleks dra, gevind nie. Dit wil dus voorkom asof hierdie genekompleks effektiewe gasheerweerstand teen die plaaslike R. secalis isolate kan bied. 'n Totaal van 20 rasse (47 isolate), gekarakteriseer in Deel 2, is geselekteer vir verdere karakterisering met behulp van DNA bandpatrone. In Deel 3 is 'n anonieme multilokus DNA peiler gebruik om deur middel van RFLP analise die genotipiese struktuur van hierdie R. secalis-isolate te bepaal. Geen assosiasie is gevind tussen DNA bandpatroon en ras, distrik, garsland of letsel nie. Die twee rasse (4 en 7) wat mees algemeen voorkom, het nie dieselfde bandpatroon vertoon nie, ook nie dié afkomstig vanuit dieselfde garsland of letsel nie. Die genotipiese diversiteit van isolate was 46.5% van die teoretiese maksimum diversiteit. Die hoë vlak van variasie waargeneem in die R. secalis populasie is soortgelyk aan variasie waargeneem in ander graanpatogene wat oor 'n geslagtelike stadium in die lewenssiklus beskik. Alhoewel geen geslagtelike stadium tot dusver geidentifiseer is nie, dui die vlak van variasie daarop dat geslagtelike rekombinasie moontlik wel plaasvind binne die plaaslike R. secalis populasie. In Suid-Afrika word blaarvlek op gars primêr deur swamdoders beheer. Die toenemende gebruik van swamdoders op graangewasse veroorsaak moontlik 'n opbou van swamdoderweerstand in die populasie. Dit kan die effektiwiteit van swamdoders verlaag. Hierdie veronderstelling is in Deel 4 ondersoek, waar die sensitiwiteit van isolate in vitro teenoor triadimenol, tebukonasool, flusilasool en propikonasool geëvalueer is. Die triasooi sensitiwiteit van R. secalis isolate wat gedurende die 1993- 1995 seisoen versamel is het gewissel en slegs vir triadimenol was daar 'n tendens na meer weerstandbiedenheid. 'n Swamdoder-evaluasie is in 'n aparte eksperiment op isolate gedoen wat versamel is vanaf twee garslande met 'n 5-6 jaar geskiedenis van triadimenol saadbehandelings en tebukonasool bespuitings. 'n Betekenisvolle positiewe korrelasie is waaJ~geneem tussen tebukonasool en triadimenol sensitiwiteit in R. secalis isolate afkomstig vanaf hierdie twee garslande. 'n Soortgelyke korrelasie is egter nie gevind in die populasie wat gedurende die 1993-1995 seisoene versamel IS me. Laasgenoemde kan moontlik toegeskryf word aan korter wisselboupatrone en die toediening van 'n verskeidenheid van swamdoders. In 'n derde eksperiment is die sensitiwiteit van plaaslike R. secalis isolate teenoor twee nuwe triasole, naamlik bromukonasool en tritikonasool getoets. Die korrelasie waargeneem tussen die twee nuwe triasole en triasooi swamdoders reeds voorheen in gebruik in die Wes-Kaap was me betekenisvol positief me. Die gebrek aan betekenisvolle kruisweerstandbiedendheid het belangrike praktiese implikasies vir die bestuur van swamdoder -weerstandbiedendheid. In Deel 5 is isolate met wisselende minimum inhiberende konsentrasies (MIKs) teenoor tebukonasool in vitro (1, 3 en 10 ug/ml) en in vivo vergelyk. Die doel van hierdie studie was om te bepaal hoe wisselende MIK-waardes virulensie (blaaroppervlakte geïnfekteer) en sporulasie sal beïnvloed. Resultate dui daarop dat alle R. secalis isolate in hierdie studie ewe fiks was om, in die afwesigheid van tebukonasool, letsels te induseer en te sporuleer. Die bevinding is dat die verlies in fiksheid nie geassosieer is met die mate van tebukonasool weerstand nie. Alle R. secalis isolate het die vermoë gehad om letsels op tebukonasool-behandelde blare te veroorsaak maar het betekenisvol verskil ten opsigte van die blaaroppervlakte geaffekteer. Isolate wat minder sensitief (MIK = 10 ug/rnl) teenoor tebukonasool in vitro is, het meer aangepastheid op tebukonasool-behandelde blare getoon. Gevolglik het hierdie isolate herhaaldelik meer letsels veroorsaak as sensitiewe isolate (MIK = 1 ug/ml), Sporulasie het nie betekenisvol verskil tussen isolate vanaf letsels op ondehandelde of tebukonsoolbehandelde blare nie. Hierdie resultate dui egter daarop dat 'n minder sensitiewe populasie tot meer siektevoorkoms in tebukonasool-bespuite lande kan lei. Die studie het die veranderlike karakter van die Suid-Afrikaanse R. secalispopulasie aangaande virulensiespektrum en genotipiese struktuur blootgelê. Dit is dus baie moontlik dat die R. secalis-populasie maklik sal aanpas by teelmateriaal met nuwe enkelgeen-weerstand. Vir volgehoue gasheerweerstand is dit egter nodig dat hoër vlakke van kwantitatiewe weerstand ingeteel moet word. In die praktyk is hierdie tipe weerstand egter baie moeiliker om te identifiseer en by nuwe teelmateriaal in te sluit as in die geval van enkelgeen-weerstand, Dit bring mee dat blaarvlekbeheer afhanklik bly van swamdodertoedienings as beheermaatreël. Die resultate van hierdie studie dui daarop dat daar tans 'n lae risiko vir die ontwikkeling van swamdoderweerstand in die plaaslike populasie is, as gevolg van die afwesigheid van kruisweerstandbiedendheid en die lae voorkoms van weerstandbiediende isolate. Ander faktore soos die wisselboustelsels wat toegepas word en die verskeidenheid van swamdoders toegedien dra ook daartoe by. Ten spyte hiervan kan die status van hierdie faktore egter oor tyd verander. Die in vivo tebukonasool studie het daarop gedui dat 'n weerstandbiedende veldpopulasie van R. secalis die potensiaal het om te vermeerder. Gevolglik is die tydige monitering van swamdodersenisitiwiteit van R. secalis isolate noodsaaklik om 'n volhoubare garsproduksie te verseker.

Maize (Zea mays L.) is the third most important cereal crops in the world after wheat and rice. In Ethiopia, maize remains the second largest food security crop after tef [Eragrostis tef (Zucc.) Trotter.]. The mid-altitude, sub-humid agro-ecology (1000 to 1800 m above sea level) is the most important maize producing environment in Ethiopia. However, productivity of maize is low, due to several biotic and abiotic constraints. Among the biotic constraints, Turcicum leaf blight disease of maize caused by Exserohilum turcicum Pass Leonard & Suggs shows high incidence of 95-100% and inflicts significant grain losses in the country. Therefore, high yielding, Turcicum leaf blight resistant and farmers-preferred maize varieties and their production technologies should be developed and made available to growers to enhance maize production and to achieve food security. The objectives of this study were to: (1) assess farmer’s preferences, and production constraints for maize in the mid-altitude, sub-humid agro-ecology of western Ethiopia, (2) determine the genetic variability among elite maize inbred lines and select promising parents for resistance to E. turcicum, (3) determine diversity among the elite germplasm lines using SSR markers, (4) determine combining ability and heterosis among elite maize inbred lines and their hybrids, and (5) investigate genotype x environment interaction and yield stability of experimental maize hybrids developed for the midaltitude sub-humid agro-ecology of Ethiopia. A participatory rural appraisal (PRA) research was conducted involving 240 maize farmers in three representative maize growing zones of western Ethiopia; West Shoa, East Wollega and West Wollega, each represented by two districts and two subdistricts. Maize was ranked number one both as food and cash crop by 82.9% of respondents. Turcicum leaf blight was ranked as number one devastating leaf disease by 46% of respondents. Breeding for improved disease resistance and grain yield, enhancing the availability of crop input and stabilizing market price during harvest time were recommended as the most important strategies to increase maize production by small-scale farmers in western Ethiopia. Fifty inbred lines were evaluated for reaction to Turcicum leaf blight during the main cropping seasons of 2011 and 2012. Inbred lines were clustered into resistant (CML202, 144-7b, 136-a, 139-5j, 30H83-7-1, ILOO’E-1-9, SZYNA-99-F2, and 142-1-e), and susceptible (CML197, CML464, A7033 , Kuleni C1-101-1-1, CML443, SC22-430 (63), (DRB-F2-60-1-2) – B-1-B-B-B, Pool9A-4-4-1-1-1). Inbred lines (CML312, CML445, Gibe-1-158-1-1-1-1, CML395, and 124-b (113)) had intermediate response to the disease. Overall, inbred lines such as CML202, 30H83-7-1, ILOO’E-1-9-1, CML312, CML395 CML445 and 142-1-e were selected with better agronomic performance and resistance to leaf blight for breeding. Twenty selected elite parental inbred lines were genotyped with 20 polymorphic SSR markers. The genotypes used were clustered into five groups consistent with the known pedigrees. The greatest genetic distance was identified between the clusters of lines CML-202 and Gibe-1-91. Eighteen selected inbred lines were crossed using the factorial mating scheme and 81 hybrids developed to determine combining ability effects and heterosis. Inbred lines with high GCA effect (CML 202, CML395, 124-b (113), ILOO’E-1-9 and CML 197) were selected as best combiners for hybrid development. Additionally five high yielding novel single cross hybrids with grain yield of > 8 t ha-1 and high SCA effects were identified such as CML395 X CML442, DE-78-Z-126-3-2-2-1-1 X CML442, ILOO’E-1-9-1-1-1-1-1 X CML312, X1264DW-1-2-2-2-2 X CML464 and SC22 X Gibe-1-91-1-1-1-1. These experimental hybrids are recommended for direct production or as hybrid testers for hybrid development. Genotype x environment interaction (GEI) effects of 81 newly developed and three check maize hybrids were evaluated across 10 locations in the mid-altitude sub-humid agro-ecologies of Ethiopia. The AMMI-3 and GGE biplot models were used to determine stability. Hybrids such as G68, G39, G37, G77, G34 and G2 were identified as the most stable and high yielding at favorable environments such as Bako, Jima, Arsi Negelle and Pawe in Ethiopia. The genotype and genotype by environment interaction (GGE) biplot clustered the 10 environments into three unique mega-environments. Environment I included Bako, Jima, Asossa, Ambo, Finote Selam, Haramaya and Pawe while environment II represented by Arsi-Negelle and environment III Areka and Hawassa. In general, the study identified valuable maize inbred lines with high combining ability for breeding and novel single cross hybrids for large-scale production or as testers for hybrid development at the mid-altitude, sub-humid agro-ecologies of Ethiopia or similar environments in sub-Saharan Africa. / Thesis (Ph.D.)-University of KwaZulu-Natal, Pietermaritzburg, 2013.

Magistrantūros studijų baigiamajame darbe pateikiami ekologiškai auginamų juodojo serbento veislių fenologinių fazių eigos, krūmo augumo, kekių ir derliaus kokybinių rodiklių bei pažeidimo grybinėmis ligomis ir serbentine erkute tyrimo duomenys. Darbo objektas – 10 ekologiškai auginamų juodojo serbento veislių: ‘Titania’, ‘Geo’, ‘Smaliai’, ‘Minaj Šmyriov’, ‘Blizgiai’, ‘Vakariai’, ‘Katiuša’, ‘Gagatai’, ‘Almiai’ ir perspektyvus selekcinis numeris ‘Aldoniai’ (Vakariai x 70-88-9-1). Darbo metodai: Grybinės ligos ir serbentinė erkutė įvertinti maksimalaus pasireiškimo metu. Atskirai apžiūrėjus kiekvieną krūmą, grybinių ligų ir serbentinės erkutės pažeidimai vertinti 6 balų skale (0 – ūgliai, lapai ir pumpurai sveiki; 5 – pažeista virš 75 proc. lapų, ūglių ir pumpurų). Krūmo augumas vertintas prasidedant ir baigiantis vegetacijai. Juodojo serbento kekės ir derliaus kokybė bei produktyvumas nustatytas įvertinus kiekvieno krūmo derlių, uogų nokimo darnumą ir kekių rodiklius. Darbo rezultatai. Veislės ‘Smaliai’ vaiskrūmiai ekologinei sodininkystei tinka dėl vėlyvo žydėjimo, pražysta tik gegužės 10 dieną, tai vertinga veislės savybė, nes vėlai žydinčių serbentų žiedai apsisaugo nuo pavasarinių šalnų pavojaus ir tuo pačiu, išsiskiria stabiliu kasmetiniu derliumi. Juodojo serbento veislės ‘Aldoniai’ (Vakariai x 70-88-9-1) augalai išaugino esmingai daugiau uogų kekėje, daugiau derančių šakų ir šios veislės augalai pasižymėjo geriausiu žiedų ir uogų kekėje santykiu (pakanka 1... [toliau žr. visą tekstą] / In the thesis of Master degree studies is presented the study data about the process of phenological phases of the species of ecologically cultivated blackcurrant, bush height, qualitative indicators of bunches and harvest, also the data about affection by diseases and blackcurrant gall mite. Object of the work – 10 species of ecologically cultivated blackcurrant: ‘Titania’, ‘Geo’, ‘Smaliai’, ‘Minaj Smyriov’, ‘Blizgiai’, ‘Vakariai’, ‘Katiusa’, ‘Gagatai’, ‘Almiai’ and perspective selection number ‘Aldoniai’ (Vakariai x 70-88-9-1). Methods of the work: Fungal diseases and blackcurrant gall mite were assessed during maximum manifestation. Affections by fungal diseases and blackcurrant gall mite after inspection of each bush separately were assessed in accordance with the scale of 6 points (0 - shoots, leaves and buds are healthy; 5 – over 75 percent of leaves, shoots and buds are affected). The bush height was assessed at the beginning and the end of vegetation. Assessment of the quality of blackcurrant bunch and harvest was performed through application of ordinary methods. The results of the work. The breed ‘Smaliai’ is suitable for ecological currant field due to its late blooming, plants bloom only on the 10th of May, this is valuable feature of the breed because the blossoms of the currants that bloom lately are protected against a danger of spring frosts and at the same time exudes a stable annual yield. The number of berries in the bunch of blackcurrant breed... [to full text]

Maize grey leaf spot (GLS), caused by Cercospora zeae-maydis, and potato late blight (LB), caused by Phytophthora infestans, are foliar diseases of maize and potato, two of the most widely grown crops in KwaZulu-Natal (KZN), after sugarcane and timber. Commercial maize in KZN accounts for just on 4.3% of the national maize crop. This is worth R563 million using an average of the yellow and white maize price for the 2001/02 season (at R1 332.87 ton(-1)). In 2003 KZN produced about 5% of the national potato crop (summer crop: 7531 300 10kg pockets from 2243 hectares). This equates to a gross value of R89.4 million based on an average price of R1 188 ton(-1) in 2001. Successful commercial production of maize and potatoes depends upon control of these diseases by translaminar fungicides with highly specific modes of action. This study extends an existing model available for timing of fungicide sprays for GLS and tests and compares two LB models for two calendar-based spray programmes. The study also evaluated the use of an early blight model which is caused by Alternaria solani, and over the single season of evaluation showed potential for use in KZN. For the GLS model it was found that a number of refinements are needed, e.g., the amount of infected maize stubble at planting and not the total amount of maize residue at planting. Based on two years' data, it was found that for the LB models there are no significant differences in levels of control between using a predicted fungicide programme and a calendar-based programme. The importance of knowing initial infection sites, and hence initial inoculum, was demonstrated. This led to the creation of a KZN LB incidence map, now being used to more accurately time the start of a preventative spray programme and to time the inclusion of systemic fungicides in the preventative spray programme. This study has contributed to the further development and expansion of the Automatic Weather Station Network (AWSN) at Cedara, which now comprises 15 automatic weather stations in KZN. The AWSN is currently used to aid farmers and advisers in decision-making regarding fungicide spray timing for GLS and LB. / Thesis (M.Sc.Agric.)-University of Natal, Pietermaritzburg, 2003.

Inoculation with free-living diazotrophic bacteria is well documented to enhance vegetative growth and yield increases of various crops coupled with suppression of sublethal pathogens. The use of microbial inoculants has been identified as an alternative or supplement to use of nitrogenous fertilizers and agrochemicals for sustainable agriculture. The search for effective free-living diazotrophic bacterial strains for formulation as biofertilizers has been on going since the 1970’s and a number of inoculant biofertilizers have been developed and are commercially available. In the current study, 250 free-living diazotrophic bacteria were isolated from soils collected from the rhizosphere and leaves of different crops in different areas within KwaZulu-Natal, province, Republic of South Africa. These were evaluated for plant growth-promotion and biological control of damping-off fungi initially by in vitro screening. The growth promotion traits tested included, phosphate-solubilization, production of indole-3-acetic acid, production of ammonia and acetylene reduction. Biocontrol traits evaluated included siderophore-production, antibiosis, and production of hydrogen cyanide (HCN). Biochemical and molecular bioassay tests were conducted to identify the twenty most promising isolates selected in the in vitro study. The twenty isolates were further tested in combination with various levels of nitrogenous fertilizer for growth-promotion of maize (Zea mays L.) and wheat (Triticum aestivum L.) under greenhouse conditions. The five most promising isolates identified for growth promotion under greenhouse conditions for each crop were assessed for their effects on the germination of maize in vitro and growth and yields of two maize and two wheat cultivars, when combined with a low dose of nitrogenous fertilizer in field trials. The five Bacillus subtilis (Ehrenberg) Cohn isolates that suppressed the growth of a wide range of pathogenic fungi in vitro were tested for their efficacy against damping-off of wheat caused by Rhizoctonia solani Kühn in the greenhouse. These isolates were further studied for their modes of action against R. solani in vitro. The modes of action tested included antibiosis, production of siderophores, extracellular enzymes, production of hydrogen cyanide (HCN) and antibiotic resistance. The twenty most promising bacterial isolates identified from the in vitro screening reduced acetylene to ethylene, produced indole-3-acetic acid and siderophores, one isolate solubilized phosphate, and 9 inhibited the growth of R. solani. These twenty isolates enhanced growth of maize and wheat above the Uninoculated Control under greenhouse conditions. The growth enhancements varied with bacterial isolate x crop species interactions, which identified five different isolates for each of the two crops. Relative to the Uninoculated Control, the best five isolates significantly (P = 0.001) enhanced the growth of maize and wheat at all fertilizer levels for a number of growth parameters: increased chlorophyll levels and heights of maize, shoot dry biomass of maize and wheat, and enhanced root development of maize in the greenhouse. Inoculation of maize and wheat with the two most promising isolates identified from the field trial for each crop, in combination with 65% and 50% of the recommended amount of nitrogenous fertilizer for maize and wheat, respectively, caused the same increases in shoot biomass as the Fully Fertilized Control. Application of a combination of the best bacterial isolates and 35% nitrogenous fertilizer resulted in the same or greater shoot dry biomass and yields of both maize and wheat under field conditions. Shoot dry biomass of wheat increased by 75% above the Uninoculated Control and 30% above the Fully Fertilized Control. The wheat yield increased by 95% above the Uninoculated Control and 43% above the Fully Fertilized Control. Seed inoculation with the best isolates combined with 35% N increased yields of maize by 41% above the Uninoculated Control and 15% above the Fully Fertilized Control. The best isolates significantly (P < 0.001) increased plant height, chlorophyll levels and shoot biomass of maize relative to the Uninoculated Control. There was a positive correlation between chlorophyll level and yield, chlorophyll level and shoot dry biomass, height and shoot dry biomass and height and yield of maize at P = 0.01 with r values of 0.87, 0.77, 0.92 and 0.81, respectively. The isolates that exhibited multiple plant-growth promoting traits in vitro, increased shoot biomass of both maize and wheat in the greenhouse and field, and caused yield increases in the two crops under field conditions. Five B. subtilis isolates inhibited the growth of some of the pathogenic fungi tested in vitro up to 95%. Seed inoculation with the same isolates significantly (P = 0.001) suppressed R. solani damping-off of wheat under greenhouse conditions and exhibited multiple mechanisms of disease control in vitro. The use of microbial inoculants in combination with low doses of nitrogenous fertilizers can enhance crop production without compromising the yields. The B. subtilis isolates obtained in this study can effectively control R. solani damping-off of wheat, fix nitrogen and enhance plant growth. The use of microbial inoculants can contribute to the integrated production of cereal crops with reduced nitrogenous fertilizer inputs, as a key component of sustainable agriculture. Key words: Free-living bacteria; plant growth-promotion; diazotrophs; biological nitrogen fixation; phosphate-solubilization; siderophores; indole-3-acetic acid; biocontrol; damping-off / Thesis (Ph.D.)-University of KwaZulu-Natal, Pietermaritzburg, 2013.